Viral and host factors in HIV-1 entry : pathogenic and therapeutic implications for HIV-1 infection
(2006)
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- Soon after HIV-1 was discovered, the cell surface CD4 molecule was considered as a critical component for HIV-1 entry. However, the interaction between HIV-1 envelope and CD4 was known to be insufficient for achieving the complete entry process. HIV-1 was also characterized by heterogeneous behaviour in vitro, with several viruses exhibiting high replication capacities and cytopathic effects (or syncytia) when cultured with lymphoblastoid cells (syncytium-inducing or SI viruses) and other viruses showing lower replication rate and no cytopathic effect (NSI viruses). In 1996, human chemokine receptors were identified as the missing piece in the human receptor complex, and were designated to as ¡§HIV-1 coreceptors¡¨, acting in concert with CD4 for HIV-1 entry. Two receptors in particular, CCR5 and CXCR4, were relevant for HIV-1 infection in vivo and clarified the heterogeneity observed in HIV-1: NSI viruses used CCR5 and were dominant early after infection while SI viruses used CXCR4 and were associated with more severe prognosis in patients. Several coreceptor polymorphisms were described, among which a 32-bp deletion in the open reading frame of CCR5 (CCR5 ´32). Individuals homozygous for the CCR5 ´32 polymorphism were almost completely resistant to HIV-1, further supporting the role of CCR5 in HIV-1 transmission. In parallel, progress was made in the molecular elucidation of the two subunits of the HIV-1 envelope complex, the exterior surface (SU) glycoprotein, gp120 and the transmembrane (TM) glycoprotein, gp41. A sequential model of HIV-1 entry is now widely accepted, based on our current understanding on viral and host factors involved in the process: first, gp120 interacts with CD4. This induces conformational changes in gp120 that exposes coreceptor binding domains. The gp120-coreceptor interaction triggers further changes in gp41, which exposes an amino-terminal fusion peptide for insertion into the target cell membrane and undergoes its transition to a state called ¡§prefusogenic intermediate¡¨. Then gp41 evolves to its ¡§fusion-active¡¨ state, which brings target cell and viral membranes in close proximity for fusion. Better understanding of HIV-1 entry has motivated the development of new inhibitors aimed at the entry step, beside reverse transcriptase and protease inhibitors. These new molecules are termed entry inhibitors (EIs) or fusion inhibitors (FIs). These agents could be classified into three different groups, based on the successive steps involved in HIV-1 entry: a first class interacts with the initial gp120-CD4 binding; a second class inhibits the gp120-coreceptor interaction and the last class, also referred to as FIs, acts at the fusion step by targeting gp41. Numerous compounds are now in pre-clinical or clinical development, with a FI, enfuvirtide (ENF or T-20), having recently been FDA-approved for clinical use. The scope of our thesis work was to develop technologies for the study of viral and host factors involved in HIV-1 entry. Using PCR-RFLP protocols, we analysed the prevalence of HIV-1 coreceptor polymorphisms in HIV-1-infected patients and uninfected volunteers in Luxembourg. Allele frequencies were in overall agreement with previous observations made in Caucasian populations, although the frequency of CCR5 ´32 was rather elevated in HIV-1 positive individuals in our cohort (allele frequency = 0.11). CCR5 ´32 was significantly associated with a minority of slow progressor patients (p = 0.006), who were characterized by stable CD4+ T cell counts for at least 10 years prior initiation of antiretroviral therapy. On a more anecdotal note, we also reported an unusual CX3CR1 genotype which contradicted previous findings on CX3CR1 haplotype distribution. We exploited a prototype technology for sequencing the V3 loop region of HIV-1 strains infecting untreated long-term non-progressors (LTNP) and late-stage patients (LSP) from Rwanda. New and rare mutations were identified in LTNP, and were significantly more frequent than in LSP (p = 0.006), in a region that is highly relevant for the host¡¦s immune response. We hypothesized that the mutations observed in LTNP could be the result of a strong immune pressure in LTNP or, alternatively, that those HIV-1 variants might represent specific patterns of attenuated HIV-1 strains infecting LTNP in Rwanda. Having developed a simple sequencing technology, we were among the first to describe the natural variation of the gp41, ENF-sensitive region within a large panel of HIV-1 B and non-B subtypes. Overall, we observed that the region was highly conserved, despite episodic variation whose impact on ENF sensitivity would deserve further evaluation. Among mutations, an unusual isoleucine to valine substitution was observed in a highly conserved tripeptide motif in the gp41 of a subtype B isolate (I37V substitution). For evaluating the impact of I37V on ENF sensitivity in vitro, we developed a recombinant virus system that allowed for the recombination of full-length HIV-1 env coding sequences within a HIV-1 proviral, eGFP-tagged plasmid. Recombination could be performed without the use of restriction enzymes and HIV-1 infection was quantified using flow cytometry. Using that system, we showed that I37V impaired the fitness of the virus but did not lead per se to ENF resistance in vitro. HIV-1 disease has become a chronic disease in countries where antiretroviral therapy is available, and factors aimed at predicting the long-term success of treatment are of prominent importance. In the case of EIs and FIs, such evaluation will likely have to combine both host and viral factors, in way that did not apply for older classes of drugs. The fact that CCR5 expression levels modulate the antiviral activity of FIs or CCR5 inhibitors in vitro supports that concept. The in vivo relevance of a comprehensive approach integrating viral and host factors when testing EI/FI resistance should be addressed in future investigations.
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Roman, F. (2006). Viral and host factors in HIV-1 entry : pathogenic and therapeutic implications for HIV-1 infection. https://hdl.handle.net/2078.5/112107