HIV-1 Env mediates fusion of viral and target cell membranes, but it can also mediate fusion of infected (producer) and target cells, thus triggering the formation of multinucleated cells, so-called syncytia. Large, round, immobile syncytia are readily observable in cultures of HIV-1-infected T cells, but these fast growing “fusion sinks” are largely regarded as cell culture artifacts. In contrast, small HIV-1-induced syncytia were seen in the paracortex of peripheral lymph nodes and other secondary lymphoid tissue of HIV-1-positive individuals. Further, recent intravital imaging of lymph nodes in humanized mice early after their infection with HIV-1 demonstrated that a significant fraction of infected cells were highly mobile, small syncytia, suggesting that these entities contribute to virus dissemination. Here, we report that the formation of small, migratory syncytia, for which we provide further quantification in humanized mice, can be recapitulated in vitro if HIV-1-infected T cells are placed into 3D extracellular matrix (ECM) hydrogels rather than being kept in traditional suspension culture systems. Intriguingly, live-cell imaging in hydrogels revealed that these syncytia, similar to individual infected cells, can transiently interact with uninfected cells, leading to rapid virus transfer without cell-cell fusion. Infected cells were also observed to deposit large amounts of viral particles into the extracellular space. Altogether, these observations suggest the need to further evaluate the biological significance of small, T cell-based syncytia and to consider the possibility that these entities do indeed contribute to virus spread and pathogenesis.

During cell-to-cell transmission of HIV-1, viral and cellular proteins transiently accumulate at the contact zone between infected (producer) and uninfected (target) cells, forming the virological synapse. Rearrangements of the cytoskeleton in producer and target cells are required for proper targeting of viral and cellular components during synapse formation, yet little is known about how these processes are regulated, particularly within the producer cell. Since ezrin-radixin-moesin (ERM) proteins connect F-actin with integral and peripheral membrane proteins, are incorporated into virions, and interact with cellular components of the virological presynapse, we hypothesized that they play roles during the late stage of HIV-1 replication. Here we document that phosphorylated (i.e., active) ezrin specifically accumulates at the HIV-1 presynapse in T cell lines and primary CD4؉ lymphocytes. To investigate whether ezrin supports virus transmission, we sought to ablate ezrin expression in producer cells. While cells did not tolerate a complete knockdown of ezrin, even a modest reduction of ezrin expression (ϳ50%) in HIV-1-producing cells led to the release of particles with impaired infectivity. Further, when cocultured with uninfected target cells, ezrin-knockdown producer cells displayed reduced accumulation of the tetraspanin CD81 at the synapse and fused more readily with target cells, thus forming syncytia. Such an outcome likely is not optimal for virus dissemination, as evidenced by the fact that, in vivo, only relatively few infected cells form syncytia. Thus, ezrin likely helps secure efficient virus spread not only by enhancing virion infectivity but also by preventing excessive membrane fusion at the virological synapse. IMPORTANCEWhile viruses, in principal, can propagate through successions of syncytia, HIV-1-infected cells in the majority of cases do not fuse with potential target cells during viral transmission. This mode of spread is coresponsible for key features of HIV-1 pathogenesis, including killing of bystander cells and establishment of latently infected T lymphocytes. Here we identify the ERM protein family member ezrin as a cellular factor that contributes to the inhibition of cell-cell fusion and thus to suppressing excessive syncytium formation. Our analyses further suggest that ezrin, which connects integral membrane proteins with actin, functions in concert with CD81, a member of the tetraspanin family of proteins. Additional evidence, documented here and elsewhere, suggests that ezrin and CD81 cooperate to prevent cytoskeleton rearrangements that need to take place during the fusion of cellular membranes. HIV-1 is transferred to uninfected cells most efficiently if infected and uninfected cells directly contact each other (for a recent review, see reference 1). Such cell-to-cell transmission requires not only successful assembly and release of viral particles from infected (producer) cells followed by their entry into adjoining uninfected (target) cells (2), but also necessitat...

HIV-1 replication normally requires Vif-mediated neutralization of APOBEC3 antiviral enzymes. Viruses lacking Vif succumb to deamination-dependent and -independent restriction processes. Here, HIV-1 adaptation studies were leveraged to ask whether viruses with an irreparable vif deletion could develop resistance to restrictive levels of APOBEC3G. Several resistant viruses were recovered with multiple amino acid substitutions in Env, and these changes alone are sufficient to protect Vif-null viruses from APOBEC3G-dependent restriction in T cell lines. Env adaptations cause decreased fusogenicity, which results in higher levels of Gag-Pol packaging. Increased concentrations of packaged Pol in turn enable faster virus DNA replication and protection from APOBEC3G-mediated hypermutation of viral replication intermediates. Taken together, these studies reveal that a moderate decrease in one essential viral activity, namely Env-mediated fusogenicity, enables the virus to change other activities, here, Gag-Pol packaging during particle production, and thereby escape restriction by the antiviral factor APOBEC3G. We propose a new paradigm in which alterations in viral homeostasis, through compensatory small changes, constitute a general mechanism used by HIV-1 and other viral pathogens to escape innate antiviral responses and other inhibitions including antiviral drugs.

Tetraspanins constitute a family of cellular proteins that organize various membrane-based processes. Several members of this family, including CD81, are actively recruited by HIV-1 Gag to viral assembly and release sites. Despite their enrichment at viral exit sites, the overall levels of tetraspanins are decreased in HIV-1-infected cells. Here, we identify Vpu as the main viral determinant for tetraspanin downregulation. We also show that reduction of CD81 levels by Vpu is not a by-product of CD4 or BST-2/ tetherin elimination from the surfaces of infected cells and likely occurs through an interaction between Vpu and CD81. Finally, we document that Vpu-mediated downregulation of CD81 from the surfaces of infected T cells can contribute to preserving the infectiousness of viral particles, thus revealing a novel Vpu function that promotes virus propagation by modulating the host cell environment. IMPORTANCEThe HIV-1 accessory protein Vpu has previously been shown to downregulate various host cell factors, thus helping the virus to overcome restriction barriers, evade immune attack, and maintain the infectivity of viral particles. Our study identifies tetraspanins as an additional group of host factors whose expression at the surfaces of infected cells is lowered by Vpu. While the downregulation of these integral membrane proteins, including CD81 and CD82, likely affects more than one function of HIV-1-infected cells, we document that Vpu-mediated lowering of CD81 levels in viral particles can be critical to maintaining their infectiousness. Tetraspanins are integral membrane proteins that span the lipid bilayer four times. The 33 members (in humans) of this protein family, by homo-and hetero-oligomerizing and by laterally interacting with other proteins and with lipids, form a web that serves as the basis for their involvement in the organization of membranes. When triggered by intra-or extracellular cues, socalled tetraspanin-enriched microdomains (TEMs) can form, and these platforms then support or modulate various membranebased processes, including cell adhesion, membrane fusion, signaling, and protein sorting. Consequently, tetraspanins play roles in a wide range of biological activities, such as fertilization, muscle formation and repair, generation of synaptic contacts at neuromuscular junctions, maintenance of skin integrity, and induction of immune responses (1-4). They are also implicated in pathologies, including cancer (e.g., metastasis [5]) and inherited disorders (6), as well as in the propagation and pathogenesis of numerous infectious agents (parasites, bacteria, and viruses) (7-11).While one member of the tetraspanin family (CD63) was shown more than 2 decades ago to be specifically acquired by HIV-1 particles released from infected cells (12)(13)(14), only during the past decade has work by several groups documented that tetraspanins play roles during different stages of the viral replication cycle (for recent reviews, see references 9 and 15). The tetraspanins CD9, CD53, CD63, CD81, CD...

Thousands of long noncoding RNAs are encoded in mammalian genomes, yet most remain uncharacterized. In this study, we functionally characterized a mouse long noncoding RNA named U90926. Analysis of U90926 RNA levels revealed minimal expression across multiple tissues at steady state. However, the expression of this gene was highly induced in macrophages and dendritic cells by TLR activation, in a p38 MAPK- and MyD88-dependent manner. To study the function of U90926, we generated U90926-deficient (U9-KO) mice. Surprisingly, we found minimal effects of U90926 deficiency in cultured macrophages. Given the lack of macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol, associates with ribosomes, and contains an open reading frame that encodes a novel glycosylated protein (termed U9-ORF), which is secreted from the cell. An in vivo model of endotoxic shock revealed that, in comparison with wild type mice, U9-KO mice exhibited increased sickness responses and mortality. Mechanistically, serum levels of IL-6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that U90926 expression is protective during endotoxic shock, potentially mediated by the paracrine and/or endocrine actions of the novel U9-ORF protein secreted by activated myeloid cells.