Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 2013 Aug;87(16):8853–8861. doi: 10.1128/JVI.00925-13

UPF1 Is Crucial for the Infectivity of Human Immunodeficiency Virus Type 1 Progeny Virions

Anna Kristina P Serquiña a, Suman R Das a,*, Elena Popova a, Ogooluwa A Ojelabi b, Christian K Roy b, Heinrich G Göttlinger a,✉
PMCID: PMC3754033  PMID: 23785196

Abstract

The SF1 helicase MOV10 is an antiviral factor that is incorporated into human immunodeficiency virus type 1 (HIV-1) virions. We now report that HIV-1 virions also incorporate UPF1, which belongs to the same SF1 helicase subfamily as MOV10 and functions in the nonsense-mediated decay (NMD) pathway. Unlike ectopic MOV10, the overexpression of UPF1 does not impair the infectivity of HIV-1 progeny virions. However, UPF1 becomes a potent inhibitor of HIV-1 progeny virion infectivity when residues required for its helicase activity are mutated. In contrast, equivalent mutations abolish the antiviral activity of MOV10. Importantly, cells depleted of endogenous UPF1, but not of another NMD core component, produce HIV-1 virions of substantially lower specific infectivity. The defect is at the level of reverse transcription, the same stage of the HIV-1 life cycle inhibited by ectopic MOV10. Thus, whereas ectopic MOV10 restricts HIV-1 replication, the related UPF1 helicase functions as a cofactor at an early postentry step.

INTRODUCTION

The putative RNA helicase MOV10 is taken up into human immunodeficiency virus type 1 (HIV-1) particles and exhibits potent antiviral activity when overexpressed in virus-producing cells (1–5). MOV10 localizes to sites of mRNA degradation and storage called cytoplasmic processing (P) bodies, and the ectopic expression of MOV10 can relocalize microRNA effectors to these sites (6, 7). Notably, the antiviral proteins APOBEC3G and APOBEC3F, which are targeted by HIV-1 Vif, also localize to P bodies, where they physically associate with MOV10 in an RNA-dependent manner (8, 9). Several groups have shown that ectopic MOV10 reduces the production and particularly the intrinsic infectivity of HIV-1 particles (2–5), and comparable effects on other retroviruses and endogenous retroelements have also been observed (2, 4, 5, 10, 11). That MOV10 is taken up into viral particles was initially suggested by a proteomic analysis of HIV-1 virions from infected monocyte-derived macrophages (12). Virions produced in the presence of high levels of MOV10 are able to fuse normally with target cells, but the production of reverse transcripts in the infected cells is severely impaired (3–5). Nevertheless, whether MOV10 controls retroviruses when expressed at endogenous levels remains controversial (2, 5). At least in certain cell lines, endogenous MOV10 does not appear to restrict HIV-1 or other exogenous retroviruses, even though the MOV10 levels in these cells are sufficient to inhibit endogenous retroelements (2).

UPF1 belongs to the same SF1 helicase subfamily as MOV10 (13). Like its Saccharomyces cerevisiae ortholog, human UPF1 is a cytosolic protein that binds RNA and exhibits a nucleic acid-dependent ATPase as well as a helicase activity (14, 15). The best-characterized function of UPF1 is in the nonsense-mediated decay (NMD) pathway, of which it is an essential component (16, 17). The NMD pathway serves as a quality control mechanism that targets mRNAs harboring premature termination codons and also suppresses transcriptional noise (17). When translation termination factors encounter a stalled ribosome due to a premature stop codon, they recruit UPF1, which subsequently engages its binding partner (UPF2) and the rest of the machinery that initiates the degradation of the defective transcript (17). Notably, there is evidence that retroviruses protect their genomic RNA against the NMD pathway (18). In the case of Rous sarcoma virus (RSV), the region immediately downstream of the gag frame harbors a stability element that inhibits recognition of the normal gag termination codon by UPF1 and the NMD pathway (19). When this stability element is deleted, the unspliced RSV RNA decays, and this decay can be inhibited by dominant negative UPF1 (19). It has been pointed out that HIV-1 full-length RNA also contains features potentially recognized by UPF1 (18). However, in contrast to what has been reported for RSV, a recent study suggests that UPF1 unexpectedly stabilizes the unspliced HIV-1 RNA (20).

In the present study, we show that the infectivity of HIV-1 virions produced in UPF1-depleted cells or in cells expressing ATPase-defective UPF1 mutants is markedly impaired. The defect is at the level of reverse transcription (RT) following entry into a new target cell. Thus, whereas ectopic MOV10 inhibits an early postentry step, the related UPF1 helicase promotes an early postentry step in HIV-1 replication.

MATERIALS AND METHODS

Plasmids.

NL4-3/env− is an env-deficient variant of HIV-1NL4-3 with a frameshift at a unique NheI site (21). The pBJ5-EnvNL4-3 vector expresses the NL4-3 envelope (Env) and Rev proteins. HXBH10-PR−, which was used to express Pr55gag, is a protease (PR)-defective and vpu-positive variant of the infectious HXB2 proviral clone of HIV-1 (22). The HXBH10-based ZWT-p6 proviral construct has the nucleocapsid (NC) domain of Gag replaced by the GCN4 leucine zipper domain, which restores particle production (22). The HXBH10-gag− proviral construct is unable to express Gag due to premature termination codons in the gag gene (23). DNAs encoding UPF1 and MOV10 with an N-terminal hemagglutinin (HA) tag were amplified from cDNA clones BC039817 and BC002548 (Open Biosystems) and cloned into the expression vector pcDNA3.1(+). UPF1 and MOV10 point mutants were made using the QuikChange mutagenesis strategy (Stratagene).

Viral particle analysis.

For the detection of virus-associated proteins, 293T cells were transfected with proviral DNAs and vectors expressing HA-tagged UPF1 or MOV10 using a calcium phosphate precipitation technique, and virions or virus-like particles released into the medium were pelleted through sucrose. To examine the incorporation of HA-UPF1, further purification in OptiPrep velocity gradients was achieved as described previously (24). Virus- and cell-associated proteins were detected by Western blotting as described previously (22). The antibodies used were 183-H12-5C (25) against the HIV-1 capsid (CA) protein, IN-2 (Santa Cruz Biotechnology) against HIV-1 integrase (IN), anti-HIV-1 gp120 (20-HG81; Fitzgerald Industries), HA.11 (Covance) against the HA epitope, and AC-40 (Sigma-Aldrich) against actin.

Analysis of virus infectivity.

Pseudovirions capable of a single round of replication were produced by transfecting 293T cells with calcium phosphate using 2 μg env-deficient HIV-1NL4-3 proviral DNA, a vector encoding HIV-1NL4-3 Env (1 μg), and empty pcDNA3.1(+) or variants encoding wild-type (WT) or mutant HA-UPF1 or HA-MOV10 (0.2 μg). Alternatively, Lipofectamine 2000 was used to transfect 293T cells with small interfering RNAs (siRNAs; 20 nM) along with 750 ng env-deficient HIV-1NL4-3 proviral DNA and 350 ng HIV-1NL4-3 Env expression vector. The siRNAs targeting endogenous UPF1 [5′-GAUGCAGUUCCGCUCCAUU-d(TT)-3′] (26), endogenous UPF2 [5′-CAACAGCCCUUCCAGAAUC-d(TT)-3′] (27), and nontargeting siRNA were purchased from Dharmacon. To confirm the effectiveness of the siRNAs, cell lysates were examined by Western blotting with anti-UPF1 (a gift from Jens Lykke-Andersen), anti-UPF2 antibody C-18 (Santa Cruz Biotechnology), and antiactin.

Virus-containing supernatants were harvested 2 or 3 days after transfection, and equivalent amounts of virus corresponding to 40 ng p24 were used to infect GHOST-CXCR4 cells (28) in T25 flasks. Two days after infection, green fluorescent protein (GFP)-positive cells were quantitated by flow cytometry.

Analysis of virion RNA.

Virions produced by transiently transfected 293T cells were pelleted through sucrose, and RNA was extracted with a QIAamp viral RNA kit (Qiagen). Aliquots were then reverse transcribed or not (for control purposes) using RNA to cDNA EcoDry premix (double primed; Clontech). After the addition of carrier DNA, quantitative PCR was performed using SYBR green (Invitrogen) and primers GagF1 and GagR1, which amplify a product specific for unspliced viral RNA (29). Copy numbers were calculated on the basis of the values obtained with known quantities of linearized pNL4-3 and adjusted for the amount of p24 measured in each sample.

RNA extracted from purified HIV-1 virions was also separated on a 1% denaturing agarose gel after normalization for p24, transferred to a nylon membrane, and blotted as described using a radioactive probe corresponding to nucleotides 8669 to 8777 of NL4-3 (30). Radioactive bands were detected by exposure of the membrane to a PhosphorImager screen and scanning with a Typhoon 9200 scanner.

Virion fusion assay.

Virions containing Blam-Vpr were produced by transfecting 293T cells with NL4-3/env−, pBJ5-EnvNL4-3, the Blam-Vpr expression vector pMM310, and vectors encoding WT or mutant HA-UPF1 or empty vector. TZM-bl cells were then inoculated for 2 h at 37°C. After extensive washing, CCF4-AM dye solution (LiveBLAzer FRET-B/G loading kit; Invitrogen), prepared according to the standard protocol recommended by the manufacturer, was added to the cells. After overnight incubation at room temperature in the dark, the cells were washed and kept in Hanks' balanced salt solution supplemented with 2 mM l-glutamine and 20 mM HEPES. Blue cells were counted in 5 random fields under a fluorescence microscope equipped with a ×20 objective and Chroma filter set 41031, and the values were normalized for the amount of p24 antigen in the virus inoculum.

Analysis of postentry events.

Virus stocks were normalized for p24 antigen, incubated with RNase-free DNase I (Roche), and used to infect GHOST-CXCR4 cells. DNA from the target cells was extracted with a QIAamp DNA blood minikit (Qiagen) and used for quantitative PCR with a 7500 real-time PCR system (Applied Biosystems) as described previously (29). Early RT products were quantified with Sybr green and primers hRU5-F2 and hRU5-R (29), whereas TaqMan probes were used to quantify late RT products (primers MH531 and MH532; probe LRT-P) and 2-long terminal repeat (2-LTR) circles (primers SS-4 and LTR-R5; probe P-HUS-SS1), as described previously (29). The standard template for early and late RT products was linearized NL4-3, and the standard template for 2-LTR circles was a plasmid containing a 2-LTR circle junction provided by Mark Sharkey (31).

RESULTS

HIV-1 virions incorporate UPF1 in an NC-dependent manner.

A proteomic analysis of Optiprep-purified, PR-defective HIV-1 particles indicated that two members of the SF1 helicase family, namely, MOV10 and UPF1, are packaged into virions (Fig. 1A). The incorporation of MOV10, which belongs to the UPF1-like subfamily of SF1 helicases (13), was recently reported (1, 3, 5). To verify the incorporation of UPF1, we coexpressed HA-tagged UPF1 with full-length HIV-1 proviral constructs expressing either no Gag or the WT Pr55 Gag polyprotein. Since the majority of UPF1 is in a complex with ribosomes (32), which may cosediment with virions through a sucrose cushion, HIV-1 particles were further separated from contaminants by ultracentrifugation through Optiprep velocity gradients (33). As shown in Fig. 1B, HA-UPF1 was readily detected in virions produced by WT HIV-1 Gag (lane 2). Furthermore, the appearance of HA-UPF1 in the particulate fraction depended on the expression of Gag (Fig. 1B, lane 1), confirming that HA-UPF1 was taken up into viral particles.

Fig 1.

Fig 1

Open in a new tab

Incorporation of UPF1 and MOV10 into HIV-1 particles. (A) Helicase superfamily 1 and 2 members identified by mass spectrometry in PR-defective HIV-1 particles produced by transiently transfected 293T cells but absent from control samples obtained by transfecting 293T cells with Gag-defective HIV-1 proviral DNA. (B) Immunoblots demonstrating the specific uptake of UPF1 into HIV-1 virions. 293T cells were transfected with full-length HIV-1 proviruses expressing no Gag or uncleaved Gag, along with a vector expressing HA-UPF1. (C) Immunoblots demonstrating that the uptake of UPF1 depends on NC. The HIV-1 provirus used in lane 1 expresses WT Gag, whereas that used in lane 2 has the NC domain precisely replaced by a leucine zipper. (D) Immunoblots showing the NC-dependent incorporation of MOV10. (E) Immunoblots showing the effect of overexpressing UPF1 on the incorporation of MOV10. 293T cells were transfected with full-length HIV-1 proviruses expressing no Gag or uncleaved Gag, along with the vector expressing HA-MOV10 and an equal molar amount of empty vector or a vector expressing native UPF1.

The NC domain of HIV-1 Gag is required for the specific encapsidation of the genomic viral RNA into nascent particles. Since it has been shown that MOV10 interacts with NC in an RNA-dependent manner (5), we examined whether the association of UPF1 or MOV10 with HIV-1 particles depends on NC. To this end, HA-UPF1 was coexpressed either with WT HIV-1 Gag or with an HIV-1 Gag construct called ZWT-p6 (22), which has the NC domain and the adjacent p1 spacer peptide precisely replaced by the GCN4 leucine zipper domain. We previously demonstrated that the GCN4 zipper domain fully rescues viral particle production by HIV-1 Gag in the absence of the NC-p1 region (22). As shown in Fig. 1C, the association of HA-UPF1 with viral particles was essentially abolished in the absence of NC-p1. Similarly, the incorporation of HA-MOV10 was prevented by the replacement of NC-p1 with the GCN4 zipper domain (Fig. 1D). Thus, UPF1 and MOV10 are both taken up by HIV-1 particles in a specific manner that depends on NC.

Since the incorporation of UPF1 and of MOV10 depends on the same region of Gag, we examined whether the uptake of HA-MOV10 was affected in the presence of increased amounts of UPF1. As shown in Fig. 1E, the overexpression of UPF1 inhibited the virion-association of HA-MOV10, suggesting at least some level of competition for uptake.

Ectopic UPF1 and MOV10 have opposite effects on HIV-1 infectivity.

It has been shown that the overexpression of MOV10 in virus producer cells inhibits HIV-1 infectivity at a postentry step (3–5). We therefore asked whether the overexpression of the related UPF1 helicase in virus-producing cells affects HIV-1 infectivity. Virions for single-cycle infections were produced by cotransfecting 293T cells with envelope (Env)-deficient HIV-1NL4-3, a vector providing NL4-3 Env in trans, and a vector expressing HA-UPF1 or the empty vector. In a separate experiment, the pcDNA3.1-based vector expressing HA-UPF1 was replaced by the same amount of a pcDNA3.1-based vector expressing HA-MOV10. Equal amounts of virions, determined by p24 antigen enzyme-linked immunosorbent assay (ELISA), were then used to infect indicator cells that express GFP upon infection with HIV-1 (28). To facilitate a comparison of relative infectivities, additional cultures were inoculated with 10-fold dilutions of some of the virus stocks. Quantitation of GFP-positive cells by flow cytometry at day 3 postinfection indicated that the overexpression of UPF1 in producer cells did not impair the infectivity of progeny virions (Fig. 2A). In contrast, the overexpression of MOV10 reduced the infectivity of progeny virions more than 10-fold (Fig. 2B), consistent with previous reports (3–5).

Fig 2.

Fig 2

Open in a new tab

Ectopic UPF1 and MOV10 have opposite effects on HIV-1 infectivity. (A) ATPase-defective UPF1DE636AA potently inhibits HIV-1 infectivity, whereas ectopic WT UPF1 has no effect. GHOST indicator cells were exposed to the indicated amounts of HIV-1 virions capable of a single round of transmission, and infected (GFP-positive) cells were quantified by flow cytometry. Virions were produced in 293T cells in the presence of vectors expressing HA-UPF1 (WT or DE636AA) or the empty vector. Two independently generated identical clones of the DE636AA mutant were examined to assess reproducibility. (B) Ectopic WT MOV10 potently inhibits HIV-1 infectivity, whereas MOV10DE645AA has no significant effect. (C) Levels of HA-tagged WT and mutant UPF1 or MOV10 expression in virus-producing cells.

Human UPF1, like its yeast ortholog, exhibits a nucleic acid-dependent ATPase activity that is abolished by the DE636AA mutation in its Walker B site (15, 34). Since it has been shown that a mutation in human UPF1 that interferes with ATP binding and hydrolysis can result in a dominant negative protein (35), we examined whether the expression of DE636AA mutant UPF1 in virus-producing cells affected HIV-1 infectivity. In contrast to HA-UPF1, HA-UPF1DE636AA expressed at comparable levels reproducibly reduced the infectivity of progeny virions 15- to 20-fold (Fig. 2A and C). Of note, the equivalent mutation in MOV10 (DE645AA) largely abolished the inhibition of HIV-1 infectivity by MOV10 (Fig. 2B). Thus, the Walker B site mutation had the opposite effect in MOV10 as in UPF1.

UPF1DE636AA binds ATP with WT efficiency (36). To determine whether ATP binding is necessary for the inhibition of HIV-1 infectivity, we also examined the effects of UPF1 mutants that are defective for ATP binding (K498A, R703A, and R865A) (36). Of these, the K498A and R703A mutants bind to RNA with WT efficiency (36), whereas the R865A mutation is in a putative RNA binding motif (34). We observed that the K498A, R703A, and R865A mutants all clearly reduced the specific infectivities of progeny HIV-1 particles (Fig. 3A and B). The inhibitory effect of HA-UPF1R865A was particularly pronounced (Fig. 3A) and approached that exhibited by HA-UPF1DE636AA (Fig. 2A). As shown in Fig. 3C, both mutants were taken up into HIV-1 particles. Thus, the ability to bind or to hydrolyze ATP was not required for the virion association of UPF1. However, mutations that prevented ATP binding or hydrolysis conferred antiviral activity.

Fig 3.

Fig 3

Open in a new tab

Inhibition of HIV-1 infectivity by UPF1 mutants defective for ATP binding. (A) The ATP binding-defective UPF1 mutants K498A, R703A, and R865A dominantly inhibit HIV-1 infectivity. Equal amounts of single-cycle virions (equivalent to 40 ng p24), produced in the presence of empty vector or of vectors expressing WT or mutant HA-UPF1, as indicated, were added to GHOST indicator cells, and infectivities were compared by quantifying the percentage of GFP-positive cells. (B) Levels of WT and mutant versions of HA-UPF1 expression in virus-producing cells. (C) Immunoblots demonstrating that UPF1 mutants defective for ATP hydrolysis or ATP binding are taken up into HIV-1 virions. In lane 1, an HIV-1 provirus with a disrupted gag gene was used, whereas the provirus used in the other lanes expresses WT Gag.

UPF1R865A does not affect the viral protein or RNA content of HIV-1 particles.

We next examined whether HA-UPF1R865A impaired the processing of HIV-1 Gag. Although HA-UPF1R865A did not affect overall Gag expression levels, it caused a moderate reduction in the level of cell-associated mature CA protein and a modest (less than 2-fold) reduction in the release of mature viral particles (Fig. 4A). However, there was no evidence for an accumulation of Gag cleavage intermediates, suggesting that virion-associated Gag was processed normally. As shown in Fig. 4B, the expression of HA-UPF1R865A in producer cells had no effect on the amount of IN detectable in progeny virions, indicating that the expression and processing of Gag-Pol were unaffected. Furthermore, HA-UPF1R865A had no effect on the virion association of the surface glycoprotein gp120 (Fig. 4B), indicating that the processing and incorporation of the Env glycoproteins occurred normally. Taken together, these data imply that virions produced in the presence of HA-UPF1R865A contained normal amounts of mature Gag, Gag-Pol, and Env products.

Fig 4.

Fig 4

Open in a new tab

HIV-1 virions produced in the presence of UPF1R865A contain normal amounts of mature Gag, Gag-Pol, and Env products. (A) UPF1R865A does not affect the processing of HIV-1 Gag. 293T cells were transfected with proviral DNA together with vectors expressing HA-tagged WT or R865A UPF1 or the empty vector, and the cell lysates and virion pellets were analyzed by Western blotting with antibodies against HIV-1 CA, HA, or actin. (B) Western blot showing that virions produced in the presence of UPF1R865A contain normal amounts of IN and of the envelope glycoprotein gp120. Virion pellets were adjusted for CA content by p24 ELISA.

To determine whether HA-UPF1R865A inhibited the encapsidation of the viral genome, viral RNA extracted from sucrose-purified virions was analyzed by SYBR green-based quantitative RT-PCR with primers specific for full-length viral nucleic acid. This analysis indicated that HA-UPF1R865A in virus-producing cells had no significant effect on the amount of virion-associated genomic RNA (Fig. 5A). As expected, no signal was obtained after the transfection of an HIV-1 mutant with a disrupted gag gene. Furthermore, virion RNA samples that were not reverse transcribed yielded negligible signals (Fig. 5A), confirming that only virion-associated RNA was detected.

Fig 5.

Fig 5

Open in a new tab

UPF1R865A does not affect the encapsidation of viral RNA. (A) Quantitative RT-PCR with primers specific for unspliced viral RNA showing normal amounts of genomic RNA in HIV-1 virions produced by transiently transfected 293T cells expressing UPF1R865A. Virion RNA samples that were not reverse transcribed [(−)RT] served as a negative control. (B) Northern blot of virion-associated RNA showing that UPF1R865A does not impair the integrity of the encapsidated genomic viral RNA. In lanes 1 to 4, RNA extracted from equal amounts of virions produced in 293T cells was analyzed. A sample from cells transfected with a provirus unable to make Gag was included as a negative control (lane 5).

Since retroviral genomes frequently contain breaks (37), we considered the possibility that HA-UPF1R865A affected the integrity of the encapsidated viral RNA. To address this possibility, virion RNA samples adjusted for equal amounts of p24 were examined by Northern blotting. As shown in Fig. 5B, intact genomic RNA of the expected size and a smear of presumably nicked viral RNAs were detected, regardless of whether the virions were produced in the presence of ectopic WT or mutant UPF1 or of the empty vector. Although the overexpression of WT HA-UPF1 caused a slight reduction in the relative intensity of the smear of nicked viral RNAs, the profiles of intact and nicked RNA species in the empty vector and HA-UPF1R865A samples did not differ. We conclude that HA-UPF1R865A did not alter the amount or integrity of encapsidated viral RNA.

Virions produced in the presence of UPF1R865A are defective for reverse transcription.

To determine whether UPF1R865A affected the ability of progeny virions to enter target cells, we used a previously described fusion assay that is based on the incorporation of a chimeric β-lactamase–Vpr (BlaM-Vpr) protein into progeny virions (38). Upon virus entry, the delivery of BlaM-Vpr into the cytosol of target cells can be detected with CCF4-AM, a cell-permeant fluorescent substrate for BlaM. In BlaM-negative cells, CCF4 emits a green fluorescence signal, whereas enzymatic cleavage of the substrate in BlaM-positive cells results in a blue signal that can be readily discerned by fluorescence microscopy (38). To limit virus transmission to a single cycle, we used env-deficient HIV-1 proviral DNA complemented with Env in trans to produce virions. As expected, no BlaM-positive cells were obtained when the Env expression vector was omitted. As shown in Fig. 6A, the exposure of TZM-bl cells to equal amounts of HIV-1 virions produced in the presence of control vector, HA-UPF1, or HA-UPF1R865A yielded comparable numbers of BlaM-positive cells, indicating that viral entry was comparable in each case.

Fig 6.

Fig 6

Open in a new tab

UPF1R865A interferes with reverse transcription. (A) Virions produced in the presence of UPF1R865A fuse normally with target cells. TZM-bl cells were exposed to virions produced in 293T cells cotransfected with vectors for BlaM-Vpr and WT or mutant HA-UPF1 or empty vector. After incubation with CCF4-AM, the numbers of BlaM-positive cells per high-power field (HPF) were counted. The numbers were adjusted for the amount of input virus used for infection. (B) Virions produced in the presence of UPF1R865A are markedly defective at the level of reverse transcription. GHOST indicator cells were exposed to equal amounts of DNase-treated virions produced in the presence of WT or mutant HA-UPF1, and the levels of early and late RT products as well as of 2-LTR circles in the target cells were compared by quantitative PCR.

Next, the reverse transcription process following infection with equal amounts of virions produced in the presence of HA-UPF1 or HA-UPF1R865A was examined by quantitative PCR using previously described primer sets (29). These were designed to amplify minus-strand strong-stop DNA or the U5 and 5′ untranslated regions of the viral DNA to quantify early and late transcription products, respectively. Additionally, we used a primer pair specific to U5 and U3 to quantify 2-LTR circle formation (29), which is considered a surrogate marker for the nuclear import of the full-length viral cDNA. We found that cells infected with HIV-1 virions produced in the presence of HA-UPF1R865A contained clearly reduced amounts of early and late reverse transcriptase products (Fig. 6B). Consistent with this finding, 2-LTR circles were also reduced (Fig. 6B). We conclude that the presence of HA-UPF1R865A during virus production led to a marked defect at an early postentry stage.

Depletion of UPF1 but not UPF2 reduces the infectivity of progeny HIV-1 virions.

The results described above revealed that mutant UPF1, when expressed during virus production, can profoundly inhibit the infectivity of HIV-1 progeny virions. We next used RNA interference to determine whether endogenous UPF1 has a role in the HIV-1 replication cycle. In addition, we targeted UPF2, which, like UPF1, is a core component of the NMD pathway. To produce virions in cells depleted of UPF1 or UPF2, 293T cells were transfected with env-deficient HIV-1NL4-3 and an expression vector for NL4-3 Env, along with control or specific siRNAs. At 48 h posttransfection, UPF1 and UPF2 were each reduced to less than 10% of the control level in cultures transfected with the specific siRNAs (Fig. 7A). Virus produced after this time point was then harvested. As shown in Fig. 7B, the depletion of UPF1 had no effect on HIV-1 particle production, as determined by p24 antigen ELISA. However, the specific infectivity of virions produced by UPF1-depeleted cells was severely compromised. Specifically, late reverse transcriptase products in GHOST-CXCR4 target cells were reduced to about 13% of the control level (Fig. 7C). Furthermore, the percentage of target cells expressing GFP, indicative of productive infection, was reduced by a comparable extent (Fig. 7D). In contrast, virus produced by cells depleted of UPF2 was as infectious as virus produced by cells transfected with the nonspecific control siRNA (Fig. 7E). We conclude that endogenous UPF1 is dispensable for virus production but crucial for the ability of progeny virions to reverse transcribe their genome in newly infected cells. Furthermore, this function of UPF1 appears to be independent of its role in NMD.

Fig 7.

Fig 7

Open in a new tab

Depletion of endogenous UPF1 but not UPF2 reduces the infectivity of HIV-1 progeny virions. (A) Western blots demonstrating efficient depletion of UPF1 or UPF2 from virus-producing cells. (B) HIV-1 p24 antigen ELISA showing that depleting UPF1 from 293T cells transfected with proviral DNA does not affect the release of HIV-1 CA protein. (C) Quantitative PCR showing that target cells exposed to HIV-1 virions produced in UPF1-depleted cells contain only low levels of reverse transcripts. GHOST cells were exposed to equal amounts of virus produced in nonsilencing control or UPF1-knockdown cells. (D) Virions produced in UPF1-depleted cells are poorly infectious. GHOST indicator cells were exposed to equal amounts of virus produced in nonsilencing control or UPF1-knockdown cells, and the percentage of infected target cells was determined by fluorescence-activated cell sorting. (E) Virions produced in UPF2-depleted cells exhibit normal infectivity. Ctrl, control.

DISCUSSION

Our results show that the SF1 helicase UPF1 is critical for the production of infectious HIV-1 virions. UPF1 is incorporated into HIV-1 particles, and the specific infectivity of virions made in cells expressing certain UPF1 mutants is substantially impaired. Likewise, depleting UPF1 markedly reduces the ability of progeny virions to initiate a further round of virus replication, indicating that the presence of UPF1 during virus assembly is necessary for subsequent steps of the HIV-1 replication cycle.

We initially identified endogenous UPF1 as a component of HIV-1 virions through a proteomics approach. This approach also yielded MOV10, another member of the UPF1-like family of SF1 helicases that strongly inhibits the specific infectivity of HIV-1 virions when overexpressed in virus-producing cells. Thus, our results imply that the related SF1 helicases UFP1 and MOV10 are simultaneously incorporated into progeny virions when expressed at endogenous levels. Like the encapsidation of MOV10 (1), the uptake of UPF1 into viral particles depends on the NC domain of HIV-1 Gag, which indicates that UPF1 associates with the viral genomic RNA. Consistent with this notion, UPF1 has been found to copurify with a model HIV-1 3′ untranslated region (39). UPF1 has also been detected in HIV-1 Gag immunoprecipitates, which led to the proposal that UPF1 is a component of the HIV-1 ribonucleoprotein (RNP) complex (20).

Unlike MOV10, ectopic UPF1 did not impair the infectivity of progeny virions in our study. However, UPF1 could be converted into a potent inhibitor of HIV-1 infectivity by mutating residues required for ATP binding or hydrolysis, which are also required for the RNA helicase activity of UPF1 (15, 34). In contrast, the equivalent mutations abolished the inhibitory activity of WT MOV10 (Fig. 2 and data not shown). Our findings thus suggest that ectopic UPF1 inhibits HIV-1 infectivity only when its helicase activity is abolished, whereas ectopic MOV10 inhibits HIV-1 only when its helicase activity is preserved.

We also note that ATP binding-defective UPF1 and ectopic WT MOV10 appear to impair the same stage of HIV-1 replication. While mutant UPF1 in virus-producing cells did not affect the ability of progeny virions to fuse with target cells, the subsequent production of reverse transcripts was significantly reduced. Several studies have shown that the overexpression of WT MOV10 in viral producer cells also blocks HIV-1 replication at the level of reverse transcription (2–5). In most of these studies, the effect of ectopic MOV10 was already clearly manifest during the early stages of reverse transcription (2, 4, 5). Similarly, our results show that HIV-1 virions assembled in the presence of ATP binding-defective UPF1 generated reduced amounts of both early and late reverse transcriptase products upon fusion with target cells.

It has been reported that the depletion of UPF1 by siRNA results in a dramatic decrease in HIV-1 Gag synthesis, apparently due to the loss of full-length HIV-1 RNA (20). However, the possibility of an off-target effect could not be excluded, because HIV-1 RNA expression was not rescued by siRNA-resistant UPF1 (20). In our hands, a siRNA that efficiently depleted cellular UPF1 levels and significantly reduced the specific infectivity of progeny virions had no effect on virus production, indicating that Gag expression levels were not affected. We also did not observe increased Gag expression levels upon UPF1 overexpression, as has been reported (20). Although we do not know the reason for these discrepancies, it is possible that the reported role for UPF1 in HIV-1 RNA metabolism and translation (20) is cell type dependent.

UPF1 is an essential component of the NMD pathway, but it also has apparently NMD-independent functions, such as in DNA replication and in telomere homeostasis (40, 41). In principle, UPF1 could be required for HIV-1 infectivity because an inhibitory factor accumulates in progeny virions if NMD is disrupted. In support of this notion, UPF1 has been shown to regulate the levels of thousands of physiological transcripts in human cells (42). Although we found that HIV-1 infectivity is insensitive to the depletion of the NMD protein UPF2, this does not strictly rule out a role for UPF1-mediated mRNA decay, because a distinct branch of the NMD pathway that depends on UPF1 but not UPF2 has been described (43). Furthermore, UPF1 is a component of the Staufen1-mediated mRNA decay pathway (44), which, similar to NMD, affects the expression of a large number of transcripts in human cells (45) but is largely insensitive to UPF2 depletion (46). Of note, Staufen1 has been detected in HIV-1 virions (47), and the depletion of Staufen1 from virus-producing cells led to a 2-fold decrease in HIV-1 infectivity (48).

It is also conceivable that UPF1 has a direct role in HIV-1 replication, for instance, in the annealing of the tRNA primer to the viral genome, which is required to initiate reverse transcription. Indeed, it has recently been established that the efficiency of tRNA annealing and its ability to prime reverse transcription can both be promoted by a cellular RNA helicase (49). Another possibility is that UPF1 serves to remodel the viral RNP to facilitate reverse transcription. The notion that UPF1 can act as an RNPase is supported by the observation that its ATPase activity is required for the removal of proteins from partially degraded NMD substrates (50). An understanding of the mechanism by which UPF1 promotes reverse transcription is likely to yield novel insights into this essential step of the HIV-1 replication cycle.

ACKNOWLEDGMENTS

We thank Scott A. Gerber and Steven P. Gygi for protein microsequencing, Jens Lykke-Andersen for anti-UPF1 antibody, and Melissa Moore for helpful discussions.

The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 p24 monoclonal antibody (183-H12-5C) from Bruce Chesebro and Kathy Wehrly and GHOST-CXCR4 cells from V. N. KewalRamani and D. R. Littman.

This work was supported by grant number R37AI029873 from the National Institute of Allergy and Infectious Diseases and by the University of Massachusetts Center for AIDS Research (P30-AI042845).

Footnotes

Published ahead of print 19 June 2013

REFERENCES

  • 1. Abudu A, Wang X, Dang Y, Zhou T, Xiang SH, Zheng YH. 2012. Identification of molecular determinants from Moloney leukemia virus 10 homolog (MOV10) protein for virion packaging and anti-HIV-1 activity. J. Biol. Chem. 287:1220–1228 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Arjan-Odedra S, Swanson CM, Sherer NM, Wolinsky SM, Malim MH. 2012. Endogenous MOV10 inhibits the retrotransposition of endogenous retroelements but not the replication of exogenous retroviruses. Retrovirology 9:53. 10.1186/1742-4690-9-53 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Burdick R, Smith JL, Chaipan C, Friew Y, Chen J, Venkatachari NJ, Delviks-Frankenberry KA, Hu WS, Pathak VK. 2010. P body-associated protein Mov10 inhibits HIV-1 replication at multiple stages. J. Virol. 84:10241–10253 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Furtak V, Mulky A, Rawlings SA, Kozhaya L, Lee K, Kewalramani VN, Unutmaz D. 2010. Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity. PLoS One 5:e9081. 10.1371/journal.pone.0009081 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Wang X, Han Y, Dang Y, Fu W, Zhou T, Ptak RG, Zheng YH. 2010. Moloney leukemia virus 10 (MOV10) protein inhibits retrovirus replication. J. Biol. Chem. 285:14346–14355 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Meister G, Landthaler M, Peters L, Chen PY, Urlaub H, Luhrmann R, Tuschl T. 2005. Identification of novel argonaute-associated proteins. Curr. Biol. 15:2149–2155 [DOI] [PubMed] [Google Scholar]
  • 7. Wulczyn FG, Smirnova L, Rybak A, Brandt C, Kwidzinski E, Ninnemann O, Strehle M, Seiler A, Schumacher S, Nitsch R. 2007. Post-transcriptional regulation of the let-7 microRNA during neural cell specification. FASEB J. 21:415–426 [DOI] [PubMed] [Google Scholar]
  • 8. Gallois-Montbrun S, Holmes RK, Swanson CM, Fernandez-Ocana M, Byers HL, Ward MA, Malim MH. 2008. Comparison of cellular ribonucleoprotein complexes associated with the APOBEC3F and APOBEC3G antiviral proteins. J. Virol. 82:5636–5642 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Gallois-Montbrun S, Kramer B, Swanson CM, Byers H, Lynham S, Ward M, Malim MH. 2007. Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules. J. Virol. 81:2165–2178 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Goodier JL, Cheung LE, Kazazian HH., Jr 2012. MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells. PLoS Genet. 8:e1002941. 10.1371/journal.pgen.1002941 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Lu C, Luo Z, Jager S, Krogan NJ, Peterlin BM. 2012. Moloney leukemia virus type 10 inhibits reverse transcription and retrotransposition of intracisternal a particles. J. Virol. 86:10517–10523 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Chertova E, Chertov O, Coren LV, Roser JD, Trubey CM, Bess JW, Jr, Sowder RC, II, Barsov E, Hood BL, Fisher RJ, Nagashima K, Conrads TP, Veenstra TD, Lifson JD, Ott DE. 2006. Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J. Virol. 80:9039–9052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Fairman-Williams ME, Guenther UP, Jankowsky E. 2010. SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol. 20:313–324 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Applequist SE, Selg M, Raman C, Jack HM. 1997. Cloning and characterization of HUPF1, a human homolog of the Saccharomyces cerevisiae nonsense mRNA-reducing UPF1 protein. Nucleic Acids Res. 25:814–821 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Bhattacharya A, Czaplinski K, Trifillis P, He F, Jacobson A, Peltz SW. 2000. Characterization of the biochemical properties of the human Upf1 gene product that is involved in nonsense-mediated mRNA decay. RNA 6:1226–1235 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Chang YF, Imam JS, Wilkinson MF. 2007. The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 76:51–74 [DOI] [PubMed] [Google Scholar]
  • 17. Nicholson P, Yepiskoposyan H, Metze S, Zamudio Orozco R, Kleinschmidt N, Muhlemann O. 2010. Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors. Cell. Mol. Life Sci. 67:677–700 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Withers JB, Beemon KL. 2011. The structure and function of the Rous sarcoma virus RNA stability element. J. Cell. Biochem. 112:3085–3092 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Weil JE, Beemon KL. 2006. A 3′ UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus. RNA 12:102–110 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Ajamian L, Abrahamyan L, Milev M, Ivanov PV, Kulozik AE, Gehring NH, Mouland AJ. 2008. Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation. RNA 14:914–927 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Pizzato M, Helander A, Popova E, Calistri A, Zamborlini A, Palu G, Gottlinger HG. 2007. Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef. Proc. Natl. Acad. Sci. U. S. A. 104:6812–6817 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Accola MA, Strack B, Gottlinger HG. 2000. Efficient particle production by minimal gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain. J. Virol. 74:5395–5402 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Dorfman T, Mammano F, Haseltine WA, Gottlinger HG. 1994. Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein. J. Virol. 68:1689–1696 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Dettenhofer M, Yu XF. 1999. Highly purified human immunodeficiency virus type 1 reveals a virtual absence of Vif in virions. J. Virol. 73:1460–1467 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Chesebro B, Wehrly K, Nishio J, Perryman S. 1992. Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism. J. Virol. 66:6547–6554 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Mendell JT, ap Rhys CM, Dietz HC. 2002. Separable roles for rent1/hUpf1 in altered splicing and decay of nonsense transcripts. Science 298:419–422 [DOI] [PubMed] [Google Scholar]
  • 27. Gehring NH, Neu-Yilik G, Schell T, Hentze MW, Kulozik AE. 2003. Y14 and hUpf3b form an NMD-activating complex. Mol. Cell 11:939–949 [DOI] [PubMed] [Google Scholar]
  • 28. Morner A, Bjorndal A, Albert J, Kewalramani VN, Littman DR, Inoue R, Thorstensson R, Fenyo EM, Bjorling E. 1999. Primary human immunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, frequently use CCR5 but show promiscuity in coreceptor usage. J. Virol. 73:2343–2349 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Mbisa JL, Delviks-Frankenberry KA, Thomas JA, Gorelick RJ, Pathak VK. 2009. Real-time PCR analysis of HIV-1 replication post-entry events. Methods Mol. Biol. 485:55–72 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Sambrook J, Russell DW. 2006. Northern hybridization. CSH Protoc. 2006:pii= pdb.prot3723. 10.1101/pdb.prot3723 [DOI] [PubMed] [Google Scholar]
  • 31. Sharkey ME, Teo I, Greenough T, Sharova N, Luzuriaga K, Sullivan JL, Bucy RP, Kostrikis LG, Haase A, Veryard C, Davaro RE, Cheeseman SH, Daly JS, Bova C, Ellison RT, III, Mady B, Lai KK, Moyle G, Nelson M, Gazzard B, Shaunak S, Stevenson M. 2000. Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy. Nat. Med. 6:76–81 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Atkin AL, Altamura N, Leeds P, Culbertson MR. 1995. The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm. Mol. Biol. Cell 6:611–625 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Cantin R, Diou J, Belanger D, Tremblay AM, Gilbert C. 2008. Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. J. Immunol. Methods 338:21–30 [DOI] [PubMed] [Google Scholar]
  • 34. Weng Y, Czaplinski K, Peltz SW. 1996. Genetic and biochemical characterization of mutations in the ATPase and helicase regions of the Upf1 protein. Mol. Cell. Biol. 16:5477–5490 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Kaygun H, Marzluff WF. 2005. Regulated degradation of replication-dependent histone mRNAs requires both ATR and Upf1. Nat. Struct. Mol. Biol. 12:794–800 [DOI] [PubMed] [Google Scholar]
  • 36. Cheng Z, Muhlrad D, Lim MK, Parker R, Song H. 2007. Structural and functional insights into the human Upf1 helicase core. EMBO J. 26:253–264 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Coffin JM. 1979. Structure, replication, and recombination of retrovirus genomes: some unifying hypotheses. J. Gen. Virol. 42:1–26 [DOI] [PubMed] [Google Scholar]
  • 38. Cavrois M, De Noronha C, Greene WC. 2002. A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat. Biotechnol. 20:1151–1154 [DOI] [PubMed] [Google Scholar]
  • 39. Hogg JR, Goff SP. 2010. Upf1 senses 3′UTR length to potentiate mRNA decay. Cell 143:379–389 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Azzalin CM, Lingner J. 2006. The human RNA surveillance factor UPF1 is required for S phase progression and genome stability. Curr. Biol. 16:433–439 [DOI] [PubMed] [Google Scholar]
  • 41. Chawla R, Redon S, Raftopoulou C, Wischnewski H, Gagos S, Azzalin CM. 2011. Human UPF1 interacts with TPP1 and telomerase and sustains telomere leading-strand replication. EMBO J. 30:4047–4058 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Mendell JT, Sharifi NA, Meyers JL, Martinez-Murillo F, Dietz HC. 2004. Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. Nat. Genet. 36:1073–1078 [DOI] [PubMed] [Google Scholar]
  • 43. Gehring NH, Kunz JB, Neu-Yilik G, Breit S, Viegas MH, Hentze MW, Kulozik AE. 2005. Exon-junction complex components specify distinct routes of nonsense-mediated mRNA decay with differential cofactor requirements. Mol. Cell 20:65–75 [DOI] [PubMed] [Google Scholar]
  • 44. Kim YK, Furic L, Desgroseillers L, Maquat LE. 2005. Mammalian Staufen1 recruits Upf1 to specific mRNA 3′UTRs so as to elicit mRNA decay. Cell 120:195–208 [DOI] [PubMed] [Google Scholar]
  • 45. Kim YK, Furic L, Parisien M, Major F, DesGroseillers L, Maquat LE. 2007. Staufen1 regulates diverse classes of mammalian transcripts. EMBO J. 26:2670–2681 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Gong C, Kim YK, Woeller CF, Tang Y, Maquat LE. 2009. SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs. Genes Dev. 23:54–66 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. Mouland AJ, Mercier J, Luo M, Bernier L, DesGroseillers L, Cohen EA. 2000. The double-stranded RNA-binding protein Staufen is incorporated in human immunodeficiency virus type 1: evidence for a role in genomic RNA encapsidation. J. Virol. 74:5441–5451 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Chatel-Chaix L, Clement JF, Martel C, Beriault V, Gatignol A, DesGroseillers L, Mouland AJ. 2004. Identification of Staufen in the human immunodeficiency virus type 1 Gag ribonucleoprotein complex and a role in generating infectious viral particles. Mol. Cell. Biol. 24:2637–2648 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Xing L, Liang C, Kleiman L. 2011. Coordinate roles of Gag and RNA helicase A in promoting the annealing of formula to HIV-1 RNA. J. Virol. 85:1847–1860 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Franks TM, Singh G, Lykke-Andersen J. 2010. Upf1 ATPase-dependent mRNP disassembly is required for completion of nonsense-mediated mRNA decay. Cell 143:938–950 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES