HIV-1 designed to use different tRNAGln isoacceptors prefers to select tRNAThr for replication

Background Previous studies have shown that infection with human immunodeficiency virus type 1 (HIV-1) causes acceleration of the synthesis of glutamine tRNA (tRNAGln) in infected cells. To investigate whether this might influence HIV-1 to utilize tRNAGln as a primer for initiation of reverse transcription, we have constructed HIV-1 proviral genomes in which the PBS and the A-loop region upstream of the PBS have been made complementary to either the anticodon region of tRNAGln,1 or tRNAGln,3 and 3' terminal 18 nucleotides of each isoacceptor of tRNAGln. Results Viruses in which the PBS was altered to be complementary to tRNAGln,1 or tRNAGln,3 with or without the A-loop all exhibited a lower infectivity than the wild type virus. Viruses with only the PBS complementary to tRNAGln,1 or tRNAGln,3 reverted to wild type following culture in SupT1 cells. Surprisingly, viruses in which the PBS and A-loop were complementary to tRNAGln,1 did not grow in SupT1 cells, while viruses in which the PBS and A-loop were made complementary to tRNAGln,3 grew slowly in SupT1 cells. Analysis of the PBS of this virus revealed that it had reverted to select tRNAThr as the primer, which shares complementarity in 15 of 18 nucleotides with the PBS complementary to tRNAGln,3. Conclusion The results of these studies support the concept that the HIV-1 has preferred tRNAs that can be selected as primers for replication.


Background
HIV-1 reverse transcription is initiated with the extension of the cellular tRNA that is bound to a specific sequence on the viral RNA genome known as the primer-binding site (PBS) [1][2][3]. The PBS is an 18-nucleotide sequence located near the 5' end of viral RNA that is complementary to the 3' terminal nucleotides of the primer tRNA used for initiation [3]. HIV-1 specifically selects tRNA Lys,3 from the intracellular milieu to be used as the primer for initiation of reverse transcription [4,5]. The mechanism of how HIV-1 specifically selects tRNA Lys,3 from the intracellular milieu is not completely understood. Previous studies have established that tRNA Lys,3 as well as tRNA Lys1,2 are enriched in HIV-1 virions [6][7][8]. The Gag-Pol polyprotein of HIV-1 is responsible, in part, for this enrichment of tRNA Lys1,2,3 into the virions [4,6,8]. Studies have also demonstrated that lysl tRNA synthetase can specifically interact with HIV-1 Gag to facilitate incorporation of tRNA Lys1,2,3 into HIV-1 virions [9][10][11]. Once this complex is incorporated into virions though, it is not clear how and why tRNA Lys,3 is specifically utilized as the primer for initiation of reverse transcription.
Previous studies from our lab and others have taken a genetic approach to understanding elements of HIV-1 primer selection [12][13][14]. For these studies, we have mutated the PBS to be complementary to tRNAs other than tRNA Lys, 3 . In general, mutation of the PBS to be complementary to other tRNAs, including tRNA Lys1,2 , results in a virus that can transiently utilize the specific tRNA but most of the time reverts back to rapidly utilize tRNA Lys, 3 following in vitro culture [12][13][14]. Stabilization of alternative tRNAs use has been accomplished through additional mutations upstream in the U5 region designated as the Aloop, which is complementary to the anticodon region of tRNA Lys,3 [15][16][17][18][19]. For some, but not all tRNAs, mutation of the A-loop region as well as the PBS to be complementary to the anticodon and 3' terminal nucleotides, respectively, of the tRNA allows this tRNA to be stably utilized by HIV-1 as a primer for reverse transcription. Using this strategy, we have generated viruses which stably utilized tRNA Lys1,2 , tRNA Met , tRNA His , and tRNA Glu [15][16][17][18][19]. A recent study has also found that HIV-1 can be forced to use tRNA Lys1,2 if mutations are made complementary to nucleotides in the TϕC loop of tRNA Lys1,2 in a second region upstream of the PBS, called the primer activation site [20].
All viruses that utilize alternative tRNAs do not replicate as efficiently as the wild type virus that utilizes tRNA Lys, 3 . This result has lead to the speculation that the availability of tRNA for primer selection might not be the same for all tRNAs. To test this it will be necessary to alter the levels of individual tRNA isoacceptors in cells. However, it is difficult to modulate the levels of tRNA in mammalian cells without leading to toxicity. Previous studies by Kuchino et al. though have found that the levels of a natural glutamine suppressor tRNA which exists as a minor species of glutamine tRNA (tRNA Gln,3 ) in normal cells is increased in murine leukemia virus (MuLV) infected cells [21,22]. In follow up studies, Muller et al. found that although the amount of the suppressor tRNA Gln,3 was only 6% of the major glutamine tRNA Gln,1 levels the amount of suppressor increased almost 20 fold while the levels of non-suppressor tRNA Gln,1 remained the same in cells infected with MuLV or HIV-1 [23,24]. Since the levels of a particular tRNA (tRNA Gln,3 ) increase following infection with HIV-1, it might be possible to force HIV-1 to use this isoacceptor of tRNA Gln as a primer for replication. To test this, we created viruses in which the PBS is complementary to the minor and major species of tRNA Gln . We also constructed viruses which contain additional mutations in the A-loop regions to determine if this will affect the stable use of these tRNAs as primers for HIV-1 reverse transcription. Results of our study show that these viruses with the PBS complementary to either tRNA Gln species were unstable and rapidly reverted back to utilize tRNA-Lys, 3 . Inclusion of the A-loop complementary to the antico-don of tRNA Gln,3 resulted in a virus that did not revert to utilize tRNA Lys,3 but selected an unexpected tRNA, tRNA-Thr . The results of these studies suggest that certain tRNAs are favored by HIV-1 for the selection as a primer for initiation of reverse transcription.

Construction of HIV-1 proviral genomes with PBS and Aloop complementary to tRNA Gln
To determine if HIV-1 can utilize tRNA Gln as a primer for reverse transcription, we mutated the PBS to be complementary to a 3' terminal nucleotide of tRNA Gln . The major isoacceptor for tRNA Gln (tRNA Gln,1 ) has an anticodon CUG. A second tRNA Gln has an anticodon UUG and is referred to as the minor tRNA Gln or tRNA Gln,3 [21,22] (Figure 1A). Previous studies have shown that in HIV-1 infected cells, the levels of tRNA Gln,3 are increased 20 fold over that of uninfected cells [23]. The 3' terminal nucleotides of tRNA Gln,1 and tRNA Gln,3 differ only by a single nucleotide ( Figure 1B). We have also constructed two additional proviruses in which the A-loop region of HIV-1 was mutated to correspond to the anticodon sequences of tRNA Gln,1 and tRNA Gln,3 , respectively ( Figure 1B).

Characterization of mutant HIV-1
The first step in the characterization of HIV-1 with the PBSs alone or PBSs in combination with A-loop modifications to be complementary to tRNA Gln was to determine the effects on the infectivity of viruses following transfection. For these studies, we transfected the proviral genomes into 293T cells and assayed the supernatants for infectious virus using the JC53βL assay. We also determined the amounts of virus in the supernatants by using a p24 antigen capture ELISA. The infectivity of the viruses is represented as the amount of infectious units divided by the p24 levels. Previous studies from our laboratory have shown that for the most part, mutations within the PBS of HIV genome results in viruses that exhibit infectivity approximately 20% (or lower) of the wild type virus [25]. Similar results were found for viruses in which the PBS was made complementary to tRNA Gln,1 or tRNA Lys,3 . No significant differences were observed between viruses with the PBS alone complementary to tRNA Gln and viruses with the PBS and A-loop complementary to tRNA Gln . The virus with a PBS and A-loop complementary to tRNA Gln,1 though had the lowest infectivity, approximately 10% of the wild type virus and half as much as the other viruses in which the PBS was altered to be complementary to tRNA Gln,3 (data not shown).
We next analyzed the replication of these viruses in SupT1 cells. Infections were established with equal amounts of infectious virus and replication was monitored by analysis of p24 in the culture supernatant. The wild type virus demonstrated a rapid increase in p24 antigen in the cul-ture supernatant, peaking at approximately 14 days following initiation of the infection; the cultures for the wild type virus were halted at day 28 post initiation of culture. In contrast, viruses in which just the PBS alone was made complementary to tRNA Gln,1 or tRNA Gln,3 exhibited slower infection compared to the wild type. The p24 levels in the culture supernatants increased slowly, reaching a maximum at days 35 to 49 post initiation of culture. The final levels of p24 antigen detected in the culture supernatants from these viruses were similar to those of the wild type virus (Figure 2A). Viruses in which the PBS and A-loop were made complementary to tRNA Gln,1 or tRNA Gln,3 had considerably different replication profiles compared to the viruses with mutations in the PBS alone. Viruses with the PBS and A-loop complementary to tRNA Gln,1 showed no increase in p24 antigen culture over the period examined (56 days of in vitro culture), indicating that the virus with this mutation in the PBS and A-loop did not undergo detectable replication and re-infection. In contrast, viruses with the PBS and A-loop complementary to tRNA Gln,3 did replicate and eventually demonstrated an increase in p24 antigen during the 56 day culture period (approximately 100 fold over the starting amount of virus (p24 antigen) ( Figure 2B).
We utilized PCR to amplify the U5-PBS region from integrated proviruses found in cellular genomic DNA to identify the PBS of viruses following in vitro culture. We analyzed cellular DNA obtained at day 42 from cultures infected with viruses in which the PBS alone was mutated to be complementary to tRNA Gln,1 or tRNA Gln,3 (Table I).
In both instances, we found that analysis of U5-PBS obtained from viruses at 42 days post initiation of culture, which corresponded to the time at which there was a rise in p24 antigen, resulted in some of the viruses containing PBS complementary to the starting tRNA Gln . Surprisingly, the major PBS recovered from analysis of both viruses was complementary to tRNA Thr , indicating both viruses had switched their preference from tRNA Gln to tRNA Thr . By day 56, though, when both cultures had plateaued with the p24 antigen and the cultured supernatant, we recovered PBS that were complementary to tRNA Lys,3 . Most probably, the process of reversion for this virus occurred through the formation of the PBS complementary to tRNA Thr followed by the subsequent conversion to a PBS complementary to tRNA Lys,3 which resulted in the high level replication observed for both of these viruses. In contrast, analysis of viruses in which the U5-PBS was complementary to tRNA Gln,3 gave a different pattern. In this case, all of the PBS recovered were complementary to tRNA Thr , suggesting that the virus had selected tRNA Thr from the intracellular milieu rather than the starting tRNA(tRNA-Gln,3 ) and was now stably using tRNA Thr as the primer for reverse transcription.

Discussion
The original intent of the experiments was to determine whether HIV-1 would accept tRNA Gln as a primer for initiation of reverse transcription. Our experiments were based on a previous study in which we found that MuLV with a PBS mutated to be complementary to tRNA Gln,1 grew well in tissue culture, even though MuLV prefers to use tRNA Pro as the primer for initiation of reverse transcription [26]. In addition to the viruses with the PBS complementary to tRNA Gln,1 , we also constructed viruses in which the PBS was complementary to the minor spe-tRNA Gln and mutated proviral genomes  Gln,3 . Note that the PBS is nearly identical with the exception of the T to C change in the PBS. NL-4 Gln3-AC refers to an HIV-1 in which the PBS was modified to be complementary to tRNA Gln,3 with additional modification in the A-loop region consisting of GAGTCAA which is complementary to the anticodon region of tRNA Gln3 .  The surprising result of this study was the reversion of viruses with the PBS complementary to tRNA Gln to utilize tRNA Thr . How this selection occurred is not clear at this time. Comparison of the PBS sequences between those complementary to tRNA Gln and tRNA Thr revealed considerable homology between the first nine nucleotides as well as the last three nucleotides (Figure 3). Previous studies from our laboratory have shown that the first nine and last three to five nucleotides can facilitate the reverse transcription of HIV-1 in which the PBS was made complementary to alternative tRNAs [27]. It is clear that following selection of tRNA Thr the virus could, through the process of reverse transcription, convert the PBS to be complementary to this tRNA and allow limited growth. Why the virus with a PBS and A-loop complementary to tRNA Gln,1 did not convert to use tRNA Thr is unknown. It is possible that the selection of tRNA Thr is passive, rather than active. Thus, if the virus happens to capture tRNA Thr , it will grow, albeit more slowly than the wild type virus. The fact that the process of conversion goes through an intermediate with a PBS complementary to tRNA Thr suggests this tRNA has a greater availability for capture than tRNA Gln . Additional studies will be needed to address this possibility.

Conclusion
In the current study, we have characterized the replication of HIV-1 in which the PBS has been altered to be complementary to tRNA Gln . Viruses were constructed in which the PBS or PBS and A-loop were modified to be complementary to either tRNA Gln,1 or tRNA Gln,3 . All viruses were found to have poor replicative capacity and the PBS was unstable following in vitro culture. However, analysis of the PBS from integrated proviruses revealed that a new tRNA, tRNA Thr was preferred by HIV-1 for replication indicating that HIV-1 prefers tRNA Thr as a primer for replication.
The results of our study re-enforces the idea that HIV-1 has preferences for the selection of certain tRNAs for replication. Obviously, the most preferred primer for selection is tRNA Lys,3

Materials and methods
Tissue culture 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), and SupT1 cells were grown in RPMI 1640 medium supplemented with 15% FBS.