Marta Calado

Macrophages (Mø) and dendritic cells (DCs) are key players in human immunodeficiency virus (HIV) infection and pathogenesis. They are essential for the spread of HIV to CD4+ T lymphocytes (TCD4+) during acute infection. In addition, they constitute a persistently infected reservoir in which viral production is maintained for long periods of time during chronic infection. Defining how HIV interacts with these cells remains a critical area of research to elucidate the pathogenic mechanisms of acute spread and sustained chronic infection and transmission. To address this issue, we analyzed a panel of phenotypically distinct HIV-1 and HIV-2 primary isolates for the efficiency with which they are transferred from infected DCs or Mø to TCD4+. Our results show that infected Mø and DCs spread the virus to TCD4+ via cell-free viral particles in addition to other alternative pathways. We demonstrate that the production of infectious viral particles is induced by the co-culture of different cell populations, indicating that the contribution of cell signaling driven by cell-to-cell contact is a trigger for viral replication. The results obtained do not correlate with the phenotypic characteristics of the HIV isolates, namely their co-receptor usage, nor do we find significant differences between HIV-1 and HIV-2 in terms of cis- or trans-infection. The data presented here may help to further elucidate the cell-to-cell spread of HIV and its importance in HIV pathogenesis. Ultimately, this knowledge is critical for new therapeutic and vaccine approaches.

The main goal of this work was to identify molecular signatures in envelope surface glycoprotein that may be correlated with coreceptor usage by different HIV-2 strains. From inspection of aligned HIV-2 sequences we verified that V1/V2 region showed the highest degree of amino acid sequence heterogeneity including polymorphisms in N-linked glycosylation sites, sequence and length. Furthermore, we did not find any correlation between the net charge and specific amino acids positions in V3 region with any particular coreceptor-usage pattern. In conclusion, we showed that for HIV-2 the genetic determinants for coreceptor usage are distinct from those of HIV-1. More specifically, we did not identify any molecular signature, based on discrete amino acid positions either in V1/V2 or V3 regions, which could be assigned to the preferential usage of a specific coreceptor.

Human immunodeficiency virus 1 and 2 (HIV-1 and HIV-2) use cellular receptors in distinct ways. Besides a more promiscuous usage of coreceptors by HIV-2 and a more frequent detection of CD4-independent HIV-2 isolates, we have previously identified two HIV-2 isolates (HIV-2MIC97 and HIV-2MJC97) that do not use the two major HIV coreceptors: CCR5 and CXCR4. All these features suggest that in HIV-2 the Env glycoprotein subunits may have a different structural organization enabling distinct - although probably less efficient - interactions with cellular receptors. By infectivity assays using GHOST cell line expressing CD4 and CCR8 and blocking experiments using CCR8-specific ligand, I-309, we show that efficient replication of HIV-2MIC97 and HIV-2MJC97 requires the presence of CCR8 at plasma cell membrane. Additionally, we disclosed the determinants of chemokine receptor usage at the molecular level, and deciphered the amino acids involved in the usage of CCR8 (R8 phenotype) and in the switch from CCR8 to CCR5 or to CCR5/CXCR4 usage (R5 or R5X4 phenotype). The data obtained from site-directed mutagenesis clearly indicates that the main genetic determinants of coreceptor tropism are located within the V1/V2 region of Env surface glycoprotein of these two viruses. We conclude that a viral population able to use CCR8 and unable to infect CCR5 or CXCR4-positive cells, may exist in some HIV-2 infected individuals during an undefined time period, in the course of the asymptomatic stage of infection. This suggests that in vivo alternate molecules might contribute to HIV infection of natural target cells, at least under certain circumstances. Furthermore we provide direct and unequivocal evidence that the usage of CCR8 and the switch from R8 to R5 or R5X4 phenotype is determined by amino acids located in the base and tip of V1 and V2 loops of HIV-2 Env surface glycoprotein.

Human immunodeficiency virus (HIV) entry into susceptible cells involves the interaction between viral envelope glycoproteins with CD4 and a chemokine receptor (coreceptor), namely CCR5 and CXCR4. This interaction has been studied to enable the discovery of a new class of antiretroviral drugs that targets the envelope glycoprotein–coreceptor interaction. However, very few data exist regarding HIV-2 susceptibility to these coreceptor inhibitors. With this work we aimed to identify this susceptibility in order to assess the potential use of these molecules to treat HIV-2-infected patients and to further understand the molecular basis of HIV-2 envelope glycoprotein interactions with CCR5 and CXCR4. We found that CCR5-using HIV-2 isolates are readily inhibited by maraviroc, TAK-779, and PF-227153, while monoclonal antibody 2D7 shows only residual or no inhibitory effects. The anti-HIV-2 activity of CXCR4-targeted molecules reveals that SDF-1α/CXCL12 inhibited all HIV-2 tested except one, while mAb 12G5 inhibited the replication of only two isolates, showing residual inhibitory effects with all the other CXCR4-using viruses. A major conclusion from our results is that infection by HIV-2 primary isolates is readily blocked in vitro by maraviroc, at concentrations similar to those required for HIV-1. The susceptibility to maraviroc was independent of CD4⁺ T cell counts or clinical stage of the patient from which the virus was obtained. These findings indicate that maraviroc could constitute a reliable therapeutic alternative for HIV-2-infected patients, as long as they are infected with CCR5-using variants, and this may have direct implications for the clinical management of HIV-2-infected patients.