Impaired nuclear import and viral incorporation of Vpr derived from a HIV long-term non-progressor

The highly conserved HIV accessory protein Viral Protein R (Vpr) is vital for HIV infection and replication in vivo, particularly within non-dividing cells, including terminally differentiated T-cells and macrophages where nuclear envelope integrity is permanently maintained [1-5]. On its own and within the context of HIV infection [6,7], Vpr has been shown to localize to the nucleus and induce G2 cell-cycle arrest through hyperphosphorylation of p34-cdc2, which provides the most optimal environment for viral replication [8-13], followed closely by apoptosis [14-21]. During HIV infection, Vpr associates with the viral cDNA containing Pre-integration Complex (PIC) increasing its affinity for components of the cellular nuclear import machinery [22-25] through the activity of 2 distinct nuclear localization signals (NLSs) within its N- [26] and C-termini [27], thus driving productive HIV infection.We previously reported [28] on a cohort spanning 1992 to 2000, comprising an HIV+ long-term non-progressor (LTNP) (donor A) and two recipients (B and C) who upon transmission (autumn and summer 1989 respectively) from donor A progressed to AIDS. Vpr sequences derived from HIV pro-viral DNA isolated from PBMCs as well as circulating virus, revealed that sequences from the progressing recipients differed markedly to those of the LTNP founder virus host, providing the first evidence for Vpr positive selection during disease progression in an epidemiologically-linked cohort (see also [29]).In this study, cohort-derived GFP-Vpr mammalian cell expression vectors were used to investigate Vpr subcellular localisation and nuclear import. In total, 4 HIV-1 vpr clones were isolated from donor A (A1, A3, A5 and A6 corresponding to years 1996, 1998, 1999 and 2000), 4 from recipient B (B3, B4, B5 and B6 corresponding to years 1997, 1998, 1998 and 1999) and 7 from recipient C (C2, C3, C5, C6, C8, C10 and C11 from 1992, 1992, 1993, 1994, 1994, 1998 and 1999 respectively) and used to generate