Recombinant CXCR4/CCR5 hybrid receptors as tools for studies of HIV-1 receptor usage

Abstract: The chemokine receptors CCR5 and CXCR4 are required, together with CD4, for the entry of HIV-1 into target cells. CCR5 using HIV-1 dominates during transmission and the asymptomatic phase of infection. During progression, virus phenotypes with the ability to use CXCR4 emerge in about 50% of infected individuals. Individuals continuously harbouring CCR5-restricted isolates still progress to AIDS. Differences among CCR5 using isolates, has been found and an evolution towards an altered mode of CCR5 coreceptor use and a reduced sensitivity to inhibition by natural CCR5 ligands has also been described. With the aim to study interactions of natural ligands and HIV-1 isolates with these chemokine receptors, a set of hybrid CXCR4/CCR5 receptors were constructed. Signalling response to their respective natural ligands, SDF-1 and RANTES were studied and prototypic R5 and X4 isolates (HIV-1BaL and HIV-1IIIB) were tested for their ability to use these chimeric receptors. The results showed that ligands and virus use different receptor epitopes which, in turn, vary between the two receptors. Further, the evolution of primary HIV-1 isolates was studied. A total of 246 sequential primary HIV-1 isolates were studied. Using our chimeric CXCR4/CCR5 receptors, we showed that R5 isolates from immunosuppressed individuals are distinct from those isolated from individuals with higher CD4+ T-cell counts, with regards to coreceptor usage. The analysis also showed that the ability to utilize chimeric receptors correlated inversely with the sensitivity to RANTES inhibition of infection. The R5 isolates used receptor chimeras to various degrees. Based on these results, the R5 viruses could be subdivided into two groups: the R5narrow phenotype and the R5broad phenotype. The R5narrow phenotype is defined as viruses that use wt CCR5 but no chimeric receptors, whereas viruses using at least one chimeric receptor in addition to CCR5, are designated R5broad viruses. The mode of coreceptor use by paired plasma and CSF isolates from HIV-1 infected individuals with varying degree of immunodeficiency and neuropathology were studied. The R5 viral phenotypes predominated both in plasma and in CSF. We were able to identify discordant plasma/CSF wt coreceptor use but also, varying R5 viral phenotypes in the paired isolates within individual patients. There were no characteristic patterns of receptor use that could distinguish CSF from plasma isolates. R5 virus use of chimera FC-2 correlated highly with immunosuppression. Efficient chimeric receptor use also correlated, with an increased resistance to inhibition by the CCR5 antagonist TAK-779. In conclusion, our findings propose that alterations in the mode of CCR5 use may be a key event in R5 virus pathogenesis. We believe that R5 virus ability to utilize these CXCR4/CCR5 chimeric receptors reflects a more flexible and more efficient CCR5 usage, which may include a reduced dependency upon interactions with the N-terminal of the receptor for infection. The findings are important, not only with regards to R5-virus pathogenesis and optimization of emerging treatment with CCR5 antagonists, but also for HIV-infection within the CNS.