Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes. Interferon-induced transmembrane proteins (IFITMs) block infection of many enveloped viruses, including the influenza A virus (IAV) that enters from late endosomes. IFITMs are thought to prevent virus hemifusion (merger of contacting leaflets without formation of a fusion pore) by altering the properties of cell membranes. Here we performed single IAV imaging and found that IFITM3 did not interfere with hemifusion, but prevented complete fusion. Also, contrary to a current view that excess cholesterol in late endosomes of IFITM3-expressing cells inhibits IAV entry, we show that cholesterol-laden endosomes are permissive for virus fusion. The ability of IFITM3 to block the formation of fusion pores implies that this protein stabilizes the cytoplasmic leaflet of endosomal membranes, either directly or indirectly, through altering its physical properties. IFITM3 may also redirect IAV to a non-productive pathway by promoting fusion with intralumenal vesicles of late endosomes instead of their limiting membrane.

Attacking HIV by stabilizing its capsid: Current HIV treatments require drugs that must be taken daily, and care would be improved with an effective drug that is long-acting. GS-6207 (Lenacapavir) is a drug developed by Gilead Sciences that shows potential for a 6-month dosing interval and is in phase 2/3 clinical trials. Bester et al. describe structural and biophysical studies that provide a basis for the potent antiviral activity of GS-6207. The HIV capsid is cone shaped, and GS-6207 binds two neighboring capsid subunits and stabilizes the curved capsid. GS-6207 also interferes with capsid binding of cofactors that play a role in viral infection. This insight into GS-6207 activity provides a platform for the rational development of improved long-acting therapies. Science , this issue p. 360

The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4⁺ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.

The host proteins, SERINC3 and SERINC5, have been recently shown to incorporate into HIV-1 particles and compromise their ability to fuse with target cells, an effect that is antagonized by the viral Nef protein. Envelope (Env) glycoproteins from different HIV-1 isolates exhibit a broad range of sensitivity to SERINC-mediated restriction, and the mechanism by which SERINCs interfere with HIV-1 fusion remains unclear. Here, we show that incorporation of SERINC5 into virions in the absence of Nef inhibits the formation of small fusion pores between viruses and cells. Strikingly, we found that SERINC5 promotes spontaneous functional inactivation of sensitive but not resistant Env glycoproteins. Although SERINC5-Env interaction was not detected by co-immunoprecipitation, incorporation of this protein enhanced the exposure of the conserved gp41 domains and sensitized the virus to neutralizing antibodies and gp41-derived inhibitory peptides. These results imply that SERINC5 restricts HIV-1 fusion at a step prior to small pore formation by selectively inactivating sensitive Env glycoproteins, likely through altering their conformation. The increased HIV-1 sensitivity to anti-gp41 antibodies and peptides suggests that SER5 also delays refolding of the remaining fusion-competent Env trimers.

Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process. HIV-1 genome and key enzymes required for establishing productive infection are encased in a cone-shaped shell made of the capsid protein (CA). After being released into the cytosol of target cells, the cone-shaped core complex undergoes a series of carefully orchestrated steps, including uncoating (loss of CA). HIV-1 uncoating remains poorly understood, due in part to the lack of direct assays enabling studies of this process in living cells. Here, we introduce a novel strategy for labeling the HIV-1 capsid without genetically modifying the CA protein. We designed a novel fluorescent cyclophilin A construct that binds the capsid with an extremely high avidity and (1) efficiently incorporates into virions without compromising infectivity; (2) remains bound to cores after viral fusion; and (3) is lost from post-fusion cores along with CA. The novel imaging assay provides new insights into the kinetics and spatial distribution of HIV-1 uncoating in living cells.