HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. We use NMR to determine an atomic structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in bicelles that mimic a lipid bilayer. The TM forms a well-ordered trimer that protects a conserved membrane-embedded arginine. An N-terminal coiled-coil and a C-terminal hydrophilic core stabilize the trimer. Individual mutations of conserved residues did not disrupt the TM trimer and minimally affected membrane fusion and infectivity. Major changes in the hydrophilic core, however, altered the antibody sensitivity of Env. These results show how a TM domain anchors, stabilizes and modulates a viral envelope spike and suggest that its influence on Env conformation is an important consideration for HIV-1 immunogen design.

Non-enveloped viruses must create a transient membrane lesion to initiate infection by transferring their genomes into a target cell 1 . Rotaviruses offer a particularly favorable opportunity to visualize the mechanism for subviral particle delivery, the principal function of their outer-layer protein, VP4 2 – 4 . We show here by electron cryomicroscopy (cryo-EM) that VP4, activated by cleavage to VP8* and VP5*, rearranges on the virion surface from an “upright” to a “reversed” conformation. The reversed structure projects an initially buried, “foot” domain outward into the host cell membrane to which the virion has attached. Analysis of cryo-tomograms of virus particles entering cells is consistent with this picture. We have stabilized with a disulfide mutant a likely intermediate in this transition. The results define molecular mechanisms for the first steps in viral membrane penetration and suggest similarities with mechanisms postulated for other viruses.

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Characterizing the cellular factors that play a role in the HIV replication cycle is fundamental to fully understanding mechanisms of viral replication and pathogenesis. Whole-genome small interfering RNA (siRNA) screens have identified positive and negative regulators of HIV replication, providing starting points for investigating new cellular factors. We report here that silencing of the deubiquitinase cylindromatosis protein (CYLD), increases HIV infection by enhancing HIV long terminal repeat (LTR)-driven transcription via the NF-B pathway. CYLD is highly expressed in CD4؉ T lymphocytes, monocyte-derived macrophages, and dendritic cells. We found that CYLD silencing increases HIV replication in T cell lines. We confirmed the positive role of CYLD silencing in HIV infection in primary human CD4 ؉ T cells, in which CYLD protein was partially processed upon activation. Lastly, Jurkat T cells latently infected with HIV (JLat cells) were more responsive to phorbol 12-myristate 13-acetate (PMA) reactivation in the absence of CYLD, indicating that CYLD activity could play a role in HIV reactivation from latency. In summary, we show that CYLD acts as a potent negative regulator of HIV mRNA expression by specifically inhibiting NF-Bdriven transcription. These findings suggest a function for this protein in modulating productive viral replication as well as in viral reactivation. IMPORTANCEHIV transcription is regulated by a number of host cell factors. Here we report that silencing of the lysine 63 deubiquitinase CYLD increases HIV transcription in an NF-B-dependent manner. We show that CYLD is expressed in HIV target cells and that its silencing increases HIV infection in transformed T cell lines as well as primary CD4 ؉ T cells. Similarly, reactivation of latent provirus was facilitated in the absence of CYLD. These data suggest that CYLD, which is highly expressed in CD4 ؉ T cells, can control HIV transcription in productive infection as well as during reactivation from latency.