P and shown to confer some level of protection against this
P and shown to confer some level of protection against this orthologue, would also make HIV-1 resistant to TRIM5hu mutants. Our data show that V86M indeed confers some level of protection against various mutants of TRIM5hu, at least in the context of single-cycle infections with HIV-1 vectors. We saw no protection in the context of HIV-spreading infections, although we tested only one HIV-1 strain (NL4-3) in one cell line (Sup-T1). Sodroski and collaborators [35] have isolated the V86M mutant in HeLa-CD4 cells, which we have not tested here. They observed modest levels (2-fold) of protection against TRIM5rh in these HeLa-derived cells when infectivity was measured in single-cycle assays, and they saw an even more modest effect in canine Cf2Th cells [35]. Altogether, V86M can confer HIV-1 protection against restriction by various TRIM5 proteins in specific replication settings. However, even in situations in which protection takes place, restriction still occurs, albeit weakened. On the basis of our data, we do not expect V86M to be highly significant in an in vivo context, although this is of course rather hazardous to predict. In order to investigate the mechanism of CA-V86M HIV-1 resistance to R332G-R335G TRIM5hu, we analyzed the role of CypA in the restriction. Altogether, our results show that restriction of V86M CA HIV-1 is largely insensitive to the presence of functional CypA, while the same virus is still inhibited in human cells devoid of CypA. In other words, HIV-1 is able to subtly alter its interactions with CypA in order to downregulate a mechanism of restriction while preserving other benefits associated with this interaction. Accordingly, our NMR data showed that the molecular interactions between CA and CypA were altered by the V86M mutation, as was the isomerisation reaction catalysed by CypA. Finally, our qPCR data correlate modifications in CypA-CA interactions with effects on nuclear transport in restrictive conditions. Interestingly, experiments predating the discovery of TRIM5 restriction had demonstrated that CsA treatment of Old World monkey cells increased nuclear transport of HIV-1 in these cells [21]. More PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27735993 recently, Lin and Emerman similarly observed that CsA treatment of the sMAGI Rhesus macaque cell line increases HIV-1 nuclear transport more than it does enhance reverse transcription [55]. Other recently published data also link nuclear transport and CA-CypA interactions in permissive human cells. Specifically, CypA knockdown and CsA treatment reduce HIV-1 dependency toward nucleopore components Nup153 and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28128382 Nup358 for its nuclear transport [56,57]. It BAY 11-7085 web should be informative to analyze whether V86M modifies the interactions between HIV-1 CA and these nucleoporins.Conclusions The V86M mutation in HIV-1 CA can confer partial resistance against restriction of HIV-1 replication by mutants of human TRIM5. V86M abrogates CypA-dependent restriction mechanisms, resulting in an increase of HIV-1 DNA nuclear transport in restrictive conditions. However, this mutation might not confer significant resistance to the restriction of a spreading HIV-1 infection.Veillette et al. Retrovirology 2013, 10:25 http://www.retrovirology.com/content/10/1/Page 10 ofMethodsPlasmid DNAsRetroviral vectors productionpMIP-TRIM5hu and pMIP-TRIM5rh express C-terminal FLAG-tagged versions of the corresponding proteins and have been extensively described before [28,38,44,58,59]. pMIP-TRIM5hu with the mutation R332G-R335G has been described [38],.
