The roles of the human immunodeficiency virus type 1 Pol protein and the primer binding site in the placement of primer tRNA(3Lys) onto viral genomic RNA

Abstract

Factors that modulate the placement of primer tRNA(3Lys) onto the viral RNA genome in human immunodeficiency virus type 1 (HIV-1) were investigated through analysis of reverse-transcribed products that are extended from the tRNA(3Lys) primer. Mutations were introduced into the HIV-1 pol gene to result in the appearance of a stop codon in the open reading frame of the reverse transcriptase (RT) gene. These constructs, BH10-RT1 and BH10-RT2, yielded viruses with truncated Pol proteins. Alternatively, we altered the sequences involved in frameshifting by generating the construct BH10-FS. With each of these mutated viruses, we found that the primer tRNA(3Lys) that was placed onto viral genomic RNA was present in an unextended state. In contrast, as expected, tRNA(3Lys) in the case of wild-type BH10 virus had been extended by 2 bases. Furthermore, the amount of tRNA(3Lys) that was placed onto viral RNA in mutated viruses was significantly less than that placed in the wild-type virus. We also generated a mutant within the polymerase-active site of RT (D185H) (Asp-->His) that eliminated RT polymerase activity. We found that the placement of primer tRNA(3Lys) onto viral genomic RNA was independent of enzyme function; however, the tRNA(3Lys) that was placed was present in an unextended state due to the loss of RT activity. In contrast, the elimination of protease activity through a D25A (Asp-->Ala) point mutation in the protease-active site (construct BH10-PR) did cause a drop in the efficiency of tRNA(3Lys) placement. In this situation, a proportion of the placed tRNA(3Lys) was found to be extended by 2 bases, although not to the extent found with wild-type virus (BH10), due to a decrease in RT activity associated with unprocessed Gag-Pol protein that could not be cleaved because of the loss of protease activity. We also investigated the role of the primer binding site (PBS) in the placement of tRNA(3Lys) through a series of 2-, 4-, and 8-nucleotide (nt) deletions at the 3' end of the PBS, i.e., BH10-PBS2, BH10-PBS4, and BH10-PBS8, respectively. In mutated viruses BH10-PBS2 and BH10-PBS4, the 2-base-extended form of tRNA(3Lys) was still detected. However, less primer tRNA(3Lys) was placed onto viral genomic RNA as more nucleotides were deleted until the percentage of placement seen with wild-type BH10 virus dropped to only 4% in the virus with 8 nt deleted (BH10-PBS8). Consistently, these mutated viruses possessed decreased initial replication capacity compared with that of the wild-type virus, with the extent of incapacity corresponding to the size of the deletion. However, after several days, an increase in replication potential was accompanied by a reversion to a wild-type PBS.

The Human Immunodeficiency Virus Type 1 Gag Polyprotein Has Nucleic Acid Chaperone Activity: Possible Role in Dimerization of Genomic RNA and Placement of tRNA on the Primer Binding Site

“…However, several observations seem to argue against this possibility. First, an early study suggested that tRNA annealing was incomplete in newly released particles of avian leukosis virus (9), and secondly, a recent report indicated that the placement was also incomplete in immature (PR-deficient) HIV-1 particles (28). The fact that the annealing is not complete at the moment of virus release suggests that it occurs simultaneously with virus assembly; perhaps under normal circumstances it is catalyzed in part by the Gag polyprotein and in part by NC.…”

HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

“…These PBS mutations, for example, M1, M2, M3, and M4 in this study, do not markedly affect HIV-1 infection of SupT1 cells, likely because viral reverse transcriptase is able to tolerate the minimal mismatch between viral RNA and tRNA 3 Lys and start reverse transcription. In theory, PBS mutations should be quickly lost in HIV-1 replication due to the regeneration of the wild-type PBS sequence by copying the first 18 nucleotides of cellular tRNA 3 Lys (32)(33)(34)(35). However, our study shows that HIV-1 takes advantage of several mechanisms to maintain these escape mutations in PBS and achieve wild-type-like replication in SupT1 cells that express Cas9 and sgPBS1 or sgPBS2.…”

“…Given that each replication cycle of HIV-1 regenerates the wild-type PBS sequence by copying cellular tRNA 3 Lys , we expected that, during long-term replication of the M1 and M2 mutants in control SupT1 cells, the wild-type PBS viruses would become the prevalent viral population in the absence of the selection pressure of sgPBS1. This mechanism has been reported for various PBS mutations that revert to the wild-type sequence after prolonged replication (32)(33)(34)(35). We thus sequenced the viruses that were harvested at the peak of long-term replication of M1 and M2 viruses in control SupT1 cells.…”