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Abstract Background Gene trap insertional mutagenesis was used as a high-throughput approach to discover cellular genes participating in viral infection by screening libraries of cells selected for survival from lytic infection with a variety of viruses. Cells harboring a disrupted ADAM10 (A Disintegrin and Metalloprotease 10) allele survived reovirus infection, and subsequently ADAM10 was shown by RNA interference to be important for replication of HIV-1. Results Silencing ADAM10 expression with small interfering RNA (siRNA) 48 hours before infection significantly inhibited HIV-1 replication in primary human monocyte-derived macrophages and in CD4+ cell lines. In agreement, ADAM10 over-expression significantly increased HIV-1 replication. ADAM10 down-regulation did not inhibit viral reverse transcription, indicating that viral entry and uncoating are also independent of ADAM10 expression. Integration of HIV-1 cDNA was reduced in ADAM10 down-regulated cells; however, concomitant 2-LTR circle formation was not detected, suggesting that HIV-1 does not enter the nucleus. Further, ADAM10 silencing inhibited downstream reporter gene expression and viral protein translation. Interestingly, we found that while the metalloprotease domain of ADAM10 is not required for HIV-1 replication, ADAM15 and γ-secretase (which proteolytically release the extracellular and intracellular domains of ADAM10 from the plasma membrane, respectively) do support productive infection. Conclusions We propose that ADAM10 facilitates replication at the level of nuclear trafficking. Collectively, our data support a model whereby ADAM10 is cleaved by ADAM15 and γ-secretase and that the ADAM10 intracellular domain directly facilitates HIV-1 nuclear trafficking. Thus, ADAM10 represents a novel cellular target class for development of antiretroviral drugs.

Abstract: CD8+ T cells are important for resolution of HSV-2 lesions from the female genital epithelium. It is uncertain whether optimal clearance of viruses such as HSV-2 that cause a limited, non-systemic infection solely requires expression of effector functions by infiltrating CD8+ T lymphocytes, or if the clearance rate is reflective of the expression level of critical effector functions. To address this, CD8+ T cells from normal OT-I mice or OT-I mice deficient in IFNγ (IFNγ−/−) or the IFNγ receptor (IFNγR−/−) were activated in vitro in the presence of IFNγ or IL-4 to generate a series of effector populations (Tc1 and Tc2-like respectively) that secreted different levels of IFNγ and expressed different levels of HSV-specific cytolytic function. Compared with Tc1 cells, Tc2-like cells produced the type 2 cytokines IL-4 and IL-5, exhibited decreased IFNγ secretion, diminished proliferation in vitro, and decreased antigen-specific cytolysis in vivo. Clearance of an ovalbumin-expressing HSV-2 strain (HSV-2 tk− OVA) by adoptively transferred Tc2-like cells was delayed relative to Tc1 cell recipients. Because donor Tc2-like cells proliferated in vivo and infiltrated the infected genital epithelium similar to Tc1 cells, the diminished virus clearance by Tc2-like effector cells correlated with reduced expression of critical effector functions. Together, these results suggest that high level expression of protective T cell functions by effector T cells is necessary for optimal clearance of HSV-2 from the genital epithelium. These results have important implications for vaccines designed to elicit CD8+ T cells against viruses such as HSV-2 that infect the genital tract. [Copyright &y& Elsevier]

Measurements of humoral immune responses to West Nile virus (WNV) infection in mouse or other animal models are valuable components of basic laboratory investigations to assess immunogenicity of candidate vaccines or to evaluate seroconversion following challenge with WNV. Here, we outline the steps for screening or titrating of total antibodies by indirect enzyme linked immunosorbent assay (ELISA) as well as assessment of neutralizing antibody titers by immunofocus detection.