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. Author manuscript; available in PMC: 2009 Jun 1.
Published in final edited form as: J Gen Virol. 2008 Jun;89(Pt 6):1421–1433. doi: 10.1099/vir.0.83464-0

Genomic and Biological Characterization of Aggressive and Docile Strains of LCMV Rescued from a Plasmid-Based Reverse Genetics System

Minjie Chen 1,$, Shuiyun Lan 1,$, Rong Ou 1,$, Graeme E Price 1,$,†, Hong Jiang 1, Juan Carlos de la Torre 2, Demetrius Moskophidis 1,*
PMCID: PMC2652504  NIHMSID: NIHMS81420  PMID: 18474558

Abstract

Arenaviruses include several causative agents of hemorrhagic fever disease in humans. In addition, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a superb model for the study of virus-host interactions, including the basis of viral persistence and associated diseases. The molecular mechanisms concerning the regulation and specific role of viral proteins in modulating arenavirus-host cell interactions associated either with an acute or persistent infection and associated disease remain little understood. Here we report the genomic and biological characterization of LCMV strains Docile (persistent) and Aggressive (not persistent) recovered from cloned cDNA via reverse genetics. Our results confirmed that the cloned viruses accurately recreated the in vivo phenotypes associated with the corresponding natural Docile and Aggressive viral isolates. In addition, we provide evidence that the ability of the Docile strain to persist is determined by the nature of both S and L RNA segments. Thus, our findings provide the foundation for studies aimed at gaining a detailed understanding of viral determinants of LCMV persistence in its natural host that may aid in the development of vaccines to prevent or treat the diseases caused by arenaviruses in humans.

MeSH Keywords: Viral infection, LCMV, Arenaviruses, RNA virus, T cell response, Reverse genetics technique, viral persistence

Introduction

The family of Arenaviridae includes 23 recognized members that have been classified into two phylogenetically distinct but related groups called the Old World and New World complexes. Arenaviruses are zoonotic pathogens that include several etiological agents of fatal hemorrhagic fever in humans, including the Lassa, Junin, Machupo, Guanarito and Sabia viruses (Emonet et al., 2006). They are rodent-associated viruses, with the single exception of Tacaribe virus, which infects bats, and their geographical distribution is determined by the range of the reservoir species. The worldwide distribution of LCMV reflects its ubiquitous natural reservoir, Mus musculus. In contrast, other arenaviruses are endemic to specific geographic regions including Sub-Sahara West Africa (Lassa virus) and South America (Junin, Machupo, Guanarito and Sabia viruses). Human infections occur from inhalation of aerosolized virus or by direct contact with contaminated material (Damonte & Coto, 2002, Jay et al., 2005, Peters, 2002). Despite the intense study of arenavirus biology since the isolation of the first family member, LCMV, in the 1930s, the molecular determinants of arenavirus-induced disease in humans and animals remain little understood.

Arenaviruses are enveloped viruses with a bi-segmented negative strand RNA genome consisting of a small (S) and a large (L) segment with approximate sizes of 3.4 and 7.2 kb, respectively (Meyer et al., 2002, Romanowski & Bishop, 1985). Each RNA segment has an ambisense coding strategy, encoding two proteins in opposite orientation separated by an intergenic region (IGR). The S RNA directs synthesis of the nucleoprotein (NP) (ca. 63 kDa) and two mature virion glycoproteins, GP1 (40–46kDa) and GP2 (35 kDa), derived from post-translational cleavage of a precursor polypeptide, GP-C (75 kDa). GP1 and GP2 make up the spikes on the virion envelope. GP1 mediates the virus interaction with its host cell receptor, identified as α-dystroglycan for Lassa and Old World viruses or transferrin receptor 1 for the New World arenaviruses (Cao et al., 1998, Kunz et al., 2002, Radoshitzky et al., 2007). The L RNA segment encodes the viral polymerase (L; ca.200 kDa) and a small polypeptide Z (ca. 11 kDa) containing a zinc-binding RING finger motif. Evidence indicates that Z is the arenavirus counterpart of the matrix (M) protein found in many negative RNA viruses (Perez et al., 2003).

LCMV provides investigators with a superb model for the investigation of viral immunology and pathogenesis (Ahmed et al., 1996, Hotchin, 1971, Lehmann-Grube, 1972, Oldstone, 2006, Traub, 1936, Zinkernagel et al., 1993). In particular, LCMV infection in the mouse has been used to study the dynamics of virus-host interactions in the context of viral persistence, uncovering two extreme scenarios regarding the interaction between the virus and the host immune system. The first is that a sustained CD8+ T cell-mediated response results in virus clearance within 2 weeks after infection. The second situation involves a transient CD8+ T cell response, in which antigen-specific CD8+ T cells are induced and proliferate, initially exhibiting antiviral function but progressively losing this ability (clonal exhaustion), which leads to viral persistence (Moskophidis et al., 1993). Such functionally deficient T cells persist in the host for long periods but may eventually be eliminated (Zajac et al., 1998, Zhou et al., 2004). Both outcomes (viral clearance or persistence) are of limited pathological consequence for the host and are determined by the strength and magnitude of the virus-specific immune response and the rate of virus replication (Leist et al., 1988, Moskophidis et al., 1995, Thomsen et al., 1996). Thus, fast growing LCMV isolates, such as Docile (Doc) strain, readily induce persistent infection, whereas the slower replicating Aggressive (Agg) isolate does not (Ahmed et al., 1984, Moskophidis et al., 1995, Pfau et al., 1982). Notably, both Doc and Agg isolates were derived from the same parental UBC strain of LCMV, suggesting high genomic similarity between these two strains and hence providing a valuable model for studying viral determinants of persistent infections. In this study to elucidate the basis of persistent infections and associated diseases caused by LCMV in its natural host, we provide detailed genetic and biological characterization of Doc and Agg strains with respect to their differential ability to cause persistent infections.

Materials and Methods

Mice and animal experiments. Mice C57BL/6 (B6), were obtained from Jackson Laboratories, Bar Harbor, ME or National Cancer Institute, Frederick, MD. Animals were maintained and experiments performed in accordance with institutional animal welfare guidelines.

Viruses. LCMV-Doc and Agg (isolated from an LCMV-UBC-carrier mouse) were obtained from Dr. C.J. Pfau (Troy, N.Y) (Pfau et al., 1982). LCMV-Armstrong was obtained from Dr, Rafi Ahmed (Emory University Vaccine Center, Atlanta, GA). LCMV titers were determined with an immunological focus assay (Battegay et al., 1991).

Sequence analysis of LCMV Docile and Aggressive (details in supplementing information).

Analyses of the S and L-segment genome of Doc or Agg 5′ terminal sequences (details in supplementing information).

Population analysis of the 5′ and 3′ genomic termini of S and L RNA fragments of LCMV strains Docile and Aggressive (details in supplementing information).

Construction of plasmids and transfection (details in supplementing information).

Verification of viruses recovered from cDNA (details in supplementing information).

Isolation of biological reassortant viruses containing the S or L-RNA segment of Doc or Agg (details in supplementing information).

Assessment of virus-specific T cell-mediated immune response. IFN-γ intracellular staining, and MHC class I tetramer staining were performed as previously described (Ou et al., 2001) (details in supplementing information).

Delayed type hypersensitivity reaction (DTH). Virus-specific DTH was determined as local swelling after subcutaneous inoculation of virus in the footpad as described previously (Moskophidis & Lehmann-Grube, 1989).

Results

Sequence comparison of the S and L RNAs of LCMV Doc and Agg strains

To analyze the genetic differences between Doc and Agg strains and to map the genetic basis of their differential ability to cause persistent infection, we determined the complete sequences of the S and L RNA segments. This analysis revealed a highly conserved genome between Doc and Agg (Fig. 1A, and supporting information (SI) Figs. 1, 2, 3, and 4) [GeneBank accession no EU480450 (Agg S segment); EU480451 (Agg L segment); EU480452 (Doc S segment); EU480453 (Doc L segment)]. We identified 14 sequence substitutions, of a total 3377 nucleotides in the S RNA that resulted in nine predicted amino-acid differences, four in GP and five in NP coding regions. In the L RNA sequence, we found 42 nucleotide substitutions, from a total 7227 nucleotides, that resulted in 13 predicted amino-acid differences within the L protein between Doc and Agg, whereas no coding substitutions were found within the Z protein. Within the non-coding (5′, 3′, and IGR) regions of the S RNA of Doc and Agg we observed only one nucleotide difference in the IGR, which was outside of the predicted RNA stem-loop structure. The alignment of the non-coding regions of Doc and Agg L RNA segments revealed identical 5′ and 3′ ends, but both viruses differed at six nucleotide positions within the IGR, including three in the predicted secondary RNA structure.

Figure 1. Schematic representations and sequence comparison of the coding regions of the genomic L and S RNA segments between Agg and Doc strains.

Figure 1

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(A) The stem-loop structures depict intergenic noncoding regions. Amino-acid differences between the Agg versus Doc strains of LCMV are indicated first by the position number, followed the amino acid abbreviation in Agg and then in Doc (for example amino-acid 47 I in GP of Agg changed to V in Doc). Nucleotide differences in the IGR of S and L RNA fragments are indicated (small letters). (B) Sequence analysis of the 5′ end termini of S and L RNA of Agg and Doc was carried out as described in the supporting information. The first-strand cDNA was T-tailed at its 3′ end and then PCR amplified and sequenced. The deduced 5′ terminal template sequence of the S and L-segment genome and nucleotide position are indicated (−2 to 9). Note the 5′ nontemplated G at position -1 and the variable presence of the additional T (A in cDNA sequence) at position -2.

Figure 2. Reverse genetics method to rescue LCMV entirely from cloned cDNA.

Figure 2

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(A) Schematic representation of the plasmid-based rescue system for LCMV (outlined for Agg). For rescue of Agg virus, 0.8μg of pol I-SAgg, 1.4μg of pol I-LAgg, 0.8μg of pC-NPAgg, and 1μg pC-LAgg were co-transfected into subconfluent BHK21 cells. At day 4, culture supernatant was collected and tested for infectivity by the immunofocus assay. Virus amplification for 72 hrs increased the virus yield considerably and was routinely used. In addition, cells were sub-cultured and virus in supernatants collected at day 8 after transfection with or without further amplification on BHK-21 for 72 hrs. (B) Schematic representation of plasmids encoding S and L-RNA species cloned in an antigenomic polarity with respect to the pol I promoter used in this study. Cis-acting elements are in box and arrows indicate protein coding regions; pol I-P, pol I promoter; pol I-T, pol I terminator; G, nontemplated G; IGR, intergenic region; 5′ UTR; 5′ untranslated region. (C) Rescued virus strains from cDNA by reverse genetics. (D) Verification of recovered viruses. Viral RNA isolated from indicated strains was RT-PCR amplified using specific primers to generate fragments within the viral S or L RNA containing unique restriction enzyme sites (BclI or BamHI) specific for Agg or Doc. PCR products subjected to restriction analysis with BclI generated two fragments of 546 bp and 146 bp for the S RNA of Agg and the original PCR fragment of 692 bp for Doc. For the L RNA, digestion of the PCR product with BamHI generated fragments of 482 and 302 bp for Doc and the original PCR fragment of 784 bp for Agg. (E) Growth in cell culture of rescued viruses (rAgg, rDoc, rArm, rSDocLAgg, and rSArm/LAgg) indicated by filled symbols (●, ■, ▲, ▼, ◆), and wild-type (authentic) viruses (Agg-wt, Doc-wt, or Arm-wt) indicated by open symbols (○, □, △). Kinetics of virus growth were determined on BHK-21 cells infected at MOI 0.1 or MOI 0.01. Data shown are mean ±SEM log10 PFU/ml and are representative of three experiments.

Figure 3. Capacity of rescued Agg or Doc to induce virus-specific CD8+ T cell response and to control an acute infection.

Figure 3

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(A) B6 mice were infected with 102 PFU i.v. of rescued (rAgg, rDoc) or wild-type (Agg-wt, or Doc-wt) virus and titers in spleen were measured at the indicated times. (B) In parallel analyses, the numbers of GP133-41 or NP396-404 peptide-specific or total (sum of GP133-41, GP2276-286 and NP396-404) CD8+ T cells were determined by staining with H-2Db tetramers (●) or staining for intracellular IFN-γ (○) after stimulation of cells with peptide or with virally infected DC2.4 cells. Data shown are mean ± SEM of log10 virus-specific T cells per spleen for 3–6 mice.

Figure 4. Distinct biological properties of rescued Doc and Agg viruses to cause persistent infection by exhaustion of virus-specific CD8+ T cells.

Figure 4

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(for details see legend for Fig. 3, except that mice were infected with 2×106 PFU).

To obtain accurate sequence information about the viral 5′ and 3′ termini, we first used an RNA ligase-based method combined with RT-PCR to amplify a DNA fragment corresponding to the 5′-3′-termini (Meyer & Southern, 1993). Our results revealed that the viral RNA population was composed of a mixture of RNAs with full-length termini and RNAs with deletions, mostly of one to four nucleotides within the conserved 5′ or 3′ end sequences as reported for LCMV Arm (Meyer & Southern, 1997). In particular we observed that a subset of truncated L RNAs exhibited larger (14 to 34 bases) deletions at their 5′ and 3′ termini. Analysis of the S and L RNA populations carried out by a 3′–5′ RNA junction-length spectranalysis, a technique broadly similar to that used to analyze the CDR3 distribution of T cell receptors in a T cell population (Pannetier et al., 1992), revealed diversity in the viral RNA population (S or L RNA) for both Doc and Agg (data not shown). Since the sizes of deletions in the S and L RNA termini were similar for both virus strains, it is unlikely that the presence of such deletion variants in the viral pool contributed to the Doc strain’s ability to cause persistence.

It been demonstrated that arenavirus S and L segments carry out a non-templated G residue at their 5′ ends (Flatz et al., 2006, Garcin & Kolakofsky, 1990, Meyer & Southern, 1997). However, it was impossible, using the RNA ligation method, to clearly identify whether the 5′ ends of the viral segments of Agg and Doc carry out a non-templated G. Therefore we analyzed the viral S and L segment 5′ ends using a 5′ RACE protocol (for details see supplementary information). This approach clearly demonstrated a G residue at position -1 on both the S and L-segment genomes of LCMV-Doc and Agg (Fig. 1B). In addition, we observed that both the S and L RNA 5′ ends contained variably an additional T residue at position -2. Likewise, sequencing of individual cDNA clones confirmed this result, but revealed that some clones contained one or two additional nucleotides at position -3 and -4. Based on our data and that in the literature the 3′ ends of viral genome segments terminates on a G residue. Thus, we suggest that in analogy to other arenaviruses (Garcin & Kolakofsky, 1990), the genome of Doc and Agg share common terminal nucleotide sequence and that intramolecular annealing of their genome into a panhandle structure does not form a flush end.

Recovery of infectious LCMV from cDNA by RNA polymerase I/II-based reverse genetics

To further explore the molecular basis underlying the ability of the Doc strain to cause a persistent infection, we use a pol-I/II-based reverse genetics system to generate isogenic infectious recombinant LCMV for the Agg, Doc or Arm strains (Fig. 2A and 2B). Co-transfection of BHK21 cells with four plasmids (pol I-SAgg, pol I-LAgg, pC-NPAgg and pC-LAgg) resulted in recovery of infectious virus from the culture supernatant within 3–4 days. Likewise, this pol I/II-driven system was efficient in recovery of Doc and Arm from cloned cDNA. We also rescued Agg and Doc recombinant viruses by co-expressing the trans-acting factors L and NP from Arm together with the corresponding pol-I S and L plasmids. This finding was consistent with the observed high frequency of reassortant viruses between genetically closely related arenaviruses. Using the same approach we generated reassortant viruses containing the same L segment of Agg but with the S segment of Doc or Arm (list of rescued virus strains in Fig. 2C). The genetic identity of the rescued Agg (rAgg), Doc (rDoc), Arm (rArm), and reassortant viruses was confirmed by sequencing RT-PCR-amplified DNA fragments from selected regions of the genome. In addition, we used also restriction enzyme digestion of RT-PCR-generated DNA fragments spanning selected regions of the viral S or L RNA segments (Fig. 2D). Cloned viruses rescued from cDNA formed plaques on Vero cells of comparable size to those formed by the authentic wild-type Agg (Agg-wt), Doc (Doc-wt), or Arm (Arm-wt) viruses. Both Arm-wt and rArm had similar growth properties in cultured cells, whereas rDoc and rAgg grew at slightly higher rates compared to Agg-wt or Doc-wt strains (Fig. 2E). This may was attributed to the identical genetic makeup of rAgg or rDoc virus (potentially free of defective interfering particles).

Biological properties of rescued rDoc and rAgg strains of LCMV

To compare the biological properties of rAgg or rDoc in vivo, we infected B6 mice with a low dose (102 PFU) of virus (Fig. 3). As anticipated, viral titers peaked between days 3 and 6, followed by a rapid decline below detectable levels by day 15 post-infection (p.i.) in all tissues examined (shown for spleen but also studied in liver and kidney). However, we noted that clearance of rAgg occurred with slightly faster kinetics compared to Agg-wt, whilst we did not observe significant differences in the kinetics of viral replication and clearance between rDoc and Doc-wt. In addition, whilst both cDNA-derived and wild-type virus strains induced efficient virus-specific CD8+ T cell responses, mice infected with rAgg exhibited higher levels (3–5 fold) of virus-specific CD8+ T cell response at days 6 and 9 p.i. compared to Agg-wt-infected mice, which may explain the faster kinetics of viral clearance. No significant difference in the magnitude of antiviral CD8+ T cell responses was observed between Doc-wt and rDoc infected mice.

Next, we tested whether the rDoc and rAgg viruses were able to cause persistent infection by subverting the virus-specific CD8+ T cell response (clonal exhaustion). Both rAgg and Agg-wt-infected with a high viral dose (2×106 PFU) mice mounted comparable virus-specific CD8+ T cell responses that resulted in rapid viral clearance, although a small fraction of the overall virus-specific CD8+ T cell population (but a greater fraction of the NP396-404-epitope-specific T cells) exhibited a functional (IFN-γ-production) deficit (Fig. 4 and data not shown). In contrast, this response was suppressed in mice persistently infected with either rDoc or Doc-wt, and in both cases infected mice exhibited similar kinetics of CD8+ T cell exhaustion.

A classic model of LCMV-induced CD8+ and CD4+ T cell-mediated inflammation is the delayed type hypersensitivity (DTH) response, detected as footpad swelling following virus inoculation in the foot (i.f.) (Hotchin, 1971, Moskophidis & Lehmann-Grube, 1989). rAgg, rDoc, and rArm viruses induced footpad swelling reactions of similar duration and magnitude, comparable to those produced by animals inoculated with Doc-wt, Agg-wt, or Arm-wt (Fig. 5). LCMV persistence is known to suppress T cell-induced local inflammation (DTH reaction) (Moskophidis et al., 1995). To test whether the cDNA-derived viruses shared the ability of their wild-type strains to cause acquired T cell suppression, we infected mice locally (i.f.) with 104 PFU of rDoc or rAgg virus followed by infection (i.v.) with increasing amounts of the same virus. rDoc-infected mice mounted a footpad swelling response that was substantially suppressed at a dose of 102 PFU and was below detection levels at a dose of 104 PFU (i.v.). In contrast, inoculation of mice with rAgg virus did not significantly affect footpad swelling at a dose of 102 PFU and only partially at a dose of 104 PFU, but the response was suppressed at high doses (106 PFU i.v.) (Fig 5B and 5C). Together our results strongly indicate that rDoc and rAgg accurately recreated accurately the phenotypic properties of the corresponding bona fide Doc-wt and Agg-wt viruses.

Figure 5. Superior capacity of rescued Doc versus Agg to subvert T cell-mediated DTH reaction.

Figure 5

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(A) Virus-specific footpad swelling reaction in B6 mice following local LCMV infection in the footpad of 104 PFU of rescued (rDoc, rAgg, or rArm) (▲) compared to wild-type (Agg-wt, Doc-wt or Arm-wt) (●) strains. (B–C) Mice were infected with 104 PFU of rDoc (in B) or rAgg (in C) in the footpad combined with an increasing intravenous dose (102, 104, 106 PFU) of the same virus isolate (rAgg or rDoc) (△). For comparison the footpad swelling of mice infected only in the footpad with 104 PFU of rDoc or rAgg is also indicated (▲). Footpad swelling reaction was monitored by measuring the increase in thickness of the infected compared to uninfected foot. Data points represent mean± the SEM of five mice.

Biological characterization of cDNA-rescued reassortant viruses

We evaluated the biological properties of the reassortant viruses with respect to their ability to cause persistent infection and to induce CD8+ T cell exhaustion. For this we infected B6 mice with a high dose (2×106 PFU) of rSDocLAgg or rSArm/LAgg virus (SI Fig. 5). For comparison, we also included infection of mice with rDoc vs. Doc-wt, rAgg vs. Agg-wt, or rArm vs. Arm-wt viruses in this analysis. As anticipated, rDoc or Doc-wt viruses readily caused viral persistence, which was associated with a rapid exhaustion of virus-specific CD8+ T cells. Mice infected with rAgg or Agg-wt viruses exhibited a marked viral spread within the liver and kidneys, but viral levels declined below detection level of the plaque assay by days 15 to 20 p.i., which was associated with a marked expansion of antigen-specific CD8+ T cells. A majority of these cells exhibited a functional phenotype, as assessed by staining for IFN-γ secretion. rArm or Arm-wt-infected mice mounted virtually unimpaired CD8+ T cell responses that resulted in rapid viral clearance by day 9 p.i. from different tissues (spleen, liver, and kidney). Notably, rSDocLAgg-infected mice mounted an unimpaired CD8+ T cell response that suppressed virus replication below detectable levels at days 15 to 20 p.i. This result revealed that the S segment of Doc does not contain all the genetic determinants responsible to cause virus-specific CD8+ T cell exhaustion and establish persistence. The fact, however that the reassortant rSArmLAgg virus exhibited a significantly attenuated phenotype compared to rAgg suggests that the S RNA structure may also influence viral properties and thus the viral persistent phenotype, a view strongly supported by the LCMV literature (Matloubian et al., 1993, Mueller et al., 2007, Salvato et al., 1991). Thus, a high dose of rArm/LAgg virus was efficiently controlled by day 9 p.i. by a robust virus-specific CD8+ T cell response that developed with kinetics comparable to that produced by infection with Arm.

To obtain a more comprehensive analysis of the in vivo phenotype of the cDNA-derived viruses, we investigated the effects of viral load on the overall outcome of infection and parameters of the virus-specific CD8+ T cell response on day 30 p.i., at a time when the outcome of infection was determined. Our data (Fig. 6) further confirmed that the phenotypic differences between Doc and Agg viruses hold true for rDoc or rAgg. Thus, rDoc exhibited a greater ability to induce functional exhaustion of virus-specific CD8+ T cell populations (see arrows), which could be observed at a moderate virus dose of 104 PFU. In contrast the suppressive capacity of rAgg was limited, and even a high dose of 2×106 PFU was efficiently cleared, despite an observed partial suppression of virus-specific CD8+ T cell response. Notably, the presence of the S RNA segment of Doc in the rSDocLAgg reassortant did not convert the non-persistent Agg to the persistent Doc. However, we noted that the rSDocLAgg virus exhibited a moderate increased ability to suppress the NP396-404-specific CD8+ T cell function. This finding suggests that the S RNA alone may exert an effect on the persistent phenotype. Consistent with the high dose infection results (SI Fig. 5), the presence of the Arm S RNA in rSArmLAgg virus abolished the ability of rAgg to induce a partial suppression of the virus-specific CD8+ T cell response even when injected at high dose, and it substantially attenuated viral spread. Taken together, the persistent phenotype is at least determined by the nature of the L RNA segment. However, a second cloned reassortant virus (rSAggLDoc) will demonstrate the importance of the S RNA segment in viral persistence. However, the recovery of this reassortant virus strain via reverse genetics has proven to be a challenging task for unknown reasons.

Figure 6. Susceptibility of mice to persistent infection is determined by the molecular structure of the L RNA segment of Doc.

Figure 6

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B6 mice were infected i.v. with different doses (102, 104, 105 or 2×106 PFU) of rArm, rDoc, rArm, rSDocLAgg or rSArmLAgg virus. Virus titers in spleen, liver, and kidney were measured at 30 days (right panels). In parallel analyses, the numbers of GP133-41 or NP396-404 peptide-specific or total (sum of GP133-41, GP2276-286 and NP396-404) CD8+ T cells were determined by staining with H-2Db tetramers (filled columns) or staining for intracellular IFN-γ after stimulation of cells with peptide or with virally infected DC2.4 cells (open columns). Significant differences in numbers of IFN-γ versus Db/tetramer-positive CD8+ T cells are indicated by arrows (p<0.05). Data shown are mean ± SEM of log10 virus-specific T cells per spleen for 3–6 mice.

Genomic difference in the S and L RNA segment of LCMV-Doc influence viral properties to persist by T cell exhaustion: studies with biological reassortants of Doc and Agg viruses. To further examine the genetic basis of the persistent phenotype we isolated biological reassortant viruses using a co-infection protocol (Fig. 7A) and performed analyses evaluating their ability to subvert an antiviral CD8+ T cell response and persist. Since the amino-acid substitution GP2280N→S in Doc, not present in the Agg strain, abolishes CD8+ T cell recognition of the GP2276-286 peptide and may contribute to the persistent phenotype of this virus, we isolated two sets of reassortant viruses (Fig. 7B): virus isolates that contain the S RNA of Agg-variant (GP280N→Y)(Agg280Y) and Doc L RNA segments and the reverse, or viruses with the S RNA of Agg with Doc revertant (GP2280S→N) (Doc280N) L RNA segments. Note that the Doc280N has been isolated from infected mice and exhibits sequence similarity with wild-type or rDoc in the coding region of GP and NP proteins except for the coding change (GP2280S→N) that restored the GP2276-286 epitope. In addition, the Agg280Y variant isolated using standard CTL selection procedures (Aebischer et al., 1991) lacks CTL recognition on the GP2276-286 epitope. The fact that both virus strain combinations (Agg280Y vs. Doc or Doc280N vs. Agg) exhibit identical recognition patterns in this major CD8+ T cell-epitope allows viral properties associated with the persistent phenotype to be more accurately mapped to viral RNA segments. The growth rates of reassortant viruses in BHK-21 cell cultures were found to be comparable to the parental viruses (Fig. 7C).

Figure 7. Characterization of biological reassortant viruses containing the S or L RNA segments of Doc vs. Agg280Y or Doc280N vs. Agg.

Figure 7

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(A) Reassortant viruses were generated using a standard co-infection procedure modified as follows: BHK-21 cells infected with Agg280Y or Agg (MOI=2) 48 hrs previously were exposed to mild heat shock (41°C, 30 min) and subsequently super-infected with Doc or Doc280N (MOI=2). Supernatant harvested 72 hrs later was plaque-purified and analyzed for recombinant viral genomes. (B) List of reassortant viruses. (C) Growth in cell culture of wild-type or reassortant viruses: Doc (●) vs. Agg280Y (▲) in left panels or Doc280N (●) vs. Agg (▲) in the fourth column panels. Different symbols in the panels for SAgg280YLDoc, SDocLAgg280Y, or SDoc280NLAgg indicate individual virus isolates. Kinetics of virus growth were determined on BHK-21 cells infected at MOI 0.1 or 0.01. Data shown are mean ±SEM log10 PFU/ml and are representative of three experiments.

In experiments shown in Fig. 8 and SI Fig. 6, we investigated the effects of viral load on the overall outcome of infection and parameters of the virus-specific CD8+ T cell response. Collectively, our data further confirmed and extended our results with rSDocLAgg virus that the presence of the S or L RNA segment of Doc in the SDocLAgg280Y or SAgg280YLDoc reassortants did not convert the non-persistent Agg to the persistent Doc. Similar analyses performed with the Doc280N, Agg and SDoc280NLAgg viruses (SI Fig. 6) confirmed the result with rSDocLAgg. However, we noted that restoration of the GP2276-286 epitope in Doc280N measurably diminished its dose-dependent ability to suppress the CD8+ T cell response causing persistent infections. This finding suggests that the substitution 280N→S in the viral GP contributes to the persistent phenotype of Doc. Collectively the data suggest that the nature of both S and L RNA segments contribute to the Doc persistent phenotype.

Figure 8. The molecular structure of both S and L-RNA segments of Doc contribute in shaping the persistent viral phenotype.

Figure 8

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B6 mice were infected i.v. with different doses (102, 104, 105 or 2×106 PFU) of wild-type Doc or Agg280Y, or reassortants SAgg280YLDoc (isolate 44-1 or 44-7) or SDocLAgg280Y. Virus titers in spleen, liver, and kidney were measured at day 6 (striped columns) to determine the in vivo growth properties of the different strains and day 30 day (filled columns) (right panels). In parallel analyses, the numbers of GP133-41 or NP396-404 peptide-specific or total (sum of GP133-41, GP2276-286 and NP396-404) CD8+ T cells were determined by staining with H-2Db tetramers (filled columns) or staining for intracellular IFN-γ after stimulation of cells with peptide or with virally infected DC2.4 cells (open columns) (left panels). Significant differences in numbers of IFN-γ versus Db/tetramer-positive CD8+ T cells are indicated by arrows (p<0.05). Data shown are mean ± SEM of log10 virus-specific T cells per spleen for 3–6 mice.

Discussion

In this report we conducted a comprehensive genomic analysis of two genetically closely related LCMV strains Doc and Agg, as a first step to map and functionally characterize LCMV genetic determinants of persistence. We provided evidence that the persistent phenotype is determined by the nature of both S and L RNA segments. The above conclusion is further supported by our studies with biological reassortant viruses, which have clearly revealed that the ability of Doc to persist in adult mice segregates with both S and L RNA segments.

Evidence indicates that amino-acid substitutions in the viral S and L RNA segments can affect the ability of LCMV to persist by different mechanisms including: 1) reduced virus recognition by T cells (Lewicki et al., 1995, Moskophidis & Zinkernagel, 1995, Pircher et al., 1990); 2) changes in virus-receptor interactions and cell tropism (Sevilla et al., 2004); and 3) changes in virus replication and overall viral load (Sevilla & de la Torre, 2006). The limited genetic diversity we found between the Doc and Agg strains facilitates studies aimed at assessing the contribution of specific amino-acid residues to the persistent phenotype associated with the Doc strain. Of particular interest is a recent report that provided a comprehensive screen of the entire LCMV Arm proteome for CD8+ T cell epitopes (Kotturi et al., 2007). Among a total of 28 CD8+ T cell epitopes identified, nine are located in the GP, four in the NP and 15 in the L protein. Whether all of these T cell epitopes are recognized in Doc- and Agg-infected cells remains to be determined. Comparison of the Doc versus Agg proteomes revealed a single amino acid difference in three of 28 epitopes (all within the GP), a result that facilitates correlation of host immune recognition patterns with the virus’ ability to persist. For example, the substitution 280N→S in the GP2276-286 T cell epitope of Doc abolishes CD8+ T cell recognition, and our data indicate that this can influence the persistent viral phenotype. To what extent additional coding changes found in the GP of Doc may influence the viral phenotype remains to be determined. The coding change 494V→I found adjacent to the WKRR sequence motif at the C terminus of GP2 may influence the structure and function of the GP ectodomain. This alteration could affect the proteolytic processing of the viral GP, which is critical for virus production, or modulate interactions of GP with cellular factors that play roles in oligomerization of the GP (Kunz et al., 2003). The contribution to the persistent Doc phenotype of the five residual differences between the NP of Doc and Agg strains remain to be determined. NP plays a critical role in formation of RNPs and control of the replication and transcription of the virus genome (Lee et al., 2000). It is therefore plausible that these changes in the NP of Doc may contribute to increase virus growth in mouse tissues, which may represent a parameter for viral persistence. The possibility that NP mutations may interfere with the innate response (e.g., type I IFN) (Martinez-Sobrido et al., 2006, Moskophidis et al., 1994) deserves particular consideration. Thus an amenable approach to address the biological implication of the IFN-inhibiting activity of NP of Doc and other LCMV strains is to generate recombinant LCMV viruses in which the NP genome is modified (mutated or replaced with NP sequence of other LCMV strains) and examine their biological properties in the context of both acute and persistent LCMV infection.

The in vivo phenotypes of the rSDocLAgg or biological reassortants support a critical contribution of the L segment to the persistent phenotype. Mutations in the L protein may modulate polymerase activity associated with viral genome replication and transcription. We cannot yet propose a specific role for any of the several amino-acid differences scattered throughout the entire sequence of the L gene of Doc versus Agg. Notably, we did not observe sequence differences between Doc and Agg for any of the nineteen CD8+ T cell epitopes described in the L protein of Arm. Finally, the IGR is a bona fide transcription termination signal and appears to be involved in viral packaging (Pinschewer et al., 2005). Therefore, it is possible that the five nucleotide differences found between the IGRs of Doc and Agg strains might contribute to the phenotypic differences between these two viruses.

The reverse genetics approach presented here and used by others to rescue Arm and Arm Cl-13 LCMV strains from cloned DNA (Flatz et al., 2006, Sanchez & de la Torre, 2006), can facilitate the investigation of complex issues about virus structure, virus-cell interactions, and viral pathogenesis and should facilitate the development of attenuated vaccines to combat arenaviral infections. Notably, the data presented here add to the list of published studies with reassortants of different arenavirus strains, which have evaluated viral determinants associated with the virulence and pathogenicity of particular virus strains (Bergthaler et al., 2007, Lukashevich, 1992, Lukashevich et al., 2005, Matloubian et al., 1993, Oldstone et al., 1990, Riviere et al., 1985, Zhang et al., 2001). Generation of recombinant Doc viruses containing coding mutations corresponding to those of the Agg virus strain and their phenotypic characterization in vivo will provide new insights into the mechanisms underlying the ability of the Doc strain to cause persistent infection by driving virus-specific T cells into distinct programs of clonal exhaustion.

Supplementary Material

1. Supplementing Information.

Figure 1: Viral genomic sense cDNA sequence of the S RNA of Doc. The coding sequence of the GP gene is in the positive sense of the viral genome, and the coding sequence of the NP gene in the negative sense. The single-letter code for amino acids is indicated. The intergenic region (IGR) of the GP and NP genes is defined from nucleotide 1575 to 1641. Numbers indicate nucleotide position in the viral segment.

Figure 2: Viral genomic sense cDNA sequence of the L RNA of Doc. The coding sequence of the Z gene is in the positive sense of the viral genome, and the coding sequence of the L gene in the negative sense. The single-letter code for amino acids is indicated. The intergenic region (IGR) of the Z and L genes is defined from nucleotide 363 to 564. Numbers indicate nucleotide position in the viral segment.

Figure 3: Viral genomic sense cDNA sequence of the S RNA of Agg. The coding sequence of the GP gene is in the positive sense of the viral genome, and the coding sequence of the NP gene is in the negative sense. The single-letter code for amino acids is indicated. The intergenic region (IGR) of the GP and NP genes is defined from nucleotide 1575 to 1641. Numbers indicate nucleotide position in the viral segment.

Figure 4: Viral genomic sense cDNA sequence of the L RNA of Agg. The coding sequence of the Z gene is in the positive sense of the viral genome, and the coding sequence of the L gene in the negative sense. The single-letter code for amino acids is indicated. The intergenic region (IGR) of the Z and L genes is defined from nucleotide 363 to 564. Numbers indicate nucleotide position in the viral segment.

Figure 5: Biological properties of rDoc, rAgg, rArm, rSDocLAgg or rSArmLAgg strains to down regulate the virus-specific CD8+ T cell response and cause chronic infection. (A) B6 mice were infected with 2×106 PFU of rescued virus (rDoc, rAgg, rArm, rSDocLAgg, or rSArmLAgg) (indicated by ▴), or wild-type virus (Doc-wt, Agg-wt, or Arm-wt) (indicated by ^) and virus titers in spleen, liver, or kidney were measured at the indicated times. (B) In parallel analyses, numbers of total virus-specific CD8+ T cells were determined by staining with H-2Db tetramers (sum of GP133-41 and NP396-404) or staining for intracellular IFN-γ after stimulation of cells with virally infected DC2.4 cells (DC-IFNγ). Analyses compare mice infected with rescued (^) or with wild-type (▴) virus strains. Data shown are mean ± SEM of log10 virus-specific T cells per spleen for 3–6 mice.

Figure 6: The molecular structure of both S- and L RNA segments of Doc contribute to shaping the persistent viral phenotype. B6 mice were infected i.v. with different doses (102, 104, 105 or 2×106 PFU) of wild-type Doc280N or Agg, or reassortants SDoc280NLAgg (isolate 4–4, 4–7 or 5-1). Virus titers in spleen, liver, and kidney were measured at day 6 (striped columns) to determine the in vivo growth properties of the different strains and day 30 (filled columns) (right panels). In parallel analyses, the numbers of GP133-41 or NP396-404 peptide-specific or total (sum of GP133-41, GP2276-286 and NP396-404) CD8+ T cells were determined by staining with H-2Db tetramers (filled columns) or staining for intracellular IFN-γ after stimulation of cells with peptide or with virally infected DC2.4 cells (open columns) (left panels). Significant differences in number of IFN-γ versus Db/tetramer-positive CD8+ T cells are indicated by arrows (p<0.05). Data shown are mean ± SEM of log10 virus-specific T cells per spleen for 3–6 mice. Note that a second reassortant virus (SAggLDoc280N) should demonstrate the importance of the S RNA segment in viral persistence in this experimental setting. However, we were unable to isolate this reassortant virus with our co-infection method, although we were successful in recovering several reassortants of Agg280Y and Doc strains.

Acknowledgments

This work was supported by NIH grant AI42114 to D.M. The authors are grateful to Dr. Rhea-Beth Markowitz for critical reading of the manuscript.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1. Supplementing Information.

Figure 1: Viral genomic sense cDNA sequence of the S RNA of Doc. The coding sequence of the GP gene is in the positive sense of the viral genome, and the coding sequence of the NP gene in the negative sense. The single-letter code for amino acids is indicated. The intergenic region (IGR) of the GP and NP genes is defined from nucleotide 1575 to 1641. Numbers indicate nucleotide position in the viral segment.

Figure 2: Viral genomic sense cDNA sequence of the L RNA of Doc. The coding sequence of the Z gene is in the positive sense of the viral genome, and the coding sequence of the L gene in the negative sense. The single-letter code for amino acids is indicated. The intergenic region (IGR) of the Z and L genes is defined from nucleotide 363 to 564. Numbers indicate nucleotide position in the viral segment.

Figure 3: Viral genomic sense cDNA sequence of the S RNA of Agg. The coding sequence of the GP gene is in the positive sense of the viral genome, and the coding sequence of the NP gene is in the negative sense. The single-letter code for amino acids is indicated. The intergenic region (IGR) of the GP and NP genes is defined from nucleotide 1575 to 1641. Numbers indicate nucleotide position in the viral segment.

Figure 4: Viral genomic sense cDNA sequence of the L RNA of Agg. The coding sequence of the Z gene is in the positive sense of the viral genome, and the coding sequence of the L gene in the negative sense. The single-letter code for amino acids is indicated. The intergenic region (IGR) of the Z and L genes is defined from nucleotide 363 to 564. Numbers indicate nucleotide position in the viral segment.

Figure 5: Biological properties of rDoc, rAgg, rArm, rSDocLAgg or rSArmLAgg strains to down regulate the virus-specific CD8+ T cell response and cause chronic infection. (A) B6 mice were infected with 2×106 PFU of rescued virus (rDoc, rAgg, rArm, rSDocLAgg, or rSArmLAgg) (indicated by ▴), or wild-type virus (Doc-wt, Agg-wt, or Arm-wt) (indicated by ^) and virus titers in spleen, liver, or kidney were measured at the indicated times. (B) In parallel analyses, numbers of total virus-specific CD8+ T cells were determined by staining with H-2Db tetramers (sum of GP133-41 and NP396-404) or staining for intracellular IFN-γ after stimulation of cells with virally infected DC2.4 cells (DC-IFNγ). Analyses compare mice infected with rescued (^) or with wild-type (▴) virus strains. Data shown are mean ± SEM of log10 virus-specific T cells per spleen for 3–6 mice.

Figure 6: The molecular structure of both S- and L RNA segments of Doc contribute to shaping the persistent viral phenotype. B6 mice were infected i.v. with different doses (102, 104, 105 or 2×106 PFU) of wild-type Doc280N or Agg, or reassortants SDoc280NLAgg (isolate 4–4, 4–7 or 5-1). Virus titers in spleen, liver, and kidney were measured at day 6 (striped columns) to determine the in vivo growth properties of the different strains and day 30 (filled columns) (right panels). In parallel analyses, the numbers of GP133-41 or NP396-404 peptide-specific or total (sum of GP133-41, GP2276-286 and NP396-404) CD8+ T cells were determined by staining with H-2Db tetramers (filled columns) or staining for intracellular IFN-γ after stimulation of cells with peptide or with virally infected DC2.4 cells (open columns) (left panels). Significant differences in number of IFN-γ versus Db/tetramer-positive CD8+ T cells are indicated by arrows (p<0.05). Data shown are mean ± SEM of log10 virus-specific T cells per spleen for 3–6 mice. Note that a second reassortant virus (SAggLDoc280N) should demonstrate the importance of the S RNA segment in viral persistence in this experimental setting. However, we were unable to isolate this reassortant virus with our co-infection method, although we were successful in recovering several reassortants of Agg280Y and Doc strains.

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