Identification of differentially expressed proteins in the cervical mucosa of HIV-1-resistant sex workers.

Novel tools are necessary to understand mechanisms of altered susceptibility to HIV-1 infection in women of the Pumwani Sex Worker cohort, Kenya. In this cohort, more than 140 of the 2000 participants have been characterized to be relatively resistant to HIV-1 infection. Given that sexual transmission of HIV-1 occurs through mucosal surfaces such as that in the cervicovaginal environment, our hypothesis is that innate immune factors in the genital tract may play a role in HIV-1 infection resistance. Understanding this mechanism may help develop microbicides and/or vaccines against HIV-1. A quantitative proteomics technique (2D-DIGE: two-dimensional difference in-gel electrophoresis) was used to examine cervical mucosa of HIV-1 resistant women ( n = 10) for biomarkers of HIV-1 resistance. Over 15 proteins were found to be differentially expressed between HIV-1-resistant women and control groups ( n = 29), some which show a greater than 8-fold change. HIV-1-resistant women overexpressed several antiproteases, including those from the serpin B family, and also cystatin A, a known anti-HIV-1 factor. Immunoblotting for a selection of the identified proteins confirmed the DIGE volume differences. Validation of these results on a larger sample of individuals will provide further evidence these biomarkers are associated with HIV-1 resistance and could help aid in the development of effective microbicides against HIV-1.

Extensive syncytium formation mediated by the reovirus FAST proteins triggers apoptosis-induced membrane instability.

The fusion-associated small transmembrane (FAST) proteins of the fusogenic reoviruses are the only known examples of membrane fusion proteins encoded by non-enveloped viruses. While the involvement of the FAST proteins in mediating extensive syncytium formation in virus-infected and -transfected cells is well established, the nature of the fusion reaction and the role of cell-cell fusion in the virus replication cycle remain unclear. To address these issues, we analyzed the syncytial phenotype induced by four different FAST proteins: the avian and Nelson Bay reovirus p10, reptilian reovirus p14, and baboon reovirus p15 FAST proteins. Results indicate that FAST protein-mediated cell-cell fusion is a relatively non-leaky process, as demonstrated by the absence of significant [3H]uridine release from cells undergoing fusion and by the resistance of these cells to treatment with hygromycin B, a membrane-impermeable translation inhibitor. However, diminished membrane integrity occurred subsequent to extensive syncytium formation and was associated with DNA fragmentation and chromatin condensation, indicating that extensive cell-cell fusion activates apoptotic signaling cascades. Inhibiting effector caspase activation or ablating the extent of syncytium formation, either by partial deletion of the avian reovirus p10 ecto-domain or by antibody inhibition of p14-mediated cell-cell fusion, all resulted in reduced membrane permeability changes. These observations suggest that the FAST proteins do not possess intrinsic membrane-lytic activity. Rather, extensive FAST protein-induced syncytium formation triggers an apoptotic response that contributes to altered membrane integrity. We propose that the FAST proteins have evolved to serve a dual role in the replication cycle of these fusogenic non-enveloped viruses, with non-leaky cell-cell fusion initially promoting localized cell-cell transmission of the infection followed by enhanced progeny virus release from apoptotic syncytia and systemic dissemination of the infection.

Identification and characterization of a baboon reovirus-specific nonstructural protein encoded by the bicistronic s4 genome segment.

All characterized orthoreoviruses encode a characteristic spike-like protein on their polycistronic S1 genome segments that mediates virus cell attachment. In the case of baboon reovirus (BRV), the polycistronic S-class genome segment corresponds to the smallest S4 segment. We recently determined that the 5'-proximal open reading frame (ORF) of the bicistronic S4 segment encodes a nonstructural protein responsible for virus-induced syncytium formation. Current analysis indicates that the p16 protein encoded by the 3'-proximal ORF of the BRV S4 genome segment shows no sequence similarity to any other protein encoded by the orthoreoviruses, including the well-characterized sigma1/sigmaC reovirus cell attachment protein. Results indicate that p16 is a BRV-specific nonstructural protein that is not required for virus infection in cell culture and is not involved in viral cell attachment. In conjunction with previous studies of the BRV S1, S2, and S3 genome segments, the current results indicate that, unlike all other orthoreoviruses, BRV does not encode a cell attachment protein in its S-class genome segments. Furthermore, cell binding and infectivity studies suggested BRV may not utilize a functional homolog of the prototypical reovirus sigma1/sigmaC cell receptor-binding protein to mediate endocytic uptake by cells.