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Journal of Virology, June 2009, p. 6067-6078, Vol. 83, No. 12
0022-538X/09/$08.00+0 doi:10.1128/JVI.02231-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Regulation of vif mRNA Splicing by Human Immunodeficiency Virus Type 1 Requires 5' Splice Site D2 and an Exonic Splicing Enhancer To Counteract Cellular Restriction Factor APOBEC3G
Dibyakanti Mandal,
Colin M. Exline,
Zehua Feng, and
C. Martin Stoltzfus*
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242
Received 22 October 2008/ Accepted 31 March 2009
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The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif is encoded by an incompletely spliced mRNA resulting from splicing of the major splice donor in the HIV-1 genome, 5' splice site (5'ss) D1, to the first splice acceptor, 3'ss A1. We have shown previously that splicing of HIV-1 vif mRNA is tightly regulated by suboptimal 5'ss D2, which is 50 nucleotides downstream of 3'ss A1; a GGGG silencer motif proximal to 5'ss D2; and an SRp75-dependent exonic splicing enhancer (ESEVif). In agreement with the exon definition hypothesis, mutations within 5'ss D2 that are predicted to increase or decrease U1 snRNP binding affinity increase or decrease the usage of 3'ss A1 (D2-up and D2-down mutants, respectively). In this report, the importance of 5'ss D2 and ESEVif for avoiding restriction of HIV-1 by APOBEC3G (A3G) was determined by testing the infectivities of a panel of mutant viruses expressing different levels of Vif. The replication of D2-down and ESEVif mutants in permissive CEM-SS cells was not significantly different from that of wild-type HIV-1. Mutants that expressed Vif in 293T cells at levels greater than 10% of that of the wild type replicated similarly to the wild type in H9 cells, and Vif levels as low as 4% were affected only modestly in H9 cells. This is in contrast to Vif-deleted HIV-1, whose replication in H9 cells was completely inhibited. To test whether elevated levels of A3G inhibit replication of D2-down and ESEVif mutants relative to wild-type virus replication, a Tet-off Jurkat T-cell line that expressed approximately 15-fold-higher levels of A3G than control Tet-off cells was generated. Under these conditions, the fitness of all D2-down mutant viruses was reduced relative to that of wild-type HIV-1, and the extent of inhibition was correlated with the level of Vif expression. The replication of an ESEVif mutant was also inhibited only at higher levels of A3G. Thus, wild-type 5'ss D2 and ESEVif are required for production of sufficient Vif to allow efficient HIV-1 replication in cells expressing relatively high levels of A3G.
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Human immunodeficiency virus type 1 (HIV-1) Vif is a 23-kDa basic protein (4, 9) that is incorporated into virus particles during productive infection (8-10). Replication of HIV-1 in some T-cell lines is dependent on the expression of a functional Vif protein. Replication of Vif-deleted HIV-1 is restricted in these cells, which are termed nonpermissive, because of the presence of several host deaminases, the most important of which for HIV-1 replication is APOBEC3G (A3G) (25, 26). Human A3G is a single-stranded DNA deaminase that inhibits the replication of HIV-1 as well as other types of retroviruses and retrotransposons (5, 12, 17, 25, 32). HIV-1 Vif forms a complex with A3G and other cellular proteins to promote A3G ubiquitination, resulting in proteasomal degradation of A3G (1, 11, 14, 18, 26). Vif-deleted HIV-1 produced in the presence of A3G packages increased levels of A3G compared to those found in the wild type (WT) and has reduced infectivity in nonpermissive T-cell lines. This reduced infectivity in the absence of Vif has been correlated with the dC-to-dU hypermutation of newly synthesized minus-strand viral DNA by A3G (6, 13, 31, 32). However, other studies have shown that A3G is also able to restrict virus replication without hypermutating viral DNA (7, 19).
It has previously been shown that the expression of Vif in infected cells is maintained at a relatively low level compared to levels of the other HIV-1 accessory proteins. One mechanism to explain this phenomenon is that Vif is degraded more rapidly than other accessory proteins by the proteasome (3). Another mechanism is that a relatively low level of vif mRNA is produced by alternative splicing (22). Alternative splicing of HIV-1 RNA results in the production of approximately 40 different mRNA species, which include three different mRNA size classes: 1.8-kb, completely spliced RNAs; 4-kb, incompletely spliced RNAs; and 9-kb, unspliced RNAs (Fig. 1A). The 4-kb mRNA class encodes Vif, Vpr, Tat, Vpu, and Env, and the completely spliced, 1.8-kb mRNA class encodes Tat, Rev, and Nef. Unspliced viral RNA is both packaged into virions as genomic RNA and used as mRNA for Gag and Gag-Pol proteins (2, 27). As shown in Fig. 1A, four different 5' splice donor sites (5'ss) and eight different 3' splice acceptor sites (3'ss), which are highly conserved among group M HIV-1 strains, are used to produce alternatively spliced HIV-1 mRNAs at different levels in infected cells (22). The efficiencies with which these 5'ss and 3'ss are used are dependent on the presence of suboptimal cis splicing elements within the 5'ss and 3'ss themselves and more-distant elements, which include exonic splicing silencers, an intronic splicing silencer, and exonic splicing enhancers (ESE) (2, 15, 27).
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FIG. 1. HIV-1 splicing pattern and elements regulating vif mRNA splicing. (A) The conserved 5'ss (D1 to D4) and 3'ss (A1 to A7) located within the 9-kb HIV-1 genome are shown. Completely and incompletely spliced HIV-1 mRNAs ( 4 kb and 1.8 kb) are shown as open boxes. Spliced mRNAs are denoted by the translated open reading frame and by the exon content. The incompletely spliced mRNAs, denoted with an I, are differentiated from completely spliced mRNAs by inclusion of the intron between 5'ss D4 and 3'ss A7. Either one or both of the noncoding exons 2 and 3 (shown as gray-shaded exons) can be differentially included within all 1.8- and 4.0-kb mRNA species, with the exception of vif mRNA (1.2I) and vpr mRNA, which can include only exon 2 (1.[2].3I). LTR, long terminal repeat. (B) Three elements regulating vif mRNA splicing are shown: positively acting enhancer ESEVif, the 5'ss D2 (underlined), and a negatively acting G4 silencer motif. The locations of noncoding exon 2 and the start site for Vif protein synthesis are also shown. (C) HIV-1 5'ss D2-down mutants used in this study are shown. Sequences were aligned and compared with that of the consensus metazoan 5'ss. The sequence of the ESEVif mutant used in this study is also aligned and compared with the WT sequence. nt, nucleotides.
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Cells. 293T cells were maintained in Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. CEM-SS and H9 T-cell lines were maintained in RPMI 1640, 10% FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin (RPMI medium). Tet-off Jurkat T cells were maintained in RPMI medium supplemented with 200 µg/ml G-418. AP3G/Jurkat cells were maintained in RPMI medium supplemented with both 200 µg/ml hygromycin B and 200 µg/ml G-418.
Plasmids and viruses.
The infectious HIV-1 molecular clones pNL4-3, pNL4-3 luc (Vpr– Env–) (pNLluc), and pHA-A3G were obtained from the NIH AIDS Research and Reference Program. Mutant plasmids derived from pNL4-3 (pNLD2up, pNLD2GC, pNLD2GC-G5, and pNL-ESEVifm) have been described previously (2). pnl
Vif is derived from pNL4-3 and has a deletion of the vif coding region. Mutants D2GG and D2GA were generated by site-directed mutagenesis of pNL4-3 using a QuikChange site-directed mutagenesis kit (Stratagene). Luciferase reporter clones (D2GCluc, D2GC-G5luc, D2GGluc, and D2GAluc) were created by QuikChange site-directed mutagenesis of pNLluc. pNLvifluc was created by insertion of an 2.6-kb AgeI/SalI fragment from pNLluc into pNL4-3. Clone U1GG was created by mutation of U1 snRNA clone pUC13-U1 by QuikChange site-directed mutagenesis using oligonucleotides U1 D2GG-F (5'-GAT CATG GTA TCT CCC CTG CCA GGG AAG TAT-3') and U1 D2GG-R (5'-ATA CTT CCC TGG CAG GGG AGA TAC CATG ATC-3') as forward and reverse primers, respectively. Plasmid pTRE2/A3G was created by PCR amplification of an 1.2-kb DNA fragment from pHA-A3G, followed by insertion of the fragment into the pTRE2-Hyg-GFP expression vector. This plasmid was modified from the original pTRE2-Hyg vector by insertion of the encephalomyocarditis virus internal ribosomal entry site element to direct translation of green fluorescent protein (GFP) (30). WT and mutant viruses were obtained by transfecting 293T cells with infectious plasmids using calcium phosphate precipitation as previously described (2). Transfection efficiencies were determined by cotransfection with lacZ expression plasmid pCMV110. Transfected cells and cell-free culture media were harvested 48 h posttransfection and assayed for β-galactosidase, protein, and RNA as described below.
RNA isolation and analysis of HIV-1 mRNA.
Total RNA was isolated from transfected cells using Tri reagent (Molecular Research Center, Inc.) according to the protocol supplied by the manufacturer. Reverse transcriptase PCR (RT-PCR) analysis of 1.8-kb-class HIV-1 mRNA species and quantitative real-time PCR analysis of vif mRNA were performed as previously described (2).
RT assays and virus production.
Cell-free supernatants were assayed for RT activity by [
-32P]dTTP incorporation as previously described (29). Incorporation of radioactivity was quantitated using an instant imager (Packard). For transfection experiments, RT data were normalized to that of the WT after correction for transfection efficiencies based on β-galactosidase assays of extracts from transfected cells (24).
Detection of viral proteins by immunoblotting. Cellular proteins (50 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane by the semidry-transfer method, and immunoblotted according to previously described methods to detect Vif and β-tubulin (2). Gag protein was detected using rabbit anti-p24 polyclonal antibody. To detect virion proteins, equal amounts of virus from the culture media, as determined by RT assays, were filtered through 0.45-µm filters and centrifuged at 18,000 x g for 90 min at 4°C. The virus pellets were then lysed in 5 mM Tris-7 mM SDS-10 mM dithiothreitol-5% glycerol (pH 6.8) at 100°C for 5 min. Virion proteins were analyzed by SDS-PAGE and immunoblotted as described above.
Generation of A3G/Jurkat cell line. Tet-off Jurkat cells (Clontech, CA) were transfected with pTRE2/A3G DNA by nucleofection using the protocol supplied by the manufacturer (Amaxa). Transfected cells were cultured for 3 to 4 days, and cells were then selected using RPMI medium containing 200 µg/ml each of hygromycin B and G-418. Growth of resistant cells was carried out for 2 weeks, after which GFP-positive cells were selected by fluorescence-activated cell sorting and were cultured for an additional 2 weeks. Single cells were then selected by dilution and cultured in the presence of hygromycin B and G-418 for an additional 1 to 2 weeks. Selected colonies were tested for A3G expression, and positive colonies were expanded. The A3G mRNA expression level in one such clone was determined by quantitative real-time PCR using primers specific for A3G (hA3G-F and hA3G-R, 5'-TCA GAG GAC GGC ATG AGA CTT AC-3' and 5'-AGC AGG ACC CAG GTG TCA TTG-3', respectively). The amount of cellular β-actin mRNA was also determined using specific primers (2), and the amounts of A3G mRNA relative to the amount of β-actin mRNA were calculated. A3G protein expressed by A3G/Jurkat cells was detected by Western blot analysis using anti-A3G and anti-hemagglutinin (HA) antibodies.
Infectivity assay. CEM-SS cells were infected with equal amounts of pNLluc or the corresponding D2 mutants produced in the absence of HA-A3G or in the presence of increasing amounts of HA-A3G. At 36 h postinfection, the cells were centrifuged, washed, and lysed with 1x lysis buffer. Cell lysates were assayed for luciferase activity using a luciferase assay system (Promega). Relative light units were determined by luminometric measurements.
Multiday replication assays. Cells were infected with equal amounts of viruses, as determined by RT assays, for 4 h at 37°C in serum-free RPMI medium. Infected cells were then centrifuged, washed, and resuspended in RPMI medium containing 10% FBS. Aliquots of cell-free media were harvested at regular intervals, and virus production was measured by RT assays.
Replication fitness assay and quantitative real-time PCR. The WTmut virus was generated by introducing two silent mutations in Gag by site-directed mutagenesis of infectious proviral plasmid NL4-3 at positions 1517 and 1520. As expected, based on the results of van Maarseveen et al. (28), there were no differences in infectivity or replication kinetics compared to those of the pNL4-3 (WT) virus. Cells were coinfected at a multiplicity of infection (MOI) of 0.005 with WTmut and 5'ss D2 mutant viruses with the WT p24 sequence. At day 12 postinfection, DNA was extracted from infected cells using a Qiagen blood and tissue kit following the protocol supplied by the manufacturer. Samples of infected-cell DNA (1 µg) were then subjected to real-time quantitative PCR using primers and probes specific for either the WTmut or the D2 mutant p24 sequence, as previously described (28).
TA cloning of PCR products and sequencing.
Viral DNA isolated from cells coinfected with WT and D2GC mutant viruses was amplified by PCR in the region containing the Gag mutations by use of primers DMp24Seq1 and DMp24Seq2 (5'-GCA TGC AGG GCC TAT TGC ACC-3' and 5'-GTC CAG AAT GCT GGT AGG GC-3', respectively). The 200-bp PCR product was then directly sequenced to determine the ratio of WT DNA to D2GC DNA. A portion of the amplified DNA was directly cloned into the TA cloning vector pPCR2.1 (Invitrogen) according to the manufacturer's protocol, and the resulting plasmids were sequenced.
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Mutations of the highly conserved U at the +2 position of 5'ss D2 reduce splicing efficiency at 3'ss A1 and exon 2 inclusion. To investigate the significance of the highly conserved 5'ss D2 downstream of 3'ss A1 for HIV-1 replication, we generated 5'ss D2 mutations within the infectious HIV-1 plasmid pNL4-3 in order to lower the strength of U1 snRNP binding and reduce splicing efficiency at 3'ss A1 (Fig. 1C). These mutations were designed to change the sequence of 5'ss D2 without changing the overlapping integrase reading frame. We have shown previously that mutations of 5'ss D2 with reduced U1 snRNP binding affinity (mutants D2GC and D2GC-G5) decrease splicing at 3'ss A1 and prevent inclusion of exon 2 (2). For the present study, we generated additional +2 mutants by replacing U with A or G (mutant D2GA or D2GG, respectively). We also compared these D2-down mutants to a previously described mutant in which ESEVif (pNL-ESEVifm) was inactivated (2).
To determine the effects of the D2-down mutants and the ESEVif mutant on HIV-1 gene expression, the mutated and WT infectious pNL4-3 plasmids were transfected into 293T cells. The transfected cells were harvested after 48 h, and total cellular RNA and protein were isolated from the cells. Analysis of the 1.8-kb, completely spliced mRNA species by RT-PCR indicated that mutation of the highly conserved U at the +2 position of 5'ss D2 to C, A, or G (mutants D2GC, D2GA, and D2GG, respectively) and mutation of ESEVif prevented detectable exon 2 inclusion, as indicated by the absence of mRNA species 1.2.5.7 and 1.2.4.7 (Fig. 2A). RT assays of supernatants from the transfected cells showed that virus particle production was not significantly affected by any of the mutations (Fig. 2B). Analysis of vif mRNA by quantitative real-time PCR indicated that all of the mutants produced decreased vif mRNA compared to levels for the WT, with the order of expression being WT > D2GC > D2GC-G5 > D2GA > D2GG 
ESEVifm (Fig. 2C). Western blot analysis showed that the levels of expression of Vif protein by the different mutants were consistent with the levels of vif mRNA expression (Fig. 2D).
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FIG. 2. Effect of 5'ss D2-down mutations on exon 2 inclusion and Vif expression. (A) Total RNA was isolated from WT-, D2 mutant-, and ESEVifm-transfected 293T cells. The 1.8-kb, completely spliced HIV-1 mRNAs were amplified by RT-PCR, and the products were analyzed by denaturing 6% PAGE (see Materials and Methods). The HIV-1 mRNA species corresponding to the PCR products are indicated on the right. Bands marked with asterisks are artifact bands. (B) Cell supernatants harvested from transfected cells were analyzed for virus production by an RT assay, and the RT activities of the D2 mutants and ESEVifm relative to that of the WT are shown. (C) Quantitative real-time PCR of vif mRNA was performed using total cellular RNA isolated from cells transfected with WT and D2-down mutant DNA, as described in Materials and Methods. The levels of vif mRNAs produced by D2-down mutants are expressed relative to that of the WT. Error bars in panels B and C indicate standard deviations. (D) Vif protein expressed by the WT and the D2 mutants was detected by Western blot analysis of extracts from transfected cells. β-Tubulin was also detected by Western blot analysis of proteins from transfected cells.
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Vifluc viruses were determined by single-cycle replication assays and compared to those of the no-A3G controls (Fig. 3A). The results indicated that mutants D2GAluc and D2GGluc exhibited reduced infectivities compared to those of mutants D2GCluc and D2GCG5luc, whose infectivities in this assay were not significantly different from that of the WT.
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FIG. 3. Infectivities and compositions of HIV-1 5'ss D2luc mutant viruses produced in the presence and absence of A3G. (A) WT NL4-3luc and 5'ss D2luc mutant viruses pseudotyped with vesicular stomatitis virus G were generated by cotransfecting 293T cells with viral plasmid DNA and increasing amounts of HA-tagged A3G DNA. As determined by RT activities, equal amounts of virus from the culture media of the transfected cells were used to infect CEM-SS cells. At 36 h postinfection, the infected cells were lysed and luciferase activities were measured. Luciferase activities at different A3G concentrations relative to luciferase activities of the no-A3G controls were determined. Error bars indicate standard deviations. (B) Pelleted virions produced from cells cotransfected with WT NL4-3luc or D2luc mutants and 2 µg A3G were disrupted and analyzed by Western blotting using anti-HA and anti-p24gag antibodies.
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Increased Vif expression and infectivity of D2GG mutant virus in the presence of U1 snRNA variant U1GG under nonpermissive conditions. We have shown that the conserved 5'ss D2 downstream of 3'ss A1 plays an important role in regulating splicing at 3'ss A1. Mutation of 5'ss D2 to a stronger or weaker splice site based on predicted binding affinities of U1 snRNA to the splice site alters the splicing of vif mRNA and expression of Vif protein. To further test the hypothesis that altered Vif production by the D2 mutants is due to decreased binding of U1 snRNA to 5'ss D2, we cotransfected D2-down mutant D2GGluc, which produces the least Vif of the D2-down mutants described above, with U1GG, a U1 snRNA clone which expresses U1 snRNA with an A-to-C change corresponding to the U-to-G change at the +2 position of 5'ss D2 (Fig. 4A). Figure 4B shows that increased Vif production comparable to that of the WT was detected in cells cotransfected with D2GGluc and increasing amounts of U1GG. Gag protein levels, on the other hand, remained unchanged. Vif protein levels of NL4-3luc transfected with increasing amounts of U1GG were not significantly changed. Analyses of vif mRNA were consistent with the Vif protein data and also showed that, as expected, there was no detectable inclusion of exon 2 in HIV-1 mRNA species in the presence of the altered U1 snRNP (data not shown).
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FIG. 4. Increased Vif production by the D2GG mutant in the presence of the U1 snRNA variant that restored U1 snRNA complementarity. (A) The sequence of U1 snRNA mutant U1GG is shown, as are the sequences of WT 5'ss D2 and the corresponding D2GG mutant. (B) The amounts of Vif protein expressed by NL4-3luc and the D2GGluc mutant in the presence or absence of U1 snRNA variant U1GG were determined as described in the legend for Fig. 2. Gag protein expressed in the transfected cells was also determined by Western blot analysis as described in Materials and Methods. (C) The infectivities of D2GGluc viruses produced in the presence or absence of U1GG under nonpermissive conditions were determined as described in the legend for Fig. 3. The infectivity of D2GGluc produced in the absence of any A3G is indicated as the control. The infectivities of D2GGluc viruses produced in the presence of 1 µg A3G and different amounts of U1GG are shown. Error bars indicate standard deviations.
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Replication kinetics of D2-down mutant viruses in permissive CEM-SS and nonpermissive H9 cells.
To further examine the importance of the highly conserved 5'ss D2 for HIV-1 replication, we studied the long-term replication kinetics of mutant viruses in permissive CEM-SS and nonpermissive H9 T-cell lines. Cells were infected at a low MOI, and virus production was monitored by an RT assay at regular time intervals for 16 days postinfection. The results in Fig. 5A indicate that the replication kinetics of all of the D2-down mutant viruses as well as the Vif-deleted HIV-1 mutant virus (NLVif) were comparable to those of the WT virus in CEM-SS cells. Thus, 5'ss D2 appears to have no significant role in virus replication under permissive conditions. We next determined the abilities of the D2-down mutant viruses to replicate in nonpermissive H9 cells. H9 cells have been shown to produce endogenous A3G, which restricts the replication of Vif-deleted HIV-1 strains (25). These results indicated that, as expected, NLVif was unable to replicate in H9 cells (Fig. 5B). In contrast, there were no significant effects on the replication of D2GC, D2GC-G5, and D2GA mutant viruses compared to the replication of WT HIV-1. Mutant D2GG, which maintains the lowest level of Vif, demonstrated an approximately 50% decrease in virus particle production over the 16-day infection period. These results suggested that, although D2GC, D2GCG5, and D2GA viruses exhibited reduced levels of Vif compared to that for the WT virus, these amounts were sufficient to counteract A3G produced by H9 cells (Fig. 5B).
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FIG. 5. Replication kinetics of the WT and 5'ss D2-down mutants in CEM-SS and H9 T cells. CEM-SS or H9 cells were infected with WT or D2-down mutant viruses at an MOI of 0.005, and virus production was determined by an RT assay of culture media harvested at various times after infection, as indicated. RT activity was determined as described in Materials and Methods and is presented as radioactive counts obtained. Error bars indicate standard deviations.
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6-fold less A3G mRNA than untreated cells, although this level of A3G was approximately twofold higher than the level in control Tet-off Jurkat cells (Fig. 6A). Attempts to lower A3G levels still further by increasing Dox levels above 2 ng/ml resulted in an increased rate of cell death (data not shown).
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FIG. 6. Relative levels of A3G expression in Tet-off Jurkat and A3G/Jurkat cell lines in the presence or absence of Dox. (A) Relative A3G mRNA levels in control Tet-off Jurkat and A3G/Jurkat cell lines without Dox or treated with the various amounts of Dox shown were determined by quantitative real-time PCR, as described in Materials and Methods. (B and C) A3G was detected by Western blot analysis of proteins isolated from Tet-off Jurkat and A3G/Jurkat cells using anti-A3G and anti-HA antibodies.
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Replication of D2-down mutant viruses is inhibited in the presence of elevated levels of A3G.
We next studied the replication kinetics of D2-down mutant viruses in A3G/Jurkat cells at elevated A3G levels. A3G/Jurkat cells were cultured in the presence of 0.1 ng, 0.25 ng, or 1 ng/ml of Dox or in the absence of Dox for 4 days, followed by infection with WT and D2 mutant viruses at an MOI of 0.005. Control Tet-off Jurkat cells were also infected with WT and D2-down mutant viruses to compare replication kinetics of these mutant viruses in the presence of the small amount of endogenous A3G produced in these cells (Fig. 7). Supernatants from infected cells were assayed for virus production by an RT assay at regular intervals after infection. The replication kinetics of WT and D2-down mutant viruses indicated that all mutant viruses were able to replicate efficiently in control Tet-off cells compared to replication of WT HIV-1, whereas replication of NLVif was restricted in these cells (Fig. 7, Tet-off cells). These results indicate that the endogenous level of A3G expressed in control Tet-off Jurkat cells is sufficient to inhibit the replication of NLVif virus but not that of any of the mutant viruses. Replication of all of the D2-down mutant viruses was inhibited compared to replication of WT HIV-1 in A3G/Jurkat cells in the absence of Dox, i.e., at the highest level of A3G (Fig. 7, No Dox). The D2GG mutant replicated with the slowest kinetics and, similarly to NLVif, produced no detectable virus over the 16-day infection period. In the presence of 0.1 ng/ml Dox, the D2GC mutant replicated similarly to WT HIV-1, whereas the other D2-down mutant viruses replicated more slowly. At higher Dox concentrations of 0.25 and 1 ng/ml, mutants D2GA and D2GC-G5 also replicated similarly to the WT. The only D2-down mutant demonstrating slower replication kinetics at the higher Dox concentrations was D2GG, which produces the least amount of Vif. These results indicated that, compared to WT HIV-1, all of the D2-down mutants are inhibited by A3G but that the magnitude of the inhibition is determined by the level of Vif.
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FIG. 7. Replication kinetics of 5'ss D2 mutants in A3G/Jurkat and control Tet-off cells. A3G/Jurkat cells that express elevated levels of A3G or control Tet-off cells were infected with WT or 5'ss D2 mutant viruses at an MOI of 0.005 in the presence of the increasing amounts of Dox shown or in the absence of Dox (No Dox). Virus production from three independent infections was monitored by RT assays performed at the times shown during the 16-day infection. Error bars indicate standard deviations.
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In the absence of Dox, i.e., at the highest level of A3G, the D2-down mutants D2GC-G5 and D2GA failed to compete efficiently with the WT virus, as can be seen by the significant differences in the respective viral DNA copy numbers under the no-Dox conditions (Fig. 8). On the other hand, in the presence of Dox or in Tet-off Jurkat cells, these two mutants were able to compete efficiently with the WT virus, and there were no significant differences in viral DNA copy numbers compared to those for the WT. The D2GG mutant virus, as expected, was much less fit in A3G/Jurkat cells in the absence of Dox, as can be seen by the accumulation of approximately 1,000-fold less viral DNA than for the WT (Fig. 8). In contrast to the results from multiday replication kinetics, shown in Fig. 7, our results did not detect a significant difference in the fitness of the D2GC mutant virus compared to that of the WT virus in the absence or presence of Dox in Tet-off Jurkat cells (Fig. 8).
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FIG. 8. Competition between WT and 5'ss D2 mutant viruses. A3G/Jurkat or control Tet-off cells were coinfected with WT and 5'ss D2 mutant viruses that produce lower levels of Vif at an MOI of 0.005 in the presence of the indicated amounts of Dox or in the absence of Dox (No Dox). At 12 days postinfection, total cell DNA was isolated and levels of viral DNA of the WT and D2 mutants were determined by quantitative real-time PCR analysis using specific TaqMan probes, as described in Materials and Methods. Results from three independent experiments are shown. Error bars indicate standard deviations.
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TABLE 1. Analysis of viral DNA from individual clones obtained from different cell lines coinfected with WT and D2GC viruses
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Vif in permissive CEM-SS and nonpermissive H9 cells (Fig. 9). All three of these viruses replicated with similar kinetics in CEM-SS cells. In nonpermissive H9 cells, on the other hand, the NLVif virus did not replicate, whereas the replication of ESEVifm after a 12-day infection period was inhibited approximately 40% compared to that of the WT.
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FIG. 9. Replication kinetics of WT and ESEVifm viruses in CEM-SS and H9 T-cells. CEM-SS and H9 T-cell lines were infected with NL4-3 or ESEVifm produced under permissive conditions at an MOI of 0.005. Virus production was measured by an RT assay of culture supernatants at the indicated times postinfection for 18 days. Error bars indicate standard deviations.
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Vif but approximately three- to fourfold slower than D2GG. We conclude that the ESEVif mutant virus, similarly to the D2-down mutants, is inhibited at higher levels of A3G than that required to inhibit the Vif mutant.
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FIG. 10. Replication of ESEVifm virus in A3G/Jurkat cell lines under different levels of A3G production. A3G/Jurkat cells treated with the indicated levels of Dox were infected with ESEVifm, NL4-3, or NL4-3Vif virus at an MOI of 0.005. Virus production was measured by an RT assay of infected-cell supernatants at the indicated times postinfection for 16 days. Parental Tet-off Jurkat cells were also monitored for infection as a low-A3G control. Error bars indicate standard deviations.
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Our results are consistent with the hypothesis that the highly conserved 5'ss downstream of the HIV-1 vif 3'ss (5'ss D2) and ESEVif exist in the HIV-1 genome to regulate the levels of vif mRNA and Vif protein in infected cells. We found that mutations that weaken 5'ss D2 affect virus fitness in nonpermissive cells under conditions where A3G is expressed but not in permissive cells, where A3G is not expressed. These results are in agreement with our previous studies and strongly suggest that the stability and function of viral mRNAs are not significantly affected by the presence or absence of the noncoding exon 2, defined by 5'ss D2 and 3'ss A1 (15). We also showed that the replication fitness of the D2-down mutant viruses expressing reduced levels of Vif was inhibited only at relatively high levels of A3G. The level of A3G required to inhibit virus replication was inversely related to the amount of Vif produced in mutant-infected cells. Similarly, the ESEVif mutant demonstrated significant effects on virus replication only at relatively high levels of A3G. Our data suggest that relatively little Vif is required to neutralize A3G in nonpermissive H9 cells or in the control Tet-off Jurkat cells. The D2GC-G5 and D2GA mutants, which expressed only 18% and 10%, respectively, of WT levels of vif mRNA in 293T cells, replicated with kinetics similar to those of the WT virus in H9 and Tet-off cells. The D2GG and ESEVif mutants, in which the intracellular vif mRNA levels were less than 5% of the WT levels in 293T cells, were inhibited only modestly in H9 and Tet-off cells, whereas replication of Vif-deleted HIV-1 is inhibited completely in both cell types.
Since our results indicated that relatively little Vif is required to overcome restriction by A3G, it might be expected that variants with mutations in 5'ss D2 and ESEVif should readily arise during HIV-1 infection. The conservation of the 5'ss D2 and ESEVif sequences in almost all HIV-1 strains isolated to date suggests that this is not the case. It is possible that some cells infected by HIV-1 in vivo are induced to express elevated levels of A3G, requiring greater levels of Vif for neutralization of A3G. Phorbol esters have been shown to induce A3G in the H9 T-cell line, and this effect is mediated by a protein kinase C/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling cascade (23). In primary CD4+ T cells and in monocyte-derived dendritic cells, it has been shown that stimulation of cell surface CCR5 or CD40 receptors with ligands to these receptors (CCL3 or CD40L, respectively) or with HSP70 treatment, which stimulates both receptors, results in a four- to sixfold upregulation of A3G (20). Thus, higher levels of Vif may be required for efficient HIV-1 replication in those cells in which A3G is upregulated.
We have shown previously that HIV-1 mutants with changes that strengthen 5'ss D2 and increase the affinity of the splice site for U1 snRNP relative to that of the WT result in increased expression of vif mRNA and Vif protein (2, 15). These mutants also exhibit an excessive RNA splicing phenotype characterized by reduced levels of unspliced RNA and reduced levels of Gag and Gag-Pol protein, resulting in reduced virus production (16). Thus, it appears that the suboptimal WT 5'ss D2 in the context of the HIV-1 genome is necessary for balancing the levels of sufficient unspliced RNA for optimal virus production and vif mRNA for neutralization of A3G. Strengthening the 5'ss D2 sequence relative to the WT sequence appears to shift RNA processing toward excessive splicing, whereas weakening the 5'ss D2 sequence relative to the WT sequence results in production of insufficient vif mRNA and Vif protein required for counteracting A3G. To further confirm that reduced splicing at 3'ss A1 and decreased Vif production by D2-down mutants is due to a decreased affinity for cellular U1 snRNA, we showed that Vif production was recovered by adding U1 snRNA with a change that restored its complementarity to a mutated 5'ss D2. The results suggest that binding of U1 snRNA to 5'ss D2 is a key to regulation of Vif production and that splicing at 5'ss D2 is not required for the effect.
We previously reported that 5'ss D3 together with hnRNP A/B-dependent exonic splicing silencers acts to regulate splicing at the HIV-1 vpr 3'ss A2 (15a). Mutations that strengthen 5'ss D3 result in increased Vpr expression but decreased virus particle production due to excessive splicing, whereas mutations that weaken 5'ss D3 result in decreased Vpr expression but WT virus production (J. M. Madsen and C. M. Stoltzfus, unpublished data). These results suggest that 5'ss D3 may play a role in regulated expression of Vpr similar to that of 5'ss D2 in regulation of Vif expression. It will be of interest to compare the levels of replication of WT HIV-1 and 5'ss D3-down mutants in macrophages, where Vpr is necessary for efficient virus infection.
Antivirals inhibiting Vif have been proposed as a possible therapeutic approach for interference with HIV-1 infection. The vif mRNA splicing mutants we have described produce a range of Vif levels in infected cells and may be useful in establishing the dose response for such potential therapeutic drugs. It will be essential to reduce Vif to very low levels, since suboptimal inactivation of A3G by Vif may result in hypermutation and emergence of drug-resistant HIV-1 variants (21).
We acknowledge the NIH AIDS Research and Reference Reagent Program for supplying HIV-1-related reagents. We thank Tom Hope, Northwestern University School of Medicine, for the pCMV110 β-galactosidase expression plasmid; Klaus Strebel, NIAID, for pNL
Vif; and Mark McNally, Medical College of Wisconsin, for the U1 snRNA expression vector pUC13-U1. We also thank J. Xiang and J. T. Stapleton, University of Iowa, for the pTRE2-Hyg-GFP vector and the Tet-off Jurkat cell line. This research was supported by PHS grant AI36073 from the National Institute of Allergy and Infectious Diseases. C.M.E. was supported by predoctoral training grant T32AI007533 from the National Institute of Allergy and Infectious Diseases.
* Corresponding author. Mailing address: Department of Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7793. Fax: (319) 335-9006. E-mail: marty-stoltzfus{at}uiowa.edu
Published ahead of print on 8 April 2009.
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Journal of Virology, June 2009, p. 6067-6078, Vol. 83, No. 12
0022-538X/09/$08.00+0 doi:10.1128/JVI.02231-08
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