Loss of a Tyrosine-Dependent Trafficking Motif in the Simian Immunodeficiency Virus Envelope Cytoplasmic Tail Spares Mucosal CD4 Cells but Does Not Prevent Disease Progression

Abstract

A hallmark of pathogenic simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections is the rapid and near-complete depletion of mucosal CD4⁺ T lymphocytes from the gastrointestinal tract. Loss of these cells and disruption of epithelial barrier function are associated with microbial translocation, which has been proposed to drive chronic systemic immune activation and disease progression. Here, we evaluate in rhesus macaques a novel attenuated variant of pathogenic SIVmac239, termed ΔGY, which contains a deletion of a Tyr and a proximal Gly from a highly conserved YxxØ trafficking motif in the envelope cytoplasmic tail. Compared to SIVmac239, ΔGY established a comparable acute peak of viremia but only transiently infected lamina propria and caused little or no acute depletion of mucosal CD4⁺ T cells and no detectable microbial translocation. Nonetheless, these animals developed T-cell activation and declining peripheral blood CD4⁺ T cells and ultimately progressed with clinical or pathological features of AIDS. ΔGY-infected animals also showed no infection of macrophages or central nervous system tissues even in late-stage disease. Although the ΔGY mutation persisted, novel mutations evolved, including the formation of new YxxØ motifs in two of four animals. These findings indicate that disruption of this trafficking motif by the ΔGY mutation leads to a striking alteration in anatomic distribution of virus with sparing of lamina propria and a lack of microbial translocation. Because these animals exhibited wild-type levels of acute viremia and immune activation, our findings indicate that these pathological events are dissociable and that immune activation unrelated to gut damage can be sufficient for the development of AIDS.

INTRODUCTION

Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have in common a tropism for activated, memory CD4⁺/CCR5⁺ T lymphocytes that are concentrated in the lamina propria of the gastrointestinal tract and in other mucosal sites (1–10). Studies of SIVmac in nonhuman primates and of HIV-1 in humans have shown that these cells are rapidly and profoundly depleted within the first 1 to 2 weeks of infection (11–14). This loss occurs concomitantly with alterations in intestinal structure, a disruption in epithelial barrier function, and microbial translocation, which have been proposed to drive chronic immune activation and disease progression (1–10). In HIV infection the degree of immune activation is a stronger predictor of disease progression than is plasma viral load (15, 16). Indeed, even when plasma viremia is below the level of detection, due either to elite control (17) or to suppression with antiretroviral therapy (18, 19), the rate of disease progression and non-AIDS related mortality is predicted by measures of T-cell and innate immune activation, independent of plasma virus load. In nonpathogenic models of SIV infection in natural hosts where viral replication occurs without disease progression, mucosal CD4⁺/CCR5⁺ T cells are transiently depleted, but without chronic immune activation, suggesting that additional factors are involved (1, 7, 8, 10, 20, 21).

Pathogenic molecular clones of SIV, such as SIVmac239, have been powerful tools for analyzing viral and host determinants of disease and host immune responses (4, 22, 23). Moreover, genetic modifications of these clones, some of which have created attenuated viruses in vivo, have provided novel insights into the viral determinants of pathogenesis and immune control (22, 24–29). Among attenuated SIVs, SIVmac239Δnef, produced on a SIVmac239 background by a 182-nucleotide deletion within the viral nef gene, has been extensively studied. In adult rhesus macaques, SIVmac239Δnef produces reduced acute plasma viremia, a low to undetectable viral set point, and delayed or absent disease progression (22, 25, 30, 31). Corresponding to its generalized reduction in systemic viral replication and acute viremia, SIVmac239Δnef infection causes little if any loss of CD4⁺ T cells in mucosal tissues (14). Chronically infected animals have also been able to resist challenges with pathogenic SIVs that are genetically homologous to SIVmac239 and represent one of the most promising models for immune protection in the vaccine field; however, the correlates for this effect have remained elusive (25, 26), and protection is considerably diminished for pathogenic heterologous challenge SIVs (27, 29, 32). The mechanism for attenuation caused by the Δnef mutation is unclear, although there are several possibilities including a loss of Nef's ability to downregulate major histocompatibility complex (MHC) class I (33), CD3 (34), and/or Bst-2/tetherin (35). Of note, a pathogenic revertant of SIVmac239Δnef reacquired the ability to downregulate Bst-2/tetherin through novel mutations in the envelope glycoprotein (Env) cytoplasmic tail (30, 35), suggesting that this function could be particularly relevant. Thus, attenuated SIVs, as well as pathogenic revertants that arise in vivo, can provide insights into viral and host correlates and determinants for disease progression and immune control.

Our laboratory has described attenuated variants of SIVmac239 containing mutations in the Env cytoplasmic tail within a highly conserved, membrane-proximal cellular trafficking motif, YxxØ, where Y is a Tyr, x is any amino acid, and Ø is an amino acid with a bulky hydrophobic side chain (36). This motif, which for primate lentiviruses also includes a proximal Gly (e.g., for SIVmac239, GYRPV at amino acids 720 to 724), is present without exception in all HIVs and SIVs (37). Either this GYxxØ motif or its associated Tyr have been shown (i) to bind to cellular adaptor proteins and mediate clathrin-dependent endocytosis of Env (37–42), (ii) to downregulate the steady-state expression of Env on the surface of infected cells (38, 40, 42, 43), and (iii) to mediate basolateral sorting of Env and viral budding in polarized epithelial cells (44). When Tyr-721 was mutated to an Ile, a change that ablated endocytic function of this motif (42), and given intravenously (i.v.) to two rhesus macaques, one animal developed high plasma viremia and progressed to AIDS, associated with rapid reversion of the Ile to a Tyr (36). In the other animal, the Ile was retained, and although the acute viral RNA peak was high, the viral set point became undetectable with no laboratory or clinical evidence of disease for up to 2 years (36). When a more extensive mutation, a 6-nucleotide deletion that removed Gly-720 and Tyr-721 (termed ΔGY), was introduced and given to three animals, two also developed a high acute viral peak followed by an undetectable viral set point and no evidence of disease. In the third animal, a high viral set point developed, with rapid progression to AIDS. Although the ΔGY mutation in this animal was retained, a flanking Ser-to-Pro substitution at amino acid 727 (S727P) occurred. Collectively, these findings, albeit in small numbers of animals, indicated that (i) there was in vivo selection pressure to maintain the YxxØ motif, (ii) loss of this motif did not prevent robust early viral replication, but did result in host control, and (iii) an S727P mutation was implicated as a possible compensatory change even though it did not recreate a recognizable YxxØ motif (36).

In the present report, we describe a comprehensive pathological and clinical evaluation of the effects of the ΔGY mutation on SIV pathogenesis in rhesus macaques while concurrently identifying the viral adaptations generated in vivo. ΔGY showed an acute viral peak that was indistinguishable from historical SIVmac239- or SIVmac251-infected controls. However, in spite of this robust early replication and abundant viral replication in organized lymphoid tissues that comprise immune inductive sites (e.g., Peyer's patches, intestinal lymphoid nodules, spleen, and tonsil), ΔGY infection in the diffuse lamina propria of the gut, the predominant site of CD4⁺ CCR5⁺ effector memory T lymphocytes, was markedly reduced with only transient infection observed. Lamina propria CD4⁺ T cells were only slightly depleted during acute infection, and there was no evidence of microbial translocation. ΔGY also exhibited reduced efficiency for vaginal transmission and did not infect central nervous system (CNS) tissues. However, although ΔGY-infected animals developed viral set points that were initially reduced compared to SIVmac239 and acutely spared gut CD4⁺ T cells, after several months plasma viral RNA increased with a progressive loss of peripheral blood central memory CD4⁺ T cells, chronic systemic immune activation, and clinical and pathological evidence of disease progression. Possible compensatory mutations in the Env cytoplasmic tail were observed, including the generation of new YxxØ motifs and novel point mutations, most notably the previously observed S727P (36). ΔGY's phenotype of robust acute viral replication with sparing of mucosal CD4⁺ T cells is unique among pathogenic models of SIV infection. In addition to highlighting novel effects of the YxxØ motif on pathogenesis, our findings indicate that immune activation and disease can occur in the absence of microbial translocation and that there are likely to be additional causes of immune activation and disease progression besides a loss of epithelial barrier function.

MATERIALS AND METHODS

Animals, viral inoculations, and sample collection.

A total of 22 Indian-origin rhesus macaques (Macaca mulatta) were used for the longitudinal study. Ten macaques were infected with SIVmac239ΔGY (ΔGY) produced from of 293T cells transfected with plasmids containing a full-length provirus, and a control group included 12 macaques infected with either SIVmac239 (n = 8) or SIVmac251 (n = 4) (Table 1). Prior to use, all of the animals were negative for SIV, STLV, and type D retrovirus. In addition, all of the animals except for four of the controls were MHC typed (Table 1) as described previously (45). Clinical findings were recorded during the course of infection, and full pathological and histopathological examinations were performed on all euthanized animals. Multiple blood samples, cerebrospinal fluid (CSF) samples, and small-intestinal biopsy specimens (from either endoscopic duodenal-pinch biopsies or a 2-cm jejunal resection biopsy) were collected under anesthesia (ketamine hydrochloride or isoflurane) at various time points from each animal, as indicated in the figures and figure legends. Animals were euthanized if they exhibited a loss of >25% of maximum body weight, anorexia for more than 4 days, or major organ failure or medical conditions unresponsive to treatment (e.g., severe and nonresponsive pneumonia or diarrhea).

Table 1.

Animals, virus strain, and experimental groups^a

Group	Animalb	Sex	Routec	Inoculum	Dose (TCID50)d	Euthanasia (wpi)e	Mamu typef
ΔGY	DT18	M	i.v.	ΔGY	100	109	A*08, B*01
	EC51	M	i.v.	ΔGY	100	103	A*08, B*17
	HF68	M	i.v.	ΔGY	100	2	DRBw201, A*02
	GH57	M	i.v.	ΔGY	100	2	DRBw201
	DT14	M	i.v.	ΔGY	100	4	A*01, A*02
	DI93	M	i.v.	ΔGY	100	4	DRBw201, A*02, A*08, B*01
	GL50	M	i.v.	ΔGY	100	7	A*02, A*11
	GP82	M	i.v.	ΔGY	100	7	A*11
	DD84*	F	i.vag.	ΔGY	350	78	A*08
	DD87†	F	i.vag./i.v.	ΔGY	350/100	77	DRBw201, A*08
	CG32‡	F	i.vag.	ΔGY/SIVmac239	350/1,000	4	A*08
	CT16‡	F	i.vag.	ΔGY/SIVmac239	350/1,000	4	A*02, B*01
Controls	CF52	M	i.v.	SIVmac239	100	69	DRBw201
	CK76	M	i.v.	SIVmac239	100	52
	CL16	F	i.v.	SIVmac239	100	24
	CT83	M	i.v.	SIVmac239	100	151	DRBw201, A*01
	DE70	M	i.v.	SIVmac239	100	30	DRBw201
	DP30	F	i.v.	SIVmac239	100	57	DRBw201, A*01
	DR43	M	i.v.	SIVmac239	100	30
	EE54	F	i.v.	SIVmac239	100	52
	DD44	M	i.v.	SIVmac251	100	72
	DE49	M	i.v.	SIVmac251	100	53
	DF49	M	i.v.	SIVmac251	100	51
	DE57	M	i.v.	SIVmac251	100	35

^aData obtained from individual rhesus macaques infected with ΔGY and control animals infected with either SIVmac239 or SIVmac251 are shown corresponding to the schematic shown in Fig. 1. Note that CG32 and CT16 were not included in the longitudinal study of ΔGY because they were not infected after serial i.vag. ΔGY inoculations.

^b*, DD84 was infected with 350 TCID₅₀ ΔGY by i.vag. infection and euthanized at week 78 due to progressive disease (see Table S1 in the supplemental material). †, DD87 was monitored for signs of infection for 3 months after serial i.vag. ΔGY inoculations and remained uninfected. It was then inoculated i.v. with ΔGY at 100 TCID₅₀ and monitored for chronic infection. ‡, CG32 and CT16 were monitored for signs of infection for 3 months after serial i.vag. ΔGY inoculations and remained uninfected. They were then inoculated i.vag. once with 1,000 TCID₅₀ of SIVmac239, became infected, and were euthanized at week 4.

^ci.v., intravenous; i.vag., intravaginal.

^dTCID₅₀, 50% tissue culture infectious dose; A shill (/) indicates “followed by.”

^ewpi, weeks postinfection. Boldface text indicates euthanasia due to progressive disease. Normal text indicates euthanasia took place at the end of the study.

^fAnimals infected with ΔGY were tested for the following Mamu types: DRBw201, A*01, A*02, A*08, A*11, B*01, B*08, B*04, and B*17. The SIVmac239-infected control group was tested for only DRBw201 and A01, except for animal CK76, which was tested for only A*01. None of the SIVmac251-infected control animals was tested. Only positive alleles are shown.

All animals were maintained at the Tulane National Primate Research Center in accordance with the standards of the Association for Assessment and Accreditation of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals prepared by the National Research Council. The Tulane Institutional Animal Care and Use Committee approved all studies.

Twelve animals were challenged intravenously (i.v.) or intravaginally (i.vag.) with ΔGY and monitored longitudinally or were euthanized at 2, 4, or 7 weeks after inoculation. Eight animals were challenged i.v. with 100 50% tissue culture infective doses (TCID₅₀) once and four animals were challenged i.vag. with ΔGY (350 TCID₅₀) at weekly intervals eight times (CG32, CT16, and DD87) or until infected (DD84). Animals were not pretreated with progestational agents to thin the vaginal mucosa for i.vag. inoculations. DD87 was subsequently inoculated i.v. with ΔGY and monitored longitudinally. CG32 and CT16 were challenged i.vag. once with SIVmac239, became infected, and were euthanized at 4 weeks after inoculation. A control group (n = 12) was inoculated i.v. with either SIVmac239 or SIVmac251 (Table 1).

Quantitation of viral load in plasma and CSF.

Plasma viral loads were determined using the bDNA assay (Siemens Diagnostics, Emeryville, CA) for all animals with limits of detection of 125 or 700 SIV RNA copies/ml, as indicated. A more sensitive quantitative reverse transcriptase PCR (RT-PCR) with a limit of detection of fewer than 70 copies/ml (Jeff Lifson, NIH, NCI-Frederick) was performed on the CSF samples from ΔGY-infected animals (46).

Lymphocyte isolation from intestinal tissues.

Cells from the intestine were collected by using either endoscopic pinch biopsies or by 2-cm surgical resections of the jejunum, from animals at various time points. The isolation of cells from jejunal tissues has been described elsewhere (47). Briefly, intestinal cells were isolated by using EDTA-collagenase digestion and Percoll density gradient centrifugation.

Detection of SIV-specific antibody production.

Serum and vaginal secretions from animals challenged intravaginally with 350 TCID₅₀ of SIVmac239ΔGY (CG32, CT16, DD84, and DD87) were assessed for the presence of SIV-specific antibodies as described previously (48).

Analysis of plasma LPS and sCD14 levels.

To measure lipopolysaccharide (LPS), plasma samples (collected in EDTA) were first diluted to 10% in endotoxin-free water, heated to 80°C for 10 min, and then quantified in duplicate with a commercially available Limulus amebocyte lysate assay (Lonza, Basel, Switzerland) performed according to the manufacturer's protocol (2, 19). To measure sCD14 in plasma samples (collected in EDTA) a commercially available enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) was used. Samples were diluted to 0.5% and tested in duplicate, as described previously (49).

Immunophenotyping and analysis of cell turnover.

T cell immunophenotyping was performed on isolated lamina propria lymphocytes (LPLs) and anticoagulated whole blood. Immunophenotyping of the LPLs was performed with the antibodies CD3 (SP34-2), CD4 (L200), and CD8 (SK1) (all from BD Biosciences, San Jose, CA). Intracellular cytokine staining was then performed on the LPLs; however, only data for the surface markers are shown here. Immunophenotyping of whole blood was performed with the antibodies CD3 (SP34-2), CD8 (SK1), CD4 (L200), CD95 (DX2), and CD28 (28.2) (all from BD Biosciences except CD28 [28.2], which was obtained from Beckman Coulter). Cells were stained for 30 min at room temperature in the dark.

CD4⁺ and CD8⁺ lymphocytes and monocytes were also analyzed for immune activation and turnover by measuring the expression of HLA-DR on the surfaces of CD4⁺ and CD8⁺ lymphocytes and BrdU (5-bromo-2′-deoxyuridine) incorporation, respectively. For turnover experiments, a preparation of BrdU (Sigma) was administered as described previously (50, 51). Briefly, a solution was made by adding BrdU to 1× phosphate-buffered saline and heated to 60°C in water bath. A single dose (60 mg/kg) of BrdU was administered i.v. Blood samples were collected for flow cytometry at 24 h after BrdU administration. The samples were collected at necropsy from animals chronically infected with ΔGY (DD87, DT18, and EC51) and compared to a control group of rhesus macaques that were not infected (n = 8 for the percent HLA-DR CD4⁺ and CD8⁺ T cells, n = 6 for the percent BrdU CD4⁺ and CD8⁺ T cells, and n = 17 for the percent BrdU CD14⁺ monocytes) and to historical control groups chronically infected with SIVmac but either not yet progressing to AIDS (n = 6 for the percent HLA-DR CD4⁺ cells, n = 15 for the percent HLA-DR CD4⁺ and CD8⁺ T cells, n = 12 for the percent BrdU CD4⁺ and CD8⁺ T cells, and n = 20 for percent BrdU monocytes) or progressing to AIDS (n = 5 for the percent BrdU CD4⁺ T cells and monocytes and n = 4 for the percent BrdU CD8⁺ T cells); these groups were from a previous study (51). Lymphocytes were analyzed for immune activation and turnover with the antibodies CD3 (SP34-2), CD8 (SK2), CD4 (L200), BrdU (3D4), and HLA-DR (L234). Monocytes were immunophenotyped by using antibodies to CD3 (SP34-2), CD8 (SK2), CD14 (M5E2), BrdU (3D4), HLA-DR (L234) (all from BD Biosciences), CD20 (B9E9; Beckman Coulter), and CD45 (MB4-6D6; Miltenyi Biotech). The cells were stained in the dark for 30 min at room temperature. Erythrocytes were lysed with 1× fluorescence-activated cell sorting (FACS) lysis buffer (BD Biosciences). BrdU staining was performed using a BrdU flow kit (BD Biosciences). The samples were analyzed by using a FACS LSRII or a FACSAria flow cytometer (BD Biosciences).

Flow cytometry data were analyzed using FlowJo software (TreeStar, Inc., Ashland, OR). Lymphocytes were analyzed by gating through side-scatter and forward-scatter lymphocytes, CD3⁺ cells, CD4⁺ cells, and, if used, CD28⁺ CD95⁺, CD28⁺ CD95⁻, and CD28⁻ CD95⁺ cells. CD4⁺ and CD8⁺ lymphocyte turnover was measured in select samples by gating through CD3⁺, CD4⁺, or CD4⁺ and then BrdU⁺. Monocytes were analyzed using the same software and identified as CD3⁻, CD8⁻, CD20⁻, HLA-DR⁺, CD14⁺, and CD45⁺. Monocyte turnover was measured in select samples by gating for monocytes (as described above) and then BrdU⁺. Absolute cell counts from whole blood were measured by multiplying complete blood count (CBC) data with the percentages of different cell subset populations as determined by flow cytometry. For CD4⁺ T-lymphocyte memory phenotypes, the number of samples collected at each time point for ΔGY was n = 3 except for weeks −3, 16, 20, 24, 28, and 55 (n = 4) and week 87 (n = 2).

In situ hybridization.

Tissue samples collected at surgery or necropsy were fixed in Z-Fix (Anatech, Battle Creek, MI) and embedded in paraffin, and 5-μm-thick sections were cut and adhered to charged glass slides. SIV RNA in situ hybridization (ISH) was then performed as described previously (52) using SIV RNA sense/antisense probes (Lofstrand Labs, Gaithersburg, MD) tagged with digoxigenin. The chromogens used were NBT/BCIP for light microscopy or liquid permanent red for fluorescence microscopy. The results were assessed by bright-field optical microscopy (Leica DM LB microscope and Spot Insight camera/software) or confocal microscopy (Leica TCS SP2 confocal microscope equipped with three lasers).

Quantification of SIV RNA ISH-positive cells was performed on SIVmac239ΔGY-infected jejunal sections at different time points postinfection and compared to SIVmac239-infected historical time-matched control samples. SIV RNA ISH-positive cells were counted in 10 high-power fields (×20) for each section of the jejunum. Images were collected using liquid crystal tunable filter multispectral imaging (Nuance multispectral imaging system; CRI, Inc., Woburn, MA) and then segmented by pattern recognition into different tissue subcompartments—lamina propria, organized lymphoid nodules, epithelium, and background—using image analysis software (Inform; CRI), and the tissue area was then quantified. SIV RNA ISH-positive cells were then counted manually in the different images, and the data are expressed as infected cells/mm² by dividing the number of infected cells counted by the tissue area.

Confocal microscopy.

Double- and triple-label confocal microscopy was performed to colocalize SIV RNA with cell type-specific markers to determine the immunophenotype of infected cells, as described previously (53). Immunofluorescence labeling for T cells (rabbit polyclonal antibody to CD3, catalog no. CP215B; Biocare Medical, Concord, CA) and macrophages (mouse IgG1 monoclonal antibody to CD68, catalog no. MO814; Dako, Carpinteria, CA) was performed after ISH as previously described (53). After incubation with the primary antibodies (anti-CD3 or anti-CD68) and subsequent washes, the following appropriate species-specific secondary antibodies were applied: Alexa Fluor 488- or 633-conjugated goat anti-rabbit (Invitrogen, Carlsbad, California) and Alexa Fluor 488- or 633-conjugated goat anti-mouse IgG1 (Invitrogen), respectively. Confocal microscopy was then performed using sequential mode to separately capture the fluorescence from the different fluorochromes (Leica Microsystems, Exton, PA). NIH Image v1.62 and Adobe Photoshop v7 software were used to correct the colors collected by the different channels (Alexa 488 [green], Alexa 568 [red], Alexa 633 [blue]) and differential interference contrast (DIC [gray scale]).

Single-genome amplification (SGA) analysis.

From each plasma specimen, ∼20,000 viral RNA copies were extracted by using a QIAamp viral RNA minikit (Qiagen). RNA was eluted and immediately subjected to cDNA synthesis. Reverse transcription of RNA to single-stranded cDNA was performed using SuperScript III RT according to the manufacturer's recommendations (Invitrogen). In brief, a cDNA reaction of 1× RT buffer, 0.5 mM concentrations of each deoxynucleoside triphosphate, 5 mM dithiothreitol, 2 U of RNaseOUT (RNase inhibitor)/ml, 10 U of SuperScript III RT/ml, and 0.25 mM antisense primer SIVEnvR1 5′-TGT AAT AAA TCC CTT CCA GTC CCC CC-3′ was incubated at 50°C for 60 min and 55°C for 60 min and then heat-inactivated at 70°C for 15 min, followed by treatment with 2 U of RNase H at 37°C for 20 min. The newly synthesized cDNA was used immediately or frozen at −80°C.

The entire env gene was sequenced from each animal at the indicated time points by using a limiting-dilution PCR so that only one amplifiable molecule is present in each reaction. This SGA approach was performed by serially diluting cDNA distributed among independent PCRs to identify a dilution where amplification occurred in <25% of the total number of reactions. PCR amplification was performed with 1× PCR buffer, 2 mM MgCl₂, 0.2 mM concentrations of each deoxynucleoside triphosphate, 0.2 μM concentrations of each primer, and 0.025 U of Platinum Taq polymerase (Invitrogen)/μl in a 20-μl reaction. First-round PCR was performed with sense primer SIVEnvF1 5′-CCT CCC CCT CCA GGA CTA GC-3′ and antisense primer SIVEnvR1 5′-TGT AAT AAA TCC CTT CCA GTC CCC CC-3′ under the following conditions: 1 cycle of 94°C for 2 min, 35 cycles at 94°C for 15 s, 55°C for 30 s, and 72°C for 4 min, followed by a final extension of 72°C for 10 min. Next, 1 μl from the first-round PCR product was added to a second-round PCR that included the sense primer SIVEnvF2 5′-TAT AAT AGA CAT GGA GAC ACC CTT GAG GGA GC-3′ and antisense primer SIVEnvR2 5′-ATG AGA CAT RTC TAT TGC CAA TTT GTA-3′ performed under the same conditions used for first-round PCR, but with a total of 45 cycles. Correct-sized amplicons were identified by agarose gel electrophoresis and directly sequenced with second round PCR primers and six SIV-specific primers using BigDye terminator technology (Applied Biosystems). To confirm PCR amplification from a single template, chromatograms were manually examined for multiple peaks, a finding indicative of the presence of amplicons resulting from PCR-generated recombination events, Taq polymerase errors, or multiple variant templates. Sequences containing two or more ambiguous sites were excluded from analysis. SGA was performed three times on each ΔGY-infected animal—DT18, DD84, and EC51—at weeks 37, 63, and 95 and on animal DD87 at weeks 7, 32, and 64.

Statistical analysis.

All statistical analyses were performed using GraphPad Prism v5.0c (GraphPad Software, Inc., La Jolla, CA). When we compared two groups, a Mann-Whitney t test was carried out. Kaplan-Meier survival curves were created and compared using the log-rank test.

Nucleotide sequence accession numbers.

All 152 SIV env sequences discussed here have been deposited in GenBank under accession numbers JQ744061 to JQ744213.

RESULTS

Protocol to evaluate ΔGY in comparison to SIVmac239.

ΔGY was created by deleting six nucleotides in the distal region of the SIVmac239 envelope glycoprotein membrane- spanning domain and proximal cytoplasmic tail, effectively removing Gly-720 and Tyr-721 (SIVmac239 numbering). Tyr-721 contributes to the consensus trafficking signal, YxxØ, which is highly conserved in all HIV and SIV envelope glycoproteins (Y = tyrosine, x = any amino acid, and Ø = an amino acid with a bulky hydrophobic side chain). Gly-720 contributes to the endocytic function of this motif in SIVmac239 (42). We previously hypothesized that ΔGY would be significantly attenuated compared to the parental virus SIVmac239. In this previous work in rhesus macaques, ΔGY resulted in infection characterized by a high acute plasma RNA peak, followed by a low to undetectable set point viremia (36). To assess more completely the impact of the ΔGY mutation on pathogenesis, rhesus macaques were inoculated intravenously (i.v.) or intravaginally (i.vag.) with virus produced from SIVmac239 containing the ΔGY mutation (Fig. 1). For i.v. infections, two animals (EC51 and DT18) received a single inoculum (100 TCID₅₀) of ΔGY and were monitored for 103 and 109 weeks, respectively (Table 1).

Fig 1.

Schematic overview of animals infected with SIVmac239ΔGY (ΔGY). Rhesus macaques infected with ΔGY are shown with animal identifiers indicated. Three animals were inoculated i.v. (100 TCID₅₀). One (DD84), inoculated weekly i.vag. (350 TCID₅₀ per inoculation), became infected after the second inoculation. Animal DD87 remained uninfected after 8 i.vag. inoculations and was subsequently infected i.v. with ΔGY. These four chronically infected animals were monitored for up to 109 weeks. Six animals were inoculated i.v. with ΔGY and euthanized at weeks 2, 4, and 7 to examine early pathological events in tissues. Not shown are the results for 12 rhesus macaques inoculated i.v. with either SIVmac239 (n = 8) or SIVmac251 (n = 4).

For i.vag. infections, four animals were inoculated weekly (350 TCID₅₀ per dose) for a maximum of eight doses (Fig. 1, Table 1). Of these, one (DD84) became infected after the second inoculation, while the other three (CG32, CT16, and DD87) remained negative for viral RNA and showed no evidence of infection for an additional 3 months, as determined by plasma viral RNA. To further confirm these findings, anti-SIV antibodies and SIV-specific CD8⁺ T-cell responses were measured in the i.vag.-challenged animals. No anti-SIV antibody responses were observed in CG32, CT16, or DD87, whereas DD84 exhibited positive SIV-specific IgG titers in both sera (1:1,700,000) and vaginal secretions (1:256). Consistent with these findings, positive antigen-specific intracellular cytokine responses by CD8⁺ T cells were only observed in DD84 in both the vaginal lamina propria and the intestinal lamina propria. All samples from the other three animals had no responses above background.

DD87 was subsequently inoculated i.v. with ΔGY (100 TCID₅₀) and followed until termination of the study at 77 weeks postinfection. The two remaining animals (CG32 and CT16) became infected after a single i.vag. inoculation of wild-type SIVmac239 (1,000 TCID₅₀) and were euthanized 4 weeks after infection (Table 1). Thus, four animals (DT18, EC51, DD84, and DD87), three infected i.v. and one infected i.vag., were available for studies of chronic ΔGY infection (Fig. 1, Table 1). To evaluate the effects of the ΔGY mutation on early pathogenic events in tissues, six additional animals were infected i.v., with two animals euthanized at 2, 4, or 7 weeks postinoculation (Fig. 1, Table 1). In addition, 12 rhesus macaques inoculated i.v. with either SIVmac239 (n = 8) or the closely related pathogenic virus SIVmac251 (n = 4) served as wild-type controls (Table 1). MHC alleles were determined for all ΔGY and SIVmac239-infected animals (Table 1). Among the four ΔGY-infected animals monitored during chronic infection, only EC51 contained an MHC allele associated with SIVmac239 control (Mamu-B*17) (54).

Viral dynamics in plasma and CSF and survival in ΔGY-infected rhesus macaques.

As expected, plasma viral RNA in SIVmac239-infected animals peaked acutely at a high level (1.46 × 10⁷ ± 5.70 × 10⁶ copies/ml, mean ± the standard error of the mean [SEM]) and was followed by a high viral set point (mean = 8.7 × 10⁶ ± 4.4 × 10⁶ copies/ml), defined as the viral load 10 weeks after infection (Fig. 2A and C). Acute RNA peaks for ΔGY-infected animals, including four chronically infected animals and four animals euthanized at weeks 4 or 7 postinoculation (Fig. 1 and Fig. 2B and C), were comparable to SIVmac239 (1.02 × 10⁷ ± 1.64 × 10⁷ copies/ml, P > 0.05) but were delayed by ∼1 week (Fig. 2A and B). This high RNA peak was followed by a reduced set point (3.9 × 10⁴ ± 1.4 × 10⁴ copies/ml, P < 0.01) (Fig. 2C). However, although plasma RNA in ΔGY-infected animals continued to decrease up to week 20, an increase was subsequently seen in all animals, with one (DD84) progressing to disease by week 79 (Fig. 2B and see Table S1 in the supplemental material).

Fig 2.

Viral loads and survival of infected animals. Plasma viral loads are shown for rhesus macaques infected with SIVmac239 (A) or SIVmac239ΔGY (ΔGY) (B). The “†” symbol indicates time points at which individual animals were euthanized because of clinical evidence of disease. The threshold of sensitivity for viral RNA (dashed line) was 125 copies/ml for the SIVmac239 group and 700 copies/ml for the ΔGY group. Animal identifiers are shown for each group. (A) All eight SIVmac239-infected animals had high acute viral peaks at 2 weeks after infection and high viral set points and progressed to disease. (B) All four ΔGY-infected animals showed high acute viral peaks at 3 weeks after infection but reduced viral set points. DD87, inoculated 3 months later than the other ΔGY-infected animals, was euthanized at week 77 of ΔGY infection with termination of the study. (C) Acute peak (left) plasma RNA levels and set points (right) are shown for individual animals (± the SEM). Acute peak values for the ΔGY and SIVmac239 groups were comparable (P > 0.05), while set points for the ΔGY group were significantly lower than for SIVmac239 group (P < 0.01). (D) Peak CSF viral loads were comparable between ΔGY (n = 5 and excludes DD84 due to blood contamination and includes two ΔGY-infected animals euthanized week 7 postinoculation) and SIVmac239 (P > 0.05). (E) CSF viral loads are shown for in four animals infected with ΔGY and eight animals infected with SIVmac239. For ΔGY-infected animals, CSF viral load peaked at week three and rapidly fell to undetectable levels, whereas for SIVmac239-infected animals the CSF viral load peaked at week 2, fell to a nadir between weeks 7 and 13, and then increased. CSF viral loads were determined for SIVmac239-infected animals using a bDNA assay (Siemens Diagnostics, limit of detection = 125 copies/ml [dashed/dotted line]) or for ΔGY-infected animals by quantitative RT-PCR (Jeff Lifson, NCI-Frederick, limit of detection = 70 copies/ml [dotted line]). (F) Kaplan-Meier survival curves. Survival for the ΔGY-infected animals was significantly longer than for SIVmac239-infected animals (P < 0.05). The median survival for the SIVmac239 group was 52 weeks; the median survival for the ΔGY-infected animals could not be determined since only one animal (i.e., DD84) died with disease during the study period.

Viral RNA in the CSF was also monitored to assess possible differences in viral entry and persistence in the CNS (Fig. 2D and E). Consistent with prior pathogenic SIV infections (55, 56), SIVmac239-infected animals showed a high CSF RNA peak that was close to the time of peak plasma viremia with subsequent viral dynamics that mirrored what was observed in blood, including increasing viral load with disease progression. For ΔGY-infected animals, the peak CSF viral RNA was similar to the acute peak of SIVmac239 (2.92 × 10⁴ ± 1.63 × 10⁴ versus 3.63 × 10⁴ ± 2.43 × 10⁴, respectively P > 0.05) (Fig. 2D) and, as in plasma, occurred 1 week later (Fig. 2E). Thereafter, virus in the CSF declined, but late in the course of the infection three animals again had detectable virus: DT18 (440 copies/ml at week 47), DD84 (220 copies/ml at week 77, and 2,900 copies/ml at necropsy), and EC51 (530 copies/ml at week 70, and 1,300 copies/ml at week 95) (Fig. 2E).

Kaplan-Meier plots for survival of SIVmac239- and ΔGY-infected animals are shown in Fig. 2F. Typical for SIVmac239, infected animals maintained high viral loads for up to 120 weeks with 7 of 8 (88%) animals progressing to disease by week 69 with declining peripheral blood CD4⁺ T cells and clinical features of simian AIDS (see below). In contrast, only one of four ΔGY-infected animals (DD84 at week 79) progressed to clinical disease during the course of the present study.

Collectively, these results indicated that ΔGY was (i) highly replication fit with an acute plasma viral peak that was comparable to SIVmac239, (ii) only transiently present in the CSF, and (iii) initially better controlled than SIVmac239 with a reduced set viral point. However, while survival was prolonged, all ΔGY-infected animals developed rising plasma viral RNA and clinical features of disease progression (see below).

Peripheral blood CD4⁺ T cells and clinical parameters of disease progression in ΔGY-infected animals.

To evaluate laboratory parameters associated with SIV-induced immunodeficiency, we determined both the percentage (Fig. 3A, C, and E) and the absolute numbers (Fig. 3B, D, and F) of peripheral blood CD4⁺ T cells. Both SIVmac239-infected and ΔGY-infected animals showed progressive decreases in these parameters, although the rate of decline for the percentage of CD4⁺ T cells was reduced for ΔGY (Fig. 3A). Individually, three of four ΔGY-infected animals developed CD4⁺ T-cell numbers of ≤200 cells/ml by week 87, with the remaining animal (DD87) being euthanized at week 77 with progressive disease (see below) (Fig. 3F). ΔGY-infected animals also exhibited a loss of both central memory and naive CD4⁺ T cells (Fig. 3G and H), although no loss was observed in the effector memory CD4⁺ T cells (Fig. 3I). Thus, in spite of the viral set points that were initially reduced compared to SIVmac239, over time, all of the ΔGY-infected animals lost peripheral blood CD4⁺ cells, a finding consistent with disease progression, albeit at a slower rate than SIVmac239 (Fig. 3A and B).

Fig 3.

Changes in the percentage and absolute number of peripheral blood CD4⁺ T cells. The percentages (A, C, and E) and absolute numbers (B, D, and F) of peripheral blood CD4⁺ T cells are shown for animals chronically infected with ΔGY or SIVmac239. Averages for each group (± the SEM) (A and B) are shown, as well as for individual animals (C, D, E, and F). The percentages of CD4⁺ T cells declined more slowly in animals infected with ΔGY compared to SIVmac239-infected animals (P < 0.02). (G to I) Absolute counts of central memory (CD95⁺ CD28⁺) (G), naive (CD95⁻CD28⁺) (H), and effector memory (CD95⁺ CD28⁻) CD4⁺ (I) T lymphocytes from the blood of SIVmac239ΔGY (ΔGY) chronically infected animals. The absolute count of central memory and naive CD4⁺ T cells declined during chronic ΔGY infection. Animals that died or were euthanized prior to the conclusion of the study are indicated (†).

Necropsy results of ΔGY-infected animals showed additional findings consistent with disease progression, including generalized lymphoid hyperplasia and dysplasia in lymph nodes, thymic atrophy, pulmonary arteriopathy, and pneumonia, possibly due to Pneumocystis jiroveci (see Table S1 in the supplemental material). At necropsy, SIV was readily detectable by RNA in situ hybridization (ISH) in lymphoid follicles in lymph nodes and spleen and in organized lymphoid tissues of gastrointestinal mucosa (see Table S2 in the supplemental material). Of note, despite an extensive search, neither SIV-infected nor multinucleated giant cells (a hallmark of SIV infection) were detectable at necropsy in the brain parenchyma of any ΔGY-infected animal.

Intestinal CD4⁺ T cells are minimally depleted during acute infection in ΔGY-infected animals.

We next evaluated CD4⁺ T cells in the lamina propria (CD4⁺ LPL) in ΔGY-infected animals and in four SIVmac251-infected controls. These cells are rapidly depleted during acute HIV-1 infection in humans (11–13) and in both pathogenic and nonpathogenic models of SIV infection (7, 10, 14, 57). As expected, all four SIVmac251-infected animals showed a profound loss of CD4⁺ LPL (mean = 43.6% ± 3.0% preinfection to <5% by week 2 postinfection) (Fig. 4A and B) corresponding to the peak of plasma viremia (Fig. 2A and C). These cells remained at this level throughout the course of infection. Remarkably, although ΔGY-infected animals exhibited acute plasma RNA peaks that were indistinguishable from SIVmac239 (Fig. 2C), CD4⁺ LPLs were unchanged or only modestly depleted at week 3, with levels ranging from 16 to 58% (Fig. 4B and D). Over time and concurrent with a decline in peripheral blood CD4⁺ T cells (Fig. 3), a gradual reduction in CD4⁺ LPL cells occurred to levels of 8, 21, and 28% (for DD84, DT18, and DD87, respectively), although one animal (EC51) euthanized at week 95 with a high plasma viral RNA (9.6 × 10⁶ copies/ml) and clinical signs of disease progression (generalized lymphoid hyperplasia and dysplasia, thymic atrophy, and pneumonia) maintained a CD4⁺ LPL level of >40% up to the time of necropsy. Thus, ΔGY-infected animals showed a striking and distinctive profile of pathogenesis in that while they exhibited high acute plasma RNA peaks and ultimately progressed to disease, they showed little acute loss of CD4⁺ LPLs. However, with increasing viral loads and disease progression, there was a gradual decline of CD4⁺ LPLs in the setting of a general decline in peripheral CD4⁺ cells, as reflected in the peripheral blood.

Fig 4.

Changes in percentage of CD4⁺ T cells in the intestine. CD4⁺ lamina propria lymphocytes (LPLs) obtained from endoscopic small-intestinal biopsy specimens are shown for animals infected with SIVmac251 or ΔGY and expressed as a percentage of CD3⁺ T cells. The averages (A) and mean percentages of CD4⁺ LPLs (B) (± the SEM) are shown at, or shortly after, the acute peak of plasma viremia (i.e., week 2 for SIVmac251 and week 3 for ΔGY). Values for individual animals infected with SIVmac251 (C) or ΔGY (D) are also shown.

Analysis of viral replication in small intestine.

Given the reduction in the magnitude and kinetics of CD4⁺ T-cell decline in the lamina propria for ΔGY-infected animals, we evaluated the distribution and kinetics of ΔGY-infection in comparison to SIVmac239 using ISH in sequential small intestine biopsy specimens or necropsy samples during early infection (i.e., up to 7 weeks) (Fig. 5 and 6). For SIVmac239, abundant viral RNA was detectable at week 1 in the lamina propria and the epithelial compartment (Fig. 5D). By week 2, corresponding to the severe depletion of CD4⁺ LPL (Fig. 4A), infection shifted to organized lymphoid nodules, including Peyer's patches (Fig. 5e). For ΔGY, virus was absent in the lamina propria, epithelia, and organized lymphoid tissues at week 1 (Fig. 5A), a finding consistent with its delayed kinetics of viremia (Fig. 5F). By week 2, RNA was detectable in the lamina propria and the epithelial compartment (Fig. 5B), although in a remarkably sparse and multifocal distribution (see Fig. S1 in the supplemental material). Strikingly, by week 4 and all time points thereafter, ΔGY RNA was undetectable in the diffuse lamina propria (Fig. 5C), despite there being an abundance of CD4⁺ T cells in this compartment (Fig. 4), whereas virus was readily detectable in organized lymphoid nodules (Fig. 5C and see Table S2 in the supplemental material).

Fig 5.

Localization of SIVmac239 and ΔGY-infected cells in the intestine. Localization of SIV RNA by in situ hybridization in jejunum of animals infected with ΔGY (A, B, and C) or SIVmac239 (D and E). The time points analyzed are shown in panel F relative to the levels of plasma RNA for each virus. At week 1 postinfection, for ΔGY-infected animals no infected cells were found (A), but for SIVmac239-infected animals infected cells were present throughout the lamina propria and epithelium (D). By week 2, ΔGY infection was in localized patches in cells in immune inductive sites (i.e., organized lymphoid nodules) (not shown) and immune effector sites (lamina propria and epithelium) (B), but this infection was sparsely distributed (see also Fig. S1 in the supplemental material). (E) In contrast, at this same time point in SIVmac239-infected animals most infected cells were in the organized lymphoid nodules corresponding to the loss of CD4⁺ T cells in the lamina propria (Fig. 4A and C). By weeks 3 and 4, ΔGY-infected cells were rare and, when present, were limited to organized lymphoid nodules (C). A single positive cell is shown in an organized lymphoid nodule (black arrow).

Fig 6.

Quantitation of SIV-infected cells in the lamina propria. Quantitation of SIVmac239- or ΔGY-infected cells in the lamina propria is shown at the indicated times postinfection. Individual infected cells were detected by in situ hybridization and cells/mm² counted as described in Materials and Methods. Each symbol represents data from a single image with the mean ± the SEM shown for each group. A highly significant increase was seen for SIVmac239 (red) infection at week 1 compared to ΔGY (blue) at week 2 (time points 1 week prior to their peaks of plasma RNA) (P < 0.0001). The decline in SIVmac239-infected cells at week 2 was associated with a loss of CD4⁺ lymphocytes from this compartment (Fig. 4A and C). In contrast, although the number of ΔGY-infected cells also declined to very low levels at weeks 4 and 7, CD4⁺ lymphocytes persisted (Fig. 4A and D).

When the number of infected cells in the intestines was quantified during acute infection (Fig. 6), the peak for SIVmac239 occurred at week 1 and was markedly increased compared to the peak for ΔGY (at week 2) (P < 0.0001) (Fig. 6). This finding was consistent with the diffuse, widespread distribution of SIVmac239-infected cells compared to the patchy, multifocal distribution for ΔGY (Fig. 5 and see Fig. S1 in the supplemental material). As noted above, ΔGY RNA declined to very low levels in the intestine at week 4 and was confined to organized lymphoid nodules despite the persistence of CD4⁺ T cells in the lamina propria (Fig. 4 and 5; see also Table S2 in the supplemental material). Thus, in marked contrast to SIVmac239, which rapidly and profoundly depleted CD4⁺ LPL, ΔGY infection in the diffuse lamina propria was transient and associated with a modest or no reduction in LPLs despite a level of acute plasma viremia that was indistinguishable from SIVmac239.

ΔGY-infected cells in tissues were CD3⁺ T cells and not CD68⁺ macrophages.

To examine the immunophentoype of infected cells in ΔGY-infected animals, multilabel confocal microscopy was performed on tissues labeled for viral RNA by ISH and stained with either anti-CD3⁺ or anti-CD68⁺ to identify T cells or macrophages, respectively. Tissues biopsied from jejunum, submandibular lymph nodes, and tonsils at weeks 2 and 4 were analyzed, and additional samples, including brain tissues, were obtained postmortem. As shown in Fig. 7, viral RNA in lymphoid tissue was detectable in CD3⁺ T cells and not in CD68⁺ macrophages. These findings were in marked contrast to our published studies in SIVmac239-infected rhesus macaques, which showed widespread infection of both macrophages and T cells within the first month of infection (58, 59). As noted previously, ΔGY RNA was also undetectable in brain macrophages or microglia from animals euthanized during acute infection or after progression to AIDS, despite the early presence of virus in the CSF (Fig. 2E). Thus, in contrast to SIVmac239, ΔGY's in vivo tropism was restricted to T lymphocytes throughout the course of infection, even in animals that progressed to AIDS.

Fig 7.

Immunophenotype of cells infected by ΔGY. Multilabel confocal microscopy was performed on tissues obtained from ΔGY-infected animals at necropsy using anti-CD3 and anti-CD68 antibodies to detect T cells and macrophages, respectively. (A) Jejunal tissue from animal GH57 at week 2 after infection. SIV-infected cells detected by SIV RNA in situ hybridization (red) are present in the lamina propria and within the epithelium (white arrow). SIV-infected cells do not colocalize with CD68⁺ cells (green). (B) Triple-label analysis of tonsil from animal DI93 at week 4 after infection. All of the SIV-infected cells detected by RNA in situ hybridization (red) colocalized with CD3⁺ cells (green), producing a yellow color in the merged image (white arrows). No SIV-infected cells colocalize with CD68⁺ macrophages (blue). In each image the individual channels are shown on the left, and the larger merged image is shown on the right.

Evaluation of microbial translocation and T-cell activation.

Chronic immune activation is closely associated with pathogenic HIV and SIV infection (2, 5, 6). Although its etiology is under intense investigation (1), the loss of CD4⁺ T cells in the lamina propria with epithelial barrier disruption and translocation of microbial products to the systemic immune system have been proposed as important contributing factors (2, 6). Given the transient nature of ΔGY's infection and early sparing of CD4⁺ LPL, we evaluated parameters of microbial translocation and immune activation. When plasma sCD14 and lipopolysaccharide (LPS) levels were measured, no change from preinfection levels was observed over 95 weeks of follow-up (Fig. 8A) despite the fact that all four ΔGY-infected animals showed progressive disease (Fig. 2 and see Table S1 in the supplemental material). These findings are in contrast to published studies of pathogenic SIVmac infection where sCD14 and LPS are typically elevated during chronic infection and progression to disease (2, 6). Thus, during ΔGY infection, consistent with its mild but transient infection in lamina propria, there was no evidence of epithelial barrier disruption and/or microbial translocation, as determined by sCD14 and plasma LPS levels.

Fig 8.

Microbial translocation, immune activation, and lymphocyte and monocyte turnover in ΔGY and SIVmac239 or SIVmac251 (SIVmac) infection. (A) Plasma LPS and soluble CD14 (sCD14) levels, as indicators of microbial translocation, are plotted for each time point (mean ± the SEM) for ΔGY-infected animals. LPS and sCD14 remained at preinfection levels up to week 95 of infection. (B and C) HLA-DR expression expressed as a percentage of peripheral CD4⁺ (B) or CD8⁺ T cells (C) is shown for naive (uninfected) animals and animals chronically infected with ΔGY or SIVmac (mean ± the SEM) with relative P values indicated. HLA-DR on CD4⁺ and CD8⁺ T cells was increased in the ΔGY group compared to naive animals (P < 0.05) and was similar to SIVmac-infected animals. (D and E) Lymphocyte turnover assessed by bromodeoxyuridine (BrdU) incorporation is shown in CD4⁺ (D) and CD8⁺ T cells (E) of ΔGY-infected animals and in SIVmac-infected animals with chronic infection and with terminal AIDS. For the ΔGY group, the percentage of cells incorporating BrdU was increased relative to naive controls (P < 0.05). (F) Monocyte turnover assessed by BrdU incorporation in the animal groups of panels D and E. ΔGY-infected animals had levels similar to those of naive animals and significantly lower than those of SIVmac animals with AIDS (P < 0.05).

To assess immune activation, we measured HLA-DR expression on CD4⁺ and CD8⁺ T cells during chronic infection. In ΔGY-infected animals, the percentage of HLA-DR⁺ CD4⁺ T cells (11.4% ± 5.6%) (mean ± the SEM) was significantly greater than in uninfected controls (1.5% ± 0.4%) (P < 0.05) and was similar to SIVmac239-infected (or SIVmac251-infected) animals (12.3% ± 2.3%) (Fig. 8B). HLA-DR⁺ CD8⁺ T cells were also elevated in ΔGY-infected animals (16.4% ± 6.0%) compared to uninfected controls (2.9% ± 0.8%) (P < 0.05), and no difference was found between ΔGY- and SIVmac239- (or SIVmac251)-infected animals (5.4% ± 1.2%) (P > 0.05) (Fig. 8C).

Because lymphocyte proliferation in general correlates with immunological markers of activation (51), we examined BrdU incorporation in CD4⁺ and CD8⁺ T cells. In ΔGY-infected animals, consistent with their increased expression of HLA-DR, BrdU incorporation was elevated in both CD4⁺ (7.3% ± 2.0%) (P < 0.05) and CD8⁺ T cells (7.4% ± 2.0%) (P < 0.05) relative to naive controls (1.2% ± 0.1% and 2.2% ± 0.1%, respectively) (Fig. 8D and E). As expected, BrdU incorporation in CD4⁺ T cells was low in SIVmac239-infected animals with fully developed AIDS where these cells were markedly depleted (Fig. 8D) but increased in CD8⁺ T cells in animals with or without AIDS (Fig. 8E) compared to naive controls. Thus, in ΔGY-infected animals, all of which had progressive disease, there was evidence of immune activation, as determined by increased HLA-DR expression and cell turnover of CD4⁺ and CD8⁺ T cells, although at no point during the course of their infection and disease progression did these animals exhibit evidence of elevated microbial translocation.

Evaluation of monocyte turnover with BrdU labeling.

We also evaluated BrdU incorporation in peripheral blood monocytes, since recent studies have shown that increased monocyte turnover is a robust correlate of SIV disease progression (50, 51). As expected, all SIVmac-infected animals that progressed to AIDS showed increased BrdU incorporation in monocytes compared to naive (uninfected) controls, whereas no increase was seen in animals that were chronically infected without AIDS (Fig. 8F). Interestingly, although ΔGY-infected animals showed increased HLA-DR expression and turnover on CD4⁺ and CD8⁺ T cells, there was no increase in monocyte BrdU incorporation (Fig. 8F). Thus, although ΔGY-infected animals showed increased immune activation and turnover in lymphocytes, they exhibited no increase in monocyte turnover.

Viral evolution in ΔGY-infected animals.

To determine the stability of the ΔGY mutation in vivo and whether possible compensatory mutations arose during disease progression, single-genome analysis (SGA) and sequence analysis of env clones from plasma was performed at the indicated time points for animals DT18, DD84, DD87, and EC51 (Fig. 9). For each animal, the timing of SGA analyses relative to the level of plasma viral RNA is shown, along with the full amino acid sequences of the cytoplasmic tail from all env clones (see Fig. 2A to D).

Fig 9.

Molecular evolution of ΔGY in chronically infected animals. Single-genome amplification (SGA) of full-length env genes was performed at the indicated time points (Wk = week postinfection) on rhesus macaques chronically infected with ΔGY (n = 4). (A) On a schematic of the SIVmac239 cytoplasmic tail showing the membrane-spanning domain (MSD) and the location of the ΔGY mutation in the cytoplasmic tail, mutations that either became the predominant change in a single animal or were seen in more than one animal are diagrammed with their sequences shown in the lower portion of the panel. The amino acid numbering (for SIVmac239) identifies the location of each mutation. (B) For each animal at the indicated time points postinfection the number of env amplicons containing the designated mutations (shown in panel A) is shown and represented as a heat map with color codes for the proportion of positive sequences shown in the figure key. Specific mutations are discussed in the text. The ΔGY mutation was retained in all sequences of ΔGY-infected animals. S727P, previously reported in a ΔGY-infected rhesus macaques, was seen (36). The R751G mutation is present in all sequences and has been reported as an “optimizing” change that is acquired in vivo in SIVmac239-infected animals (30). Full amino acid sequences of the cytoplasmic tails of env clones from chronic ΔGY-infected animals are shown in Fig. S2A to D in the supplemental material.

The ΔGY mutation was detected in all clones analyzed, which is consistent with the evidence that this change did not preclude robust replication in vivo (Fig. 2B and C). However, in each animal additional changes were observed. Figure 9a shows a diagram of mutations that occurred in at least 40% of the env clones in the cytoplasmic tail in any of the four ΔGY-infected animals. These included (i) a duplication and insertion of AKL (ins-AKL) at the end of the predicted membrane-spanning domain with or without an L-to-V mutation in the insert, (ii) R722G flanking the ΔGY mutation, (iii) more distal point mutations S727P, Q738K, R746K, H831Y, and VW to FR at amino acid positions 837 to 838, and (iv) a deletion of QTH at residues 734 to 736. For all clones, including those from a time point as early as 7 weeks postinoculation (i.e., for DD87 [see Fig. S2D in the supplemental material]), an Arg-to-Gly change at position 751 (R751G) occurred that has been well described during SIVmac239 replication in vivo, an observation consistent with the view that this mutation or an associated coding change in the overlapping second exon of rev optimizes SIVmac239 for replication (30).

Figure 9B shows the relative frequency of these mutations expressed as a heat map as they arose over time in each animal. Although some changes were only seen in a single animal, e.g., ins-AKL and VW(837-338)FR, several others were more common, e.g., R722G, S727P, and ΔQTH. One common mutation, S727P, which became the predominant sequence in DT18 and DD87, was previously described in a single ΔGY-infected rhesus macaque that developed a high viral load and AIDS (36). Of note, ΔQTH, with or without a flanking I732L (occurring in DT18 and DD84), and H831Y created new Tyr-based motifs that conformed to the consensus YxxØ that the ΔGY mutation disrupted (i.e., YFQI for DT18, YFQL for DD84, and YEAV for EC51). Of note, H831Y occurred in EC51, a Mamu-B*17 animal, and has been previously described during SIVmac251 infection as a CTL escape mutation for this allele (54). R722G, which flanked the ΔGY mutation, although failing to create a recognizable YxxØ motif, was seen in all four animals at various frequencies and occurred in up to 80% of amplicons (i.e., 27 of 34 amplicons) for animal DD84. Other changes shown in Fig. 9 (e.g., R722G, ΔQTH, and ins-AKL) have not been described in SIVmac239- or SIVmac251-infected rhesus macaques in the GenBank sequence database, indicating they were likely selected for as a result of the ΔGY mutation. There were also distinct patterns in which these changes occurred within Env clones in that S727P and R722G, although present in single animals at the same time (i.e., DT18 and DD87), were never present in the same amplicon (see Fig. S2A and B in the supplemental material), suggesting that their effects, if compensatory, were not additive or complementary in vivo.

Overall, these findings indicated that during ΔGY replication, mutations in the Env cytoplasmic tail evolved that may reflect changes compensating for disruption of the conserved GYRPV motif. Although some mutations created new YxxØ motifs, there were clearly other mutations that did not generate an identifiable YxxØ motif but occurred in more than one animal (i.e., R722G, S727P, and ΔQTH), signifying evolutionary convergence to repair whatever defect the ΔGY mutation had caused.

DISCUSSION

Pathogenic models of SIV infection in Asian macaques recapitulate key features of human HIV infection, including high levels of acute and steady-state viremia, tropism for CD4⁺ CCR5⁺ T lymphocytes with a progressive loss of these cells from peripheral and mucosal immune sites, and chronic immune activation (1–10). In nonpathogenic SIV infection in African natural hosts, acute and chronic replication occur to high levels and CD4⁺ CCR5⁺ T cells are also targeted, but these animals fail to develop chronic immune activation and disease (4, 7, 8, 10, 20, 21, 30). It has therefore become of central importance to understand the causes of chronic immune activation in pathogenic infection, since it is a critical component of disease progression. Common to both pathogenic and nonpathogenic models is the rapid infection and depletion of CD4⁺ CCR5⁺ effector memory T cells in mucosal tissues, particularly the gastrointestinal tract, although at this site the depletion of absolute cell numbers is significantly less in nonpathogenic models (7, 10, 14, 20). In pathogenic infection these cells are massively and irreversibly depleted by direct viral infection and possibly activation-induced apoptosis, associated with the local release of proinflammatory cytokines, robust and sustained type 1 interferon responses, and a preferential loss of Th17 cells from the lamina propria with a disruption of epithelial barriers (1, 60). In nonpathogenic infection, local immune responses occur in the mucosa but are transient, and epithelial barrier function remains intact (4, 6). Damage to epithelial barriers in pathogenic SIV infection and in human AIDS has been associated with translocation of microbial products from the intestinal lumen to regional lymphoid tissues and the systemic circulation and has been implicated as a direct cause of immune activation (2, 6). However, while virally induced gut damage has closely correlated with disease, a number of additional explanations for chronic immune activation have been proposed (see below) (1). It is therefore, unclear whether infection of gut lymphoid tissue, epithelial damage, and microbial translocation are true causes or close correlates of disease evolution.

In the present report we describe a novel alteration in SIV pathogenesis resulting from the deletion of two amino acids from a highly conserved, Tyr-dependent trafficking motif (GYxxØ) in the cytoplasmic tail of the SIVmac239 Env. Consistent with a prior report, which showed that this ΔGY mutation or a Y721I substitution in the GYRPV motif produced little effect on in vitro growth of SIVmac239 in rhesus macaque peripheral blood mononuclear cells (36), in vivo ΔGY replicated acutely to a viral peak that was comparable to SIVmac239. Although the time to peak viremia was delayed by ∼7 days, ΔGY clearly established robust early infection of multiple peripheral lymphoid tissues, as determined by an abundance of infected cells in the spleen, tonsils, intestines, and mesenteric lymph nodes. However, in contrast to SIVmac239, which, as reported by our group as well as many others, causes profound acute depletion of CD4⁺ CCR5⁺ T cells in the lamina propria, ΔGY infection in this compartment was patchy and transient, producing only a mild acute reduction in CD4⁺ T cells. Correspondingly, no microbial translocation was observed in ΔGY-infected animals throughout the course of their infection, as determined by plasma LPS and sCD14. However, these animals still developed chronic immune activation with increased HLA-DR expression and BrdU incorporation in CD4⁺ and CD8⁺ T cells, and all developed progressive disease, albeit at a rate slower than SIVmac239- or SIVmac251-infected animals, with increasing plasma viral RNA, gradually declining peripheral and mucosal CD4⁺ T cells, and pathological findings at necropsy indicative of disease progression. Of note, the prototypic live attenuated SIV, SIVmac239Δnef, exhibits a markedly reduced peak viral replication compared to the parental SIVmac239 (14). The finding that ΔGY, unlike SIVmac239Δnef, replicated to high levels in plasma and peripheral lymphoid tissues during early infection and yet spared intestinal CD4⁺ T cells is novel but more generally demonstrates that gut infection with associated disruption of epithelial barrier function may not be an absolute requirement for chronic immune activation and disease progression.

Although gut damage and microbial translocation have been proposed to drive chronic immune activation in both humans (2, 61) and macaques in pathogenic models of SIV infection (2, 6), there are other possible causes including immunomodulatory effects of viral proteins and/or host responses, bystander activation from proinflammatory cytokines, and ineffective regulation of innate immunity (1). In addition, pathogenic HIV and SIV infections have been closely correlated with a decline in CD4⁺ central memory T cells consistent with direct infection, bystander killing, and/or exhaustion of their regenerative capacity (3). Recently, in a nonpathogenic model of SIV infection in sooty mangabeys, central memory CD4⁺ T cells were shown to express low levels of surface CCR5 upon activation that restricted SIV infection, suggesting that host adaptation to prevent infection of these critical cells in vivo could limit infection and disease progression (8). Alternatively, host-specific differences in the kinetics, magnitude, and anatomic sites of infection and coordination between early innate and later adaptive immune responses have been proposed to play key roles leading to the tolerance of viral replication seen in nonpathogenic hosts in contrast to the sustained and ultimately deleterious immune responses of the pathogenic host (49, 62–65). As noted above, given that ΔGY-infected animals progressed to AIDS with chronic immune activation in the absence of early gut damage, it is likely that alternative drivers of this sustained host immune response are responsible.

Aside from what is the cause of immune activation in ΔGY-infected animals, the in vivo consequences of this two amino acid deletion are striking. Moreover, because in the present report, the ΔGY mutation was introduced on a full-length SIVmac239 genome in contrast to our earlier study where the virus contained a stop codon in nef (36) that rapidly reverted in vivo, it is clear that the observed alterations in pathogenesis were the direct result of GY deletion. Why should the disruption of the GYxxØ trafficking motif lead to an anatomical redistribution of infection that spares immune effector sites in the lamina propria without affecting infection in peripheral immune tissues? Although for SIV and HIV-1, this motif binds to adaptor AP2 complexes at the plasma membrane and mediates highly efficient, clathrin-dependent endocytosis (37–39, 41, 42, 66), it also binds to AP1 and AP3 in the trans-Golgi and endosomes and could participate in other pathways involved in Env intravesicular trafficking, processing, or delivery to sites of viral assembly (37, 41). For HIV-1, a region encompassing the analogous Tyr-721 of SIVmac239 (Tyr-712 in HXB numbering) has been shown to direct Env sorting and virion budding to the basolateral surface of polarized cells (67). It is possible that within the lamina propria microenvironment, viral infection of CD4⁺ T cells and spread could require directional budding of virus, which has been described in lymphoid cells (68). Alternatively, diffuse infection at this site could be more dependent on cell-to-cell spread, which requires the formation of a virological synapse between infected cells and target cells (69). Env is critical for this synapse to function (70, 71), and it is possible that the ΔGY mutation disrupts Env delivery to and/or formation of this structure. Dendritic cells have also been proposed to play a key role in targeting virions to mucosal sites (72, 73) and alterations in the incorporation or expression of Env trimers on virions could impact interactions with dendritic cells and impair this process. In addition, HIV-1 Env has been shown to bind to the α4β7 integrin on CD4⁺/CCR5⁺ effector memory cells in lamina propria, possibly directing virions and/or virally infected cells to this site (72, 74, 75), and quantitative or qualitative alterations in Env on particles or infected cells caused by the ΔGY mutation could impede this interaction as well. Notably, for both SIVmac and HIV-1, there are additional but less well defined regions outside the proximal GYxxØ motif that can mediate endocytosis, which would not be eliminated by a ΔGY mutation (37, 42, 66, 76). Thus, given that endocytic function is a redundant feature of the Env cytoplasmic tail, it is likely that additional functions are involved in gut-sparing phenotype of ΔGY in vivo.

ΔGY exhibited additional alterations in tropism in vivo. Despite an extensive search in diverse lymphoid tissues using in situ hybridization and multilabel confocal microscopy, only CD3⁺ T cells and not CD68⁺ macrophages were infected throughout the course of infection. These findings are in contrast to SIVmac239, where despite the limited ability of this virus to infect macrophages in vitro (23, 77, 78), abundant infection of cells in the monocyte/macrophage lineage is typically seen (58, 59, 79). These findings could not be explained by differences in CD4 tropism or coreceptor utilization, since the ΔGY Env showed no changes in CD4 or CCR5 utilization in cell-cell fusion assays or as an infectious virus (J. Hoxie, unpublished observations). Given that SIV- and HIV-infected monocytes likely traffic to brain as “Trojan horses” to initiate CNS infection (80), the lack of monocyte/macrophage infection in ΔGY-infected animals could also explain the remarkable sparing of CNS tissues from infection. Despite high level of plasma viremia with an associated acute but transient CSF viremia, no virus was found in brain parenchyma in animals euthanized at 14, 28, and 50 days postinfection or at necropsy after signs of disease progression. These findings are in marked contrast to SIVmac239 where virus is readily detectable in diverse anatomic regions of brain in fully developed disease and early in infection (80–83). Interestingly, transmission of ΔGY across a mucosal barrier may also be impaired. Despite a rigorous mucosal challenge protocol in which animals were inoculated intravaginally every week for 8 weeks, only one of four animals (DD84) became infected. Although these numbers are small, this finding is consistent with the possibility that ΔGY is impaired in the ability to spread across barriers (i.e., vaginal mucosa and the blood brain barrier) and within certain tissue compartments such as the intestinal mucosa. There is abundant evidence that cell-cell interactions involving lymphocytes, dendritic cells, and epithelial cells play roles in transmission of HIV and SIV across epithelial and mucosal barriers (84, 85). Efforts to understand qualitative and quantitative alterations in Env on virions and on infected cells, as well as its interactions with dendritic cells and other migratory cells within these compartments, are currently in progress.

Although ΔGY was initially controlled to a set point that was 1 to 2 logs less than SIVmac239, plasma viral RNA increased in all animals with declining peripheral and lamina propria CD4⁺ T cells as they progressed to disease. Furthermore, although the ΔGY mutation persisted, changes outside this region appeared, raising the possibility that they could be compensatory. Among the more striking changes were those creating new Tyr-based motifs corresponding to the consensus YxxØ that was ablated by ΔGY. In animals DT18 and DD84, a 9-nucleotide deletion arose in different reading frames deleting amino acids QTH at positions 734 to 736 to produce YFQI (in DT18) and YFQL (in DD84) (see Fig. S2A and C in the supplemental material). These changes are particularly striking given that they also ablated a highly conserved splice acceptor site for the second exons of tat and rev (86). Efforts are in progress to determine the fate of transcripts for these essential viral genes, but it is clear that a strong selection pressure must have been required for such a change to occur in vivo in two different animals. In animal EC51 an H831Y mutation occurred creating the sequence YEAV (see Fig. S2B in the supplemental material). However, this animal also expressed the Mamu-B*17 allele, and given that this region lies within an epitope for Mamu-B*17 cytotoxic T lymphocytes and that H831Y has been described in SIVmac239-infected macaques (54), it is unclear whether this change was related to the ΔGY mutation or simply arose to enable escape from CTL responses. It remains to be determined whether any of these new YxxØ motifs restore endocytic function or the ability to engage cellular adaptor proteins.

In addition to these changes, point mutations in the cytoplasmic tail also occurred in different animals, including the same change in multiple animals. Among these, S727P, which occurred in three of four animals (DT18, DD87, and EC51), was of particular interest. This mutation was described in our previous report of a ΔGY-infected rhesus macaque that rapidly progressed to AIDS with a high viral load. Although not creating a recognizable endocytosis signal, S727P was proposed to represent a novel compensatory change (36). Ser-727 flanks the sequence PPSY, and given the role of PPxY motifs in binding WW domains of cellular proteins, which include Nedd4 family members and ubiquitin E3 ligases that can facilitate assembly of retroviruses through interactions with Gag proteins (87, 88), it is intriguing to speculate that this change could have promoted an interaction with cellular factors involved with Env processing and/or transport to sites of virion assembly. Remarkably, this same change was also reported in a pathogenic variant of SIVmac239Δnef (30). Although mutations acquired in the Env cytoplasmic tail of this virus were shown to reestablish an interaction with rhesus BST-2/Tetherin, which had been lost with the nef deletion, S727P was not directly involved in this effect (35). It seems likely that this change conferred a growth advantage to ΔGY- and SIVmac239Δnef-infected animals (30), although the basis for this apparent convergence in viral evolution is unclear. Another mutation seen in at least one time point in all four ΔGY-infected animals was an R722G flanking the GY deletion. This change also does not create a recognizable endocytosis signal but could provide clues in revealing underlying mechanisms for the altered pathogenesis of ΔGY. Interestingly, although R727G and S727P were observed in multiple Env clones from different animals and even within the same animal, they were never observed together in same clone, suggesting that any putative gain of function that they conferred was not additive.

Our findings with the ΔGY mutant reveal a critical role for the GYxxØ motif in vivo. This motif is also conserved throughout the phylogeny of all known lentiviruses with the exception of equine infectious anemia virus, suggesting that it has played an important role in their evolution (not shown). For SIVmac239, we have found little effect in vitro by disrupting this motif with either ΔGY or a Y721I mutation (36), although defects in viral replication have been reported for these changes in HIV-1 (89). Although SIVmac239 with a Y721I mutation could replicate at wild-type levels in vitro and showed no evidence of reversion after multiple passages in T-cell lines (36; unpublished observations), reversion of this mutation was documented in an infected rhesus macaque that subsequently progressed to AIDS (36). Although in the present report differences in the in vivo spread of the ΔGY mutant suggest an alteration in viral assembly, replication, and/or transmission, it is also possible that ΔGY could be more susceptible to host immune responses. Indeed, the initially low set points in all ΔGY-infected animals that followed high acute peaks suggest that, at least initially, this virus was more susceptible to host control. Preliminary results of ΔGY infection in pigtail macaques, a species that is highly susceptible to SIVmac239-induced disease (90), have shown that ΔGY also replicates acutely to wild-type levels, but with the onset of host immune responses viral load is profoundly suppressed to a level 2 to 3 logs lower than in rhesus macaques (Hoxie, unpublished). Studies to identify the defects in ΔGY replication, as well as host responses that are relevant to its control in different species of nonhuman primates, will be the subject of future reports.

In summary, we have shown that the ΔGY mutation in the GYxxØ trafficking motif of SIVmac239 Env produces a novel alteration in pathogenesis with (i) acute sparing of lamina propria CD4⁺ T cells despite wild-type levels of replication, (ii) a lack of microbial translocation, and (iii) the absence of infection in macrophages and the CNS. Studies that address viral, cellular, and host determinants for this anatomical redistribution of infection will be of considerable interest. Most importantly for this study, because ΔGY-infected rhesus macaques still developed immune activation and disease, this model demonstrates that chronic immune activation, acute loss of mucosal CD4⁺ T cells, and gut damage can be dissociated and that alternative causes of systemic immune activation outside the gut can contribute to disease progression.