Background: During the retroviral assembly process, the HIV-1 Gag structural protein specifically encapsidates two copies of full-length viral genomic RNA into the newly forming virion. We previously showed that HIV-1 mutant viruses containing deletions in the 5' untranslated region of the viral RNA genome (i.e. the dimerization initiation site; DIS) have decreased viral replication capacity, but over long-term culture generated compensatory point mutations (CMs) in Gag that restored replication to these viruses. More recently, we demonstrated that these DIS mutants had a packaging specificity defect, in that they encapsidate large quantities of spliced viral RNA compared to wild-type. Interestingly, one of these CMs (a T12I substitution in p2) was able to correct this packaging defect.

Methods: To test the effects of this CM on other related mutant viruses, we generated HIV-1 mutants by PCR mutagenesis of the BH10-HXB2 plasmid. Virus replication was monitored based on infectivity assays in T cell lines using COS-7-derived virus, and RNase protection assays (RPA) were performed on virion-associated RNA from purified virus particles.

Results: We now show that a series of HIV-1 mutants containing mutations in the poly(A) stem-loop and the U5-PBS stem, as well as stem-loop 3 and adjacent GA-rich sequences, all display similar packaging specificity defects, based on RPA anaylses. Surprisingly, T12I in p2 can correct packaging specificity defects in all of these mutants, suggesting that this CM acts through some broad mechanism that provides an overall increased affinity of Gag for the viral genomic RNA, while at the same time excluding encapsidation of the spliced RNA. To extend these findings we also tested whether other amino acids in p2 can affect packaging specificity in the context of the wild-type 5' RNA sequences. We found that mutations at positions 2, 7 and 14 impacted on packaging specificity, as seen by increased levels of spliced viral RNA in the virions.

Conclusions: These results help to elucidate the mechanism employed by our CMs to restore replication capacity to our mutant viruses, and also contribute to the general understanding of the events that take place during HIV-1 packaging and virus assembly. In light of previous studies reporting that p2 affects Gag multimerization and virion morphogenesis, this work furthers the possibility that p2 could be a potential target for novel HIV-1 assembly inhibitors.

Keywords: Packaging; Assembly; Dimerization