Evolution of DC-SIGN use revealed by fitness studies of R5 HIV-1 variants emerging during AIDS progression

<p>Abstract</p> <p>Background</p> <p>At early stages of infection CCR5 is the predominant HIV-1 coreceptor, but in approximately 50% of those infected CXCR4-using viruses emerge with disease progression. This coreceptor switch is correlated with an accelerated progression. However, those that maintain virus exclusively restricted to CCR5 (R5) also develop AIDS. We have previously reported that R5 variants in these "non-switch virus" patients evolve during disease progression towards a more replicative phenotype exhibiting altered CCR5 coreceptor interactions. DC-SIGN is a C-type lectin expressed by dendritic cells that HIV-1 may bind and utilize for enhanced infection of T cells in <it>trans</it>. To further explore the evolution of the R5 phenotype we analyzed sequential R5 isolates obtained before and after AIDS onset, i.e. at the chronic stage and during end-stage disease, with regard to efficiency of DC-SIGN use in <it>trans</it>-infections.</p> <p>Results</p> <p>Results from binding and <it>trans</it>-infection assays showed that R5 viruses emerging during end-stage AIDS disease displayed reduced ability to use DC-SIGN. To better understand viral determinants underlying altered DC-SIGN usage by R5 viruses, we cloned and sequenced the HIV-1 <it>env </it>gene. We found that end-stage R5 viruses lacked potential N-linked glycosylation sites (PNGS) in the gp120 V2 and V4 regions, which were present in the majority of the chronic stage R5 variants. One of these sites, amino acid position 160 (aa160) in the V2 region, also correlated with efficient use of DC-SIGN for binding and <it>trans</it>-infections. In fitness assays, where head-to-head competitions between chronic stage and AIDS R5 viruses were setup in parallel direct and DC-SIGN-mediated infections, results were further supported. Competitions revealed that R5 viruses obtained before AIDS onset, containing the V2 PNGS at aa160, were selected for in the <it>trans</it>-infection. Whereas, in agreement with our previous studies, the opposite was seen in direct target cell infections where end-stage viruses out-competed the chronic stage viruses.</p> <p>Conclusion</p> <p>Results of our study suggest R5 virus variants with diverse fitness for direct and DC-SIGN-mediated <it>trans</it>-infections evolve within infected individuals at end-stage disease. In addition, our results point to the importance of a glycosylation site within the gp120 V2 region for efficient DC-SIGN use of HIV-1 R5 viruses.</p

Increased Sensitivity to Broadly Neutralizing Antibodies of End-Stage Disease R5 HIV-1 Correlates with Evolution in Env Glycosylation and Charge

BACKGROUND: Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset. PRINCIPAL FINDINGS: HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase. The increased sensitivity correlated with low CD4(+) T cell count at time of virus isolation and augmented viral infectivity. Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb). Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to the 2G12 epitope. CONCLUSIONS: Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies, including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of adaptive molecular Env changes, including loss of glycosylation and gain of positive charge

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

Co-evolution of the HIV-1 R5 phenotype and the viral envelope glycoproteins

To gain entry into target cells, HIV-1 binds to CD4 via the viral glycoprotein gp120. This interac-tion initiates a series of events including binding of a coreceptor, CCR5 and/or CXCR4, and ulti-mately leads to gp41-mediated fusion of the viral and cell membranes. CCR5-restricted (R5) vi-ruses predominate in early, asymptomatic stages of the infection. Emergence of viruses able to use CXCR4 instead of, or in addition to, CCR5 for cell entry has been correlated with rapid progres-sion to AIDS. Importantly, approximately 50% of HIV-1 infected individuals progress to AIDS while maintaining viruses that are restricted to CCR5. The major reason for today’s inadequate treatment of HIV-1 infection and failure to develop a potent vaccine is the fast evolution of the virus. Due to an error-prone reverse transcriptase en-zyme (RT), mutations are continuously introduced into the viral genome, which result in pheno-typic variation of the virus. Thus, the intra-host virus evolution can be traced by analyzing the changes in the viral genome over time. We have studied the intra-patient evolution of the R5 phe-notype and the viral envelope glycoproteins in patients that maintain an exclusive CCR5-restricted virus population. We used a panel of sequentially isolated viruses from patients who all progressed to AIDS. Isolations were done at the chronic, asymptomatic phase and after AIDS onset, at which time the patients were all severely immunodeficient. Our results revealed emergence of virus vari-ants with enhanced viral fitness during end-stage disease. After AIDS onset viruses with higher infectivity and replicative capacity dominated. These viruses were also more resistant to inhibition by several entry inhibiting compounds, which suggests a central role of the viral Env (gp120/gp41 complex) in the evolution of viral properties towards increased fitness. To dissect the evolution of the viral Env at the molecular level, we cloned, sequenced and analyzed full length gp160 env clones. We discovered that the Env of end-stage R5 viruses were less glycosylated and had higher net charge. These changes were all mapped to gp120 and specifi-cally to the variable regions, i.e., the outer surface of the Env. Furthermore, the reduced glycan shield correlated with a decreased ability to infect target cells in DC-SIGN-mediated trans-infection and increased sensitivity to MAb neutralization. Viruses with increased Env charge are selected for during severe immunodeficiency and correlated with enhanced cell attachment and properties of viral fitness. Both reduced glycosylation and increased net charge of Env correlated with reduced CD4+ T cell count at time of R5 virus isolation. In summary, during end-stage disease, R5 virus variants emerge with increased viral fitness. The elevated fitness is a result of altered phenotypic properties. The underlying mechanisms can be mapped to the molecular properties of the viral Env. Thus, after AIDS onset, viruses with al-tered phenotypic properties may evolve, in an opportunistic manner, and result in the emergence of HIV-1 R5 variants with enhanced pathogenicity

Reduced baseline sensitivity to Maraviroc inhibition among R5 HIV-1 isolates from individuals with severe immunodeficiency.

Supplemental Digital Content is Available in the Text