Probing the importance of tRNA anticodon: human immunodeficiency virus type 1 (HIV-1) RNA genome complementarity with an HIV-1 that selects tRNA(Glu) for replication

Abstract

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at the primer binding site (PBS) that is complementary to the 3'-terminal nucleotides of tRNA(3)(Lys). Why all known strains of HIV-1 select tRNA(3)(Lys) for replication is unknown. Previous studies on the effect of altering the PBS of HIV-1 on replication identified an HIV-1 with a PBS complementary to tRNA(Glu). Since the virus was not initially designed to use tRNA(Glu), the virus had selected tRNA(Glu) from the intracellular pool of tRNA for use in replication. Further characterization of HIV-1 that uses tRNA(Glu) may provide new insights into the preference for tRNA(3)(Lys). HIV-1 constructed with the PBS complementary to tRNA(Glu) was more stable than HIV-1 with the PBS complementary to tRNA(Met) or tRNA(His); however, all of these viruses eventually reverted back to using tRNA(3)(Lys) following growth in SupT1 cells or peripheral blood mononuclear cells (PBMCs). New HIV-1 mutants with nucleotides in U5 complementary to the anticodon of tRNA(Glu) remained stable when grown in SupT1 cells or PBMCs, although the mutants grew more slowly than the wild-type virus. Sequence analysis of the U5 region and the PBS revealed additional mutations predicted to further promote tRNA-viral genome interaction. The results support the importance of the tRNA anticodon-genome interaction in the selection of the tRNA primer and highlight the fact that unique features of tRNA(3)(Lys) are exploited by HIV-1 for selection as the reverse transcription primer.

FIG. 1.

Diagrams of tRNAs and U5-PBS RNA. (A) and tRNA^Glu. The cloverleaf diagrams of and tRNA^Glu are presented with the modified bases in both tRNAs noted. The PBS of HIV is complementary to the 3′-terminal nucleotides of ; a second HIV genome was constructed in which the PBS was complementary to the 3′-terminal 18 nucleotides of tRNA^Glu. Those nucleotides circled in the TφC-loop of and tRNA^Glu are complementary to regions downstream of the PBS. Abbreviations: S, 5-methoxycarbonylmethyl-2-thiouridine; ψ, pseudouridine; D, dihydrouridine; T_m, 2′-O-methyl-5-methyluridine; A^ml, 1-methyladenosine; C^5m, 5-methylcytidine; G^m2, N²-methylguanosine; G_7m, 7-methylguanosine. (B) The U5-PBS region from HXB2 and that from HXB2(Glu) in which the PBS has been altered to be complementary to tRNA^Glu are shown. The PBS is part of a stem-loop structure in which adenines are displayed at the crown of the stem-loop.

FIG. 2.

Replication of HIV-1 with altered PBS in SupT1 cells and PBMCs. (A) The replication of the wild-type virus (WT) and viruses with PBSs complementary to tRNA^Glu (Glu), tRNA^Met (Met), and tRNA^His (His) was analyzed in SupT1 cells. Cultures were initiated as described in Materials and Methods, and the supernatants were analyzed for p24 antigen production during a 24-day culture period. The values represented are from a single experiment; repeat experiments gave similar replication profiles. The arrow denotes the sample time at which PBSs from both viruses which used tRNA^Met and tRNA^His reverted to PBSs complementary to (Table 1). (B) Replication of HIV-1 with the PBS complementary to tRNA^His or tRNA^Glu in PBMCs. Cultures were initiated as described in Materials and Methods, and virus replication was monitored by analysis of p24 antigen in culture supernatants. The arrow denotes the sample time at which the PBSs complementary to tRNA^His and tRNA^Glu were found to revert to PBSs complementary to (Table 2). Results presented are from a single experiment; repeat experiments gave similar replication profiles for each virus.

FIG. 3.

HIV-1 with the U5-PBS region complementary to tRNA^Glu. HIV-1 genomes in which the PBS and A-loop regions have been altered to be complementary to the 3′-terminal nucleotides and anticodon of tRNA^Glu are presented. The PBS region is noted in bold. The circled nucleotides are those which were changed to be complementary to the anticodon of tRNA^Glu (CUC). (A) Wild-type HIV-1 genome in which the PBS has been altered to be complementary to tRNA^Glu. (B) HIV-1 genome with additional nucleotides making it complementary to the anticodon of tRNA^Glu [HXB2(Glu Loop 1)]. An additional nucleotide change was added to enhance the stem of the U5-PBS stem-loop (A, circled). (C) HIV-1 genome in which the PBS and A-loop have been altered to correspond to the 3′-terminal nucleotides of tRNA^Glu and the anticodon region. Additional nucleotides inserted within the stem destabilized the stem-loop, creating a bulge region; virus with this genome is designated HXB2(Glu Loop 2).

FIG. 4.

Replication of HIV genomes with U5-PBS regions optimized for use of tRNA^Glu. (A) Replication in SupT1 cells. Virus cultures were initiated as described in Materials and Methods. The supernatants from the cultures were analyzed for p24 antigen production at various days postinitiation of culture. For comparison, results for viruses in which only the PBS was altered to correspond to tRNA^Glu (Glu) are shown. Results for two additional viruses with both the U5 and the PBS altered to correspond to tRNA^Glu (Glu-Loop 1 and Glu-Loop 2) are presented. WT, wild type. (B) Replication of viruses in PBMCs. The replication of one of the viruses represented in panel A (Glu-Loop 2) was analyzed in PBMCs. For comparison, results for the virus with only the PBS altered to be complementary to tRNA^Glu (Glu) are presented. The arrow denotes the sample time at which the virus with only the PBS complementary to tRNA^Glu had a PBS complementary to . Results are from a single experiment; the cultures were performed in duplicate with similar replication profiles.

FIG. 5.

Analysis of intracellular and HIV-1 virion-associated and tRNA^Glu. (A) Analysis of tRNA from SupT1 cells. Total RNA from SupT1 cells was isolated as described in Materials and Methods. Following electrophoresis and transfer onto nitrocellulose membrane, equal amounts were probed with oligonucleotides specific for or tRNA^Glu. Specificity controls included equal amounts (1 ng) of in vitro RNA transcription products; note that the in vitro transcript (xscript) for tRNA^Glu contains extra nucleotides that retard the migration in the agarose gels used for this analysis. The densities of the probes reacting with the cognate tRNA for both and tRNA^Glu were similar (i.e., the density of the probe with 1 ng of in vitro-transcribed versus the density of the tRNA^Glu probe with 1 ng of in vitro-transcribed tRNA^Glu). The use of scanning densitometry to determine the cellular amounts of tRNA^Glu and revealed an approximate 10:1 ratio. This analysis was repeated three times, and a representative blot is shown. The closed arrow denotes migration of full-length tRNA^Lys or tRNA^Glu. The open arrow denotes migration of the tRNA^Glu in vitro transcript containing additional nucleotides from cloning of the tRNA gene into the transcription plasmid. (B) Analysis of tRNA from HIV-1 virions. Total virion RNA collected from viruses isolated from supernatant of SupT1 cells infected with HXB2 or HXB2(Glu Loop 2). HIV-1 virions were isolated by ultracentrifugation, and RNA was extracted and prepared as described in Materials and Methods. The blot was probed for tRNA^Glu and as indicated. Lane 1, 1 μg of RNA isolated from virions; lane 2, 0.1 μg of RNA isolated from virions.

FIG. 6.

Summary of sequence changes found in U5-PBS regions following virus growth in SupT1 cells and PBMCs. (A) Changes found in the U5 region of the virus HXB2(Glu Loop 1) following replication in SupT1 cells. The arrows denote the major changes observed following replication. An additional change of G to A within the A-loop region (circled) was also found. Note that we did not see reversion of the PBS back to the wild-type PBS complementary to . (B) Changes found in the virus HXB2(Glu Loop 2) following in vitro culture in SupT1 cells or PBMCs. The major changes were noted within the stem region of the U5 PBS stem-loop. In this case, C-to-G, G-to-U, and G-to-A changes were noted in viruses grown in SupT1 cells; a G-to-A change was noted in viruses grown in PBMCs and SupT1 cells (PBMC + SupT1). These changes will be predicted to maintain a bulge region reduced from that of the initial HXB2(Glu Loop 2). Note again that reversion of the PBS to the wild-type PBS complementary to was found.