Study Of Coreceptor Switching In HIV-1CRF07_BC

Background and objectiveThe envelope glycoproteins of human immunodeficiency virus type1(HIV-1) serve to mediate viral entry into cells. This process requires the sequential interaction of gp120with the receptor CD4and coreceptors, such as CCR5or CXCR4. Viruses that only use either CCR5or CXCR4coreceptors are termed R5-or X4-tropic, respectively, while R5X4dual-tropic viruses can use both. HIV-1infection is almost always established with R5viruses, which predominate during the acute and asymptomatic phases of infection. The appearance of X4and/or R5X4viruses is associated with a more rapid decline of CD4+cells in peripheral blood and faster progression to AIDS. Although R5viruses typically persist into late stages of disease, CXCR4-or dual-tropic viruses emerge in approximately50%of individuals infected with subtype B viruses, and coreceptor switching is also reported in subtype A, C, D, CRF01_AE, and CRF02_AG infections. So far, CRF07_BC CXCR4-using viruses have not been found in nature and its characteristics were unknown. So, we have three key questions about CRF07_BC coreceptor switch. First, why there is no CXCR4-tropic virus in CRF07_BC up to now? Second, will CRF07_BC X4virus emerge in future? Third, if yes, what slow CRF07_BC coreceptor switching? We have a hypothesis about CRF07_BC coreceptor switching. We think the genetic barrier is much higher for CRF07_BC than other subtype virus to switch coreceptor-usage. The gp120structure and function also restrict coreceptor switching. Besides, circulating time and evolutionary characteristics may also correlate with HIV-1coreceptor switching.Almost all the studies about coreceptor switching were based on subtype B virus, and recently there were some reports also in subtype C. Dose the coreceptor prediction method developed on other subtype also work in CRF07_BC? Do CRF07_BC CXCR4-tropic viruses share the same characteristics in the V3region with X4variants in other subtypes? Can we construct CRF07_BC CXCR4-tropic variants by mutagenesis according to the V3characteristics of subtype B and C X4viruses? Is there any correlation between HIV-1coreceptor switching and envelope evolution? If yes, what kind of relationship is it and can we use it to predict the CRF07_BC X4virus emerging time in nature? Knowing coreceptor switching in HIV-1will help us to better understand the pathogenesis and improve efficiency of HRRTA treatment. A functional CRF07_BC CXCR4-tropic molecular clone will definitely accelerate the study in this subtype and will be a powerful tool for therapeutics and vaccine research. In this study, firstly we tested the impact of X4-related V3features found in subtype B and C on CRF07_BC. Secondly, according to rational design, we constructed two CRF07_BC X4viruses before its emerging in nature. This makes us better understand the genetic barrier for HIV-1coreceptor switching. Thirdly, we determined the relationship between HIV-1coreceptor switching and envelope evolutionary characteristics and then predicted the CRF07_BC X4virus emerging time in nature. Systematic analysis of HIV-1envelope will show more detailed relationship between viral evolution and HIV-1transmission, fitness, coreceptor switching and so on. This study has special meaning for CRF07_BC pathogenesis study, HRRTA treatment efficiency, therapeutics and vaccine research. Methods and Results 1. Impact of X4-related V3features found in subtype B and C on CRF07_BC coreceptor switchingAll the V3sequences of subtype B, C and CRF07_BC were retrieved from Los Alamos HIV database and coreceptor-specific features were analyzed by VESPA tool. According to VESPA results and X4-related V3characteristics reported previously, we have designed and constructed a panel of V3mutants based on CRF07_BC CCR5-tropic molecular clone pXJDC13. The coreceptor usage of35mutants were predicted by C-PSSM, geno2pheno and CoRSeqV3-C. In combination of these three prediction results, there were3/35R5,17/35R5X4and15/35X4. Twenty-three CRF07BC V3mutants harbor subtype B X4-related features, including S11R, Q18R, A22T, G24R and D25Q/E/H. Twenty one out of23were infectious, but all of them were still CCR5-tropic. Twelve mutants harbor subtype C X4-related V3features, including S11R,112V, P16R, Q18R, D25Q and two amino acids IG or MG insertion between position13and14. Only mutants M55containing IG insertion was poor in infectivity and still CCR5-tropic, while the rest mutants were non-infectious.2. Construction and identification of HIV-1CRF07_BC CXCR4-tropic virusIn this part of work, according to rational design, we constructed two CRF07_BC CXCR4-tropic variants. There were two steps to construct functional X4virus. In the first step, we determined the CRF07_BC X4-related V3features in pNL4-3based chimeric virus system. The CRF07_BC mutated V3region constructed in first part were transferred to pNL4-3backbone by fusion PCR and then the viral coreceptor usage were tested. Twenty-one chimeric viruses were constructed and all of them were infectious. One of ten chimeras containing subtype B X4-related features was dual-tropic and the rest were still CCR5-tropic. Among ten chimeras containing subtype C X4-related features, three were CCR5-tropic and the other seven were dual-tropic. Two kinds of mutations within CRF07_BC V3were correlated with coreceptor switching, IG insertion and P16R or MG insertion and S11R. But these two kinds of mutations deprived infectivity of mutants M45and M61in the CRF07_BC molecular backbone. Similarly, these mutations also have deleterious effect on infectivity of other CRF07_BC (CH70, CH91, CH110, CH119, CH120) and subtype C (Dul56.12, Du172.17, ZM55F.PB28a, ZM109F.PB4) envelope clones. In the second step, based on non-infectious CRF07_BC molecular clone M45and M61, we have successfully constructed two factional CXCR4-tropic variants. The V1V2region comes from subtype C CXCR4-tropic Env clone1109-F-30rescued M45and M61, and conferred them CXCR4-using ability. These two CXCR4-tropic variants grow well on PBMCs and could induce syncitium formation on MT-2cells, and they were also sensitive to CCR5and CXCR4antagonists.3. Quantitive analysis of HIV-1coreceptor switching and envelope evolutionIn this part, we characterized the relationship between HIV-1coreceptor switching and viral envelope evolution. The full-length envelope sequences of nine subtypes were retrieved from Los Alamos HTV databaes, including subtype A, B, C, D, F, G, CRF01_AE, CRF02_AG and CRF07_BC. The tMRCA and envelope evolutionary rate were calculated using BEAST software. The envelope evolutionary rate were between1.97X10-3～7.54×10-3substitutions/site/year. Also, the envelope subregions evolutionary rate were also determined and from faster to slower were V4, V5, V1V2, C3, V3, C2, C4, C1and C5respectively. The tMRCA of each subtype were CRF01_AE1971/1959, CRF02_AG1965/1968, CRF07_BC1990/1987, subtype Al1959/1939, subtype B1956/1965, subtype C1932/1939, subtype D1887/1912, subtype F11960/1952and1966/1969. According to V1V2V3evolutionary rate, tMRCA and emerging time of CXCR4-tropic virus in each subtype, we could determine the mean accumulated mutations needed for coreceptor switching and then predict CRF07_BC CXCR4-tropic virus emerging time in nature. The CXCR4-tropic variants emerging time in CRF07_BC were2032(2018-2045) predicted by V1V2V3accumulated mutations. We also tested this prediction using the two CXCR4-tropic variants constructed in part two. The vlv2v3sequences including two CXCR4-tropic variants created were used to construct ML phylogenetic tree to analyze these variants emerging time in nature. We found that these two CRF07_BC CXCR4-tropic variants would emerge in approximately2026and this is consistent with the predicted time.ConclusionsAccording to rational design, we have successfully created two CRF07_BC CXCR4-tropic variants before its emerging in nature. Subtype C X4-related features, but not subtype B, could confer CRF07_BC CXCR4-using ability. So the coreceptor-using prediction methods developed on subtype C may work well for CRF07_BC. But subtype C X4-related features within V3deprived viral infectivity in CRF07_BC backbone and this deletetious mutations need compensate changes within V1V2to maintain a functional CXCR4-using variant. In consistent with its fuction, the envelope trimer structure revealed that V1V2region just covered on V3loop and they had a close interaction. Besides, we firstly showed that HIV-1coreceptor switching correlated with its prevalance time and evolutionary rate, especially V1V2V3rate. The accumulated mutations within V1V2V3was used to predict CRF07_BC CXCR4-tropic variants emerging time in nature and tested by two CXCR4-tropic variants constructed in this study. In conclusion, at present the genetic barrier is very high for coreceptor switching in CRF07_BC. According to rational design, we have successfully created two CRF07_BC CXCR4-tropic variants. The divergence within V1V2and V3were firstly used to predict and test CRF07_BC CXCR4-tropic variants emergence in nature. This study will help us to improve HRRTA treatment efficiency and provide a powerful tool for therapeutics and vaccine research in HIV-1, especially for CRF07_BC.