HIV has evolved two independent activities for Nef to downregulate the receptor complex and to facilitate its efficient replication and spread. This evasion strategy likely represents a mechanism by which the pathogenicity factor Nef elevates viral replication in vivo and thus promotes AIDS pathogenesis.

The Nef protein is a key determinant of human immunodeficiency virus (HIV) pathogenicity that, among other activities, sensitizes T-lymphocytes for optimal virus production. The initial events by which Nef modulates the T-cell receptor (TCR) cascade are poorly understood.TCRengagementtriggersactinrearrangementsthatcontrol receptor clustering for signal initiation and dynamic organization of signaling protein complexes to form an immunological synapse. Here we report that Nef potently interferes with cell spreading and formation of actin-rich circumferential rings in T-lymphocytes upon surface-supported TCR stimulation. These effects were conserved among Nef proteins from different lentiviruses and occurred in HIV-1-infected primary human T-lymphocytes. This novel Nef activity critically depended on its Src homology 3 domain binding motif and required efficient association with Pak2 activity. Notably, whereas overall signaling microcluster formation immediately following TCR engagement occurred normally in Nef-expressing cells, the viral protein inhibited the concomitant activation of the actin organizer N-Wasp. During the subsequent maturation phase of the stimulatory contact, Nef interfered with the translocation of N-Wasp to the cell periphery, the overall induction of tyrosine phosphorylation, and the selective recruitment of phosphorylated LAT to stimulatory contacts. Consistent with such a critical role of N-Wasp in this process, Nef also blocked morphological changes induced by the known N-Wasp regulators Rac1 and Cdc42. Together, our results demonstrate that Nef alters both the amount and composition of signaling microclusters. We propose modulation of actin dynamics as an important mechanism for Nef-induced alterations of TCR signaling.The Nef protein of the primate lentiviruses HIV-1/-2 2 and simian immunodeficiency virus is a crucial pathogenicity factor and substantially increases virus replication in vivo (1-4). Whereas the underlying molecular mechanisms remain to be fully elucidated, Nef facilitates immune evasion of infected cells and directly boosts virus spread. These effector functions probably reflect the ability of Nef to serve as a protein-interaction adaptor that modulates cellular vesicle transport and signal transduction machineries (5, 6). In CD4 ϩ T-lymphocytes, one of the major HIV-1 target cell populations in vivo, Nef lowers the threshold of TCR activation possibly to induce an intermediate activation state that is permissive for HIV-1 replication (7-12). Several protein assemblies, including the association with the Nef-associated kinase complex, the guanine exchange factors Vav and DOCK2-ELMO1, and the TCR chain, are involved in the modulation of TCR signaling by Nef (13-16). However, it has not been addressed how Nef affects early events of TCR signal initiation. Physiologically, TCR triggering occurs in the context of a close contact between a T-cell and antigenpresenting cell referred to as the immunological synapse (IS). Within this highly dynamic structure, spatial redistribut...

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Background: In vivo studies on HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Since native rodents are nonpermissive, we developed transgenic rats that selectively express the HIV-1 receptor complex, hCD4 and hCCR5, on relevant target cells. These animals display a transient low-level plasma viremia after HIV-1 YU-2 infection, demonstrating HIV-1 susceptibility in vivo. However, unlike macrophages, primary CD4 T-cells from double-transgenic animals fail to support viral spread ex vivo. To identify quantitative limitations or absolute blocks in this rodent species, we quantitatively assessed the efficiency of key steps in the early phase of the viral replication cycle in a side-by-side comparison in infected cell lines and primary T-cells from hCD4/hCCR5-transgenic rats and human donors.

DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. For immunotherapy of HPV-16-associated diseases the E7 protein is considered a prime candidate, as it is expressed in all HPV-16-positive tumors. Unfortunately, the E7 protein is a very poor inducer of a cytotoxic T-cell response, when being used as antigen in DNA vaccination. Here we demonstrate that after fusion to protein export/import signals such as the herpes simplex virus ferry protein VP22, E7 can translocate in vitro from VP22-E7-expressing cells to neighboring cells that do not carry the VP22-E7 gene. In vivo, the VP22-E7 fusion shows significantly increased efficiency in inducing a cytotoxic T-cell response. Our data suggest that the export function of VP22 plays a major role in this phenomenon, since VP22 can be replaced by classical protein export signals, without impairing the induction of the E7-specific cellular immune response. However, all E7 fusion constructs showed significantly elevated protein steady-state levels, which might also account for the observed boost in immunogenicity.