BackgroundDetailed local HIV-1 sequence data are essential for monitoring the HIV epidemic, for maintaining sensitive sequence-based diagnostics, and to aid in designing vaccines.ResultsReported here are full envelope sequences derived from 38 randomly selected HIV-1 infections identified at a Gambian clinic between 1991 and 2009. Special care was taken to generate sequences from circulating viral RNA as uncloned products, either by limiting dilution or single genome amplification polymerase chain reaction (PCR). Within these 38 isolates, eight were subtyped as A and 18 as CRF02_AG. A small number of subtype B, C, D viruses were identified. Surprising, however, was the identification of six isolates with subtype J-like envelopes, a subtype found normally in Central Africa and the Democratic Republic of the Congo (DRC), with gag p24 regions that clustered with subtype A sequences. Near full-length sequence from three of these isolates confirmed that these represent a novel circulating recombinant form of HIV-1, now named CRF49_cpx.ConclusionsThis study expands the HIV-1 sequence database from the Gambia and will provide important data for HIV diagnostics, patient care, and vaccine development.
Background'Intrinsic' resistance to retroviral infection was first recognised with the Friend virus susceptibility gene (Fv1), which determines susceptibility to murine leukaemia virus (MLV) infection in different murine species. Similarly, the tripartite motif (TRIM) family of proteins determine lentiviral restriction in a primate host-species specific manner. For example rhesus TRIM5α (rhTRIM5α) can potently restrict HIV-1 infection while human TRIM5α (huTRIM5α) only has a mild effect on SIVmac and HIV-1 infectivity (Lv1). Human TRIM5α is able to restrict MLV-N virus replication, but is ineffective against MLV-B or MLV-NB virus infection. Lv2 restriction of some HIV-2 viruses is seen in human cells. Like Lv1, Lv2 is a post-entry restriction factor, whose viral determinants have been mapped to the viral capsid (CA). Unlike Lv1, however, Lv2 is determined by envelope (Env) in addition to CA. Here we present evidence of a novel Env determined post entry restriction to infection in human cells of pseudotyped MLV-B and MLV-NB cores.ResultsWe generated retroviral vectors pseudotyped with various gamma and lentiviral Envs on MLV-B and -NB CAs containing a green fluorescent protein (GFP) reporter. Flow cytometry was used to determine transduction efficiencies in NP2/CD4/CXCR4 (glioma cell line stably transduced with the HIV receptors) and HeLa/CD4 cell lines. The HeLa/CD4 cell line restricted both MLV CAs in an Env dependent manner, compared to NP2/CD4/CXCR4 cells. Quantitative polymerase chain reaction (QT-PCR) analysis of reverse transcription (RT) transcripts demonstrates that this restriction occurs at a post entry and RT level. siRNA knockdown of huTRIM5α ruled out a direct role for this cellular component in mediating this restriction. We describe a previously unobserved Env determined restriction of MLV-B and MLV-NB CAs in HeLa/CD4 cells when pseudotyped with HIV-2 and RD114 Envs, but not gibbon ape leukaemia virus (GALV), HIV-1 or Amphotrophic (Ampho) Envs.ConclusionsOur data further demonstrate the variability of Env and CA mediated susceptibility to post entry host cell restriction. We discuss the relevance of these findings in light of the growing evidence supporting the complexities involved in innate host immunity to retroviral infection.
Nuclear export of unspliced and incompletely spliced human immunodeficiency virus type 1 mRNA is mediated by the viral Rev protein. Rev binds to a structured RNA motif known as the Rev-response element (RRE), which is present in all Rev-dependent transcripts, and thereby promotes entry of the ribonucleoprotein complex into the nuclear-export pathway. Recent evidence indicates that a dimerization interface and a genetically separable 'trimerization' interface are required for multimeric assembly of Rev on the RRE. In this report, the effect of mutations within the trimerization interface on Rev function was examined in mammalian cells. All trimerization-defective Rev molecules had profoundly compromised Rev function and a range of localization defects was observed. However, despite the potential for formation of heterodimers between functional and non-functional Rev proteins, trimerization-defective Rev mutants were unable to inhibit wild-type Rev function in a trans-dominant-negative manner.The human immunodeficiency virus (HIV) rev gene product is a prototypic example of a class of functionally diverse, arginine-rich, sequence-specific RNA-binding proteins. Rev binds to an elaborate RNA structure, the Rev-response element (RRE), and promotes nuclear export of RRE-containing mRNAs that encode gag/pol and env (Pollard & Malim, 1998). Regions required for RNA binding, trans-activation and multimerization have been mapped within Rev (Daly et al., 1989;Malim & Cullen, 1991;Malim et al., 1989 Malim et al., , 1990Olsen et al., 1990;Pollard & Malim, 1998), but the precise features that determine the specificity and stability of the Rev-multimerization process have yet to be defined fully.An elegant genetic selection was used to identify Rev mutants with deficiencies in the Rev multimeric-assembly pathway (Jain & Belasco, 2001). Three classes of multimerization defect were resolved. Class one Rev mutants bind to the RRE as monomers, but are defective in their ability to form dimers; consequently, these mutants do not form multimers readily on the RRE. Moreover, class three mutants exhibit defects at all stages of RRE binding and Rev dimerization and multimerization and are probably structurally defective. In contrast, and perhaps most interestingly, class two mutants are competent for dimerization and RNA binding, but show greatly reduced multimerization properties. Thus, Jain & Belasco (2001) were able to genetically separate the process of dimerization from the subsequent process of multimeric Rev assembly. The refined molecular model (Jain & Belasco, 2001) suggests that there are two Rev-interaction surfaces; one surface is required for Rev-Rev dimerization, whereas the second is required for trimerization and higher-order assembly (Fig. 1a).Considerable evidence suggests that the amino-terminal half of Rev adopts a helix-loop-helix motif (Hope et al., 1990;Jain & Belasco, 2001;Madore et al., 1994;Pollard & Malim, 1998;Thomas et al., 1998;Zapp et al., 1991). Amino acid residues 11-32 of the amino-terminal helix cons...
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