Preferences for the selection of unique tRNA primers revealed from analysis of HIV-1 replication in peripheral blood mononuclear cells

Abstract

Background: All human immunodeficiency virus (HIV-1) uses a host tRNALys,3 as the primer for reverse transcription. The tRNALys,3 is bound to a region on the HIV-1 genome, the primer-binding site (PBS), that is complementary to the 18 terminal nucleotides of tRNALys,3. How HIV-1 selects the tRNA from the intracellular milieu is unresolved.

Results: HIV-1 tRNA primer selection has been investigated using viruses in which the primer-binding site (PBS) and a sequence within U5 were altered so as to be complementary to tRNAMet, tRNAPro or tRNAIle. Analysis of the replication of these viruses in human peripheral blood mononuclear cells (PBMC) revealed preferences for the selection of certain tRNAs. HIV-1 with the PBS altered to be complementary to tRNAMet, with and without the additional mutation in U5 to be complementary to the anticodon of tRNAMet, stably maintains the PBS complementary to tRNAMet following extended in vitro culture in PBMC. In contrast, viruses with either the PBS or PBS and U5 mutated to be complementary to tRNAIle were unstable during in vitro replication in PBMC and reverted to utilize tRNALys,3. Viruses with the PBS altered to be complementary to tRNAPro replicated in PBMC but reverted to use tRNALys,3; viruses with mutations in both the U5 and PBS complementary to tRNAPro maintained this PBS, yet replicated poorly in PBMC.

Conclusion: The results of these studies demonstrate that HIV-1 has preferences for selection of certain tRNAs for high-level replication in PBMC.

Figure 1

U5-PBS sequence and infectivity levels of HIV-1 NL4-3 viral mutants at start of PBMC infection. Panel A. HIV-1 U5 and PBS sequence shown (from 5' to 3'). Viral primer binding site (PBS) sequence was altered to be complementary to the 3' terminal 18 nucleotides of tRNA^Ile, tRNA^Met and tRNA^Pro. The PBS sequence is shadowed. Panel B. Comparison of infectivity of NL4-3 PBS mutants. HIV-1 NL4-3 proviral clones were transfected into 293T cells, incubated for 48 hours, and supernatants were measured for infectious units. For a given sample, the number of infectious units per microliter is equal to the number of blue cells in a well divided by the dilution factor for that well and represents the average of at least two wells. Wild type infectivity levels were set at 100% and mutant virus infectivity was reported as a percentage of wild type. All viruses with altered PBS sequences showed reduced levels of infectivity as compared to wild type. Results presented are representative of three experiments.

Figure 2

Replication of HIV-1 with PBS sequence altered to be complementary to the 3' 18 nucleotides of tRNA^Ile, tRNA^Met and tRNA^Pro in human peripheral blood mononuclear cells (PBMC). Infections were initiated with transfection supernatant containing approximately 200 pg of p24 antigen in a volume of 10 mLs of media, giving a final p24 level of 20 pg/mL on day zero. At 14 day intervals, 5 × 10⁶ fresh PHA stimulated PBMC were added to each culture. Supernatants were assayed for p24 viral antigen using an ELISA. Two additional separate infections produced similar replication patterns for each virus. Squares are wild type NL4-3; diamonds are NL4-3-Met; open circles are NL4-3-Ile and closed circles are NL4-3-Pro.

Figure 3

U5-PBS sequence and Infectivity levels of HIV-1 NL4-3 viral mutants with altered U5 and PBS sequences at start of PBMC infection. Panel A. HIV-1 U5 and PBS sequence (shown 5' to 3'). Viral primer binding site (PBS) and U5 A-loop sequences were altered to be complementary to the 3' terminal 18 nucleotides and anticodon loop of tRNA^Ile, tRNA^Met, tRNA^Pro, and tRNA^Trp, respectively. U5 A-loop sequence and PBS are shadowed. Panel B. Comparison of the infectivity of U5-PBS mutant NL4-3 viruses. HIV-1 NL4-3 proviral clones were transfected into 293T cells, incubated for 48 hours, and supernatants were measured for infectious units. For a given sample, the number of infectious units per microliter is equal to the number of blue cells in a well divided by the dilution factor for that well and represents the average of at least two wells. Wild type infectivity levels were set at 100%, and mutant virus infectivity was reported as a percentage of wild type. All viruses with altered U5 and PBS sequences had reduced levels of infectivity as compared to wild type virus. The data presented are representative for three independent experiments.

Figure 4

Replication of HIV-1 with U5 and PBS sequence altered to be complementary to the anticodon loop and 3' 18 nucleotides of tRNA^Ile, tRNA^Met, tRNA^Pro and tRNA^Trp in PBMC. Infections were initiated with transfection supernatant containing approximately 200 pg of p24 antigen in a volume of 10 mLs of media, giving a final p24 level of 20 pg/mL on day zero. Supernatants were assayed for p24 viral antigen every 7 days for a period of 42 days. At 14-day intervals, 5 × 10⁶ fresh PHA stimulated PBMC were added to each culture. Two additional separate infections produced very similar replication patterns for each virus (data not shown). Squares are NL4-3 wild type; diamonds are NL4-3-Met-AC; open circles are NL4-3-Ile-AC; closed circles are NL4-3-Pro-AC.