Background: Laboratory of genetics and physiology 2 (LGP2) is a cytoplasmic RNA receptor required for innate antiviral signaling. Results:LGP2 uses ATP hydrolysis to diversify RNA recognition and enhance antiviral signaling. Conclusion:LGP2 mediates antiviral responses by ATP-enhanced RNA recognition. Significance: This study reveals a novel property of LGP2 providing a mechanistic basis for its positive role in antiviral signaling.

Following cell entry, the RNA genome of HIV-1 is reverse transcribed into double-stranded DNA that ultimately integrates into the host cell genome to establish the provirus. These early phases of infection are notably vulnerable to suppression by a collection of cellular anti-viral effectors, called restriction or resistance factors. The host anti-viral protein APOBEC3G (A3G) antagonizes the early steps of HIV-1 infection through the combined effects of inhibiting viral cDNA production, and cytidine-to-uridine driven hypermutation of this cDNA. In seeking to address the underlying molecular mechanism for inhibited cDNA synthesis, we developed a deep sequencing strategy to characterize nascent reverse transcription products and their precise 3'-termini in HIV-1 infected T cells. Our results demonstrate site- and sequence-independent interference with reverse transcription, which requires A3G’s specific interaction with reverse transcriptase (RT) itself. This approach also established, contrary to current ideas, that cellular uracil base excision repair (UBER) enzymes target and cleave A3G-edited uridine-containing viral cDNA. Together, these findings yield further insights into the regulatory interplay between RT, A3G and the cellular DNA repair machinery, and identify the suppression of HIV-1 RT by a directly interacting host protein as a new cell-mediated anti-viral mechanism.

bThe Vif protein of human immunodeficiency virus type 1 (HIV-1) promotes viral replication by downregulation of the cell-encoded, antiviral APOBEC3 proteins. These proteins exert their suppressive effects through the inhibition of viral reverse transcription as well as the induction of cytidine deamination within nascent viral cDNA. Importantly, these two effects have not been characterized in detail in human CD4؉ T cells, leading to controversies over their possible contributions to viral inhibition in the natural cell targets of HIV-1 replication. Here we use wild-type and Vif-deficient viruses derived from the CD4 ؉ T cells of multiple donors to examine the consequences of APOBEC3 protein function at natural levels of expression. We demonstrate that APOBEC3 proteins impart a profound deficiency to reverse transcription from the initial stages of cDNA synthesis, as well as excessive cytidine deamination (hypermutation) of the DNAs that are synthesized. Experiments using viruses from transfected cells and a novel method for mapping the 3= termini of cDNAs indicate that the inhibition of reverse transcription is not limited to a few specific sites, arguing that APOBEC3 proteins impede enzymatic processivity. Detailed analyses of mutation spectra in viral cDNA strongly imply that one particular APOBEC3 protein, APOBEC3G, provides the bulk of the antiviral phenotype in CD4 ؉ T cells, with the effects of APOBEC3F and APOBEC3D being less significant. Taken together, we conclude that the dual mechanisms of action of APOBEC3 proteins combine to deliver more effective restriction of HIV-1 than either function would by itself.

Interferons (IFNs) mediate cellular defence against viral pathogens by upregulation of interferon-stimulated genes (ISGs) whose products interact with viral components or alter cellular physiology to suppress viral replication ( 1 – 3 ). Among the ISGs that can inhibit influenza A virus (IAV) ( 4 ) are the myxovirus resistance 1 (MX1) GTPase ( 5 ) and IFN-induced transmembrane protein 3 (IFITM3) ( 6 , 7 ). Here we use ectopic expression and gene knock-out to demonstrate that the IFN-inducible 219 amino acid short isoform of human nuclear receptor coactivator 7 (NCOA7) is an inhibitor of IAV as well as other viruses that enter the cell by endocytosis, including hepatitis C virus (HCV). NCOA7 interacts with the vacuolar H + -ATPase (V-ATPase) and its expression promotes cytoplasmic vesicle acidification, lysosomal protease activity and the degradation of endocytosed antigen. Step-wise dissection of the IAV entry pathway demonstrates that NCOA7 inhibits fusion of the viral and endosomal membranes and subsequent nuclear translocation of viral ribonucleoproteins (vRNPs). NCOA7, therefore, provides a mechanism for immune regulation of endo-lysosomal physiology that not only suppresses viral entry into the cytosol from this compartment but may also regulate other V-ATPase-associated cellular processes such as physiological adjustments to nutritional status, or the maturation and function of antigen presenting cells.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-stimulated gene (ISG) and was recently identified as a late postentry suppressor of human immunodeficiency virus type 1 (HIV-1) infection, inhibiting the nuclear accumulation of viral cDNAs. Although the HIV-1 capsid (CA) protein is believed to be the viral determinant of MX2-mediated inhibition, the precise mechanism of antiviral action remains unclear. The MX family of dynamin-like GTPases also includes MX1/MXA, a well-studied inhibitor of a range of RNA and DNA viruses, including influenza A virus (FLUAV) and hepatitis B virus but not retroviruses. MX1 and MX2 are closely related and share similar domain architectures and structures. However, MX2 possesses an extended N terminus that is essential for antiviral function and confers anti-HIV-1 activity on MX1 [MX1(NMX2)]. Higher-order oligomerization is required for the antiviral activity of MX1 against FLUAV, with current models proposing that MX1 forms ring structures that constrict around viral nucleoprotein complexes. Here, we performed structure-function studies to investigate the requirements for oligomerization of both MX2 and chimeric MX1(NMX2) for the inhibition of HIV-1 infection. The oligomerization state of mutated proteins with amino acid substitutions at multiple putative oligomerization interfaces was assessed using a combination of covalent cross-linking and coimmunoprecipitation. We show that while monomeric MX2 and MX1(NMX2) mutants are not antiviral, higher-order oligomerization does not appear to be required for full antiviral activity of either protein. We propose that lower-order oligomerization of MX2 is sufficient for the effective inhibition of HIV-1.IMPORTANCE Interferon plays an important role in the control of virus replication during acute infection in vivo. Recently, cultured cell experiments identified human MX2 as a key effector in the interferon-mediated postentry block to HIV-1 infection. MX2 is a member of a family of large dynamin-like GTPases that includes MX1/MXA, a closely related interferon-inducible inhibitor of several viruses, including FLUAV, but not HIV-1. MX GTPases form higher-order oligomeric structures, and the oligomerization of MX1 is required for inhibitory activity against many of its viral targets. Through structure-function studies, we report that monomeric mutants of MX2 do not inhibit HIV-1. However, in contrast to MX1, oligomerization beyond dimer assembly does not seem to be required for the antiviral activity of MX2, implying that fundamental differences exist between the antiviral mechanisms employed by these closely related proteins.

Type I interferons (IFNs), including IFN-α, upregulate an array of IFN-stimulated genes (ISGs) and potently suppress Human immunodeficiency virus type 1 (HIV-1) infectivity in CD4+ T cells, monocyte-derived macrophages, and dendritic cells. Recently, we and others identified ISG myxovirus resistance 2 (MX2) as an inhibitor of HIV-1 nuclear entry. However, additional antiviral blocks exist upstream of nuclear import, but the ISGs that suppress infection, e.g., prior to (or during) reverse transcription, remain to be defined. We show here that the HIV-1 CA mutations N74D and A105T, both of which allow escape from inhibition by MX2 and the truncated version of cleavage and polyadenylation specific factor 6 (CPSF6), as well as the cyclophilin A (CypA)-binding loop mutation P90A, all increase sensitivity to IFN-α-mediated inhibition. Using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, we demonstrate that the IFN-α hypersensitivity of these mutants in THP-1 cells is independent of MX2 or CPSF6. As expected, CypA depletion had no additional effect on the behavior of the P90A mutant but modestly increased the IFN-α sensitivity of wild-type virus. Interestingly, the infectivity of wild-type or P90A virus could be rescued from the MX2-independent IFN-α-induced blocks in THP-1 cells by treatment with cyclosporine (Cs) or its nonimmunosuppressive analogue SDZ-NIM811, indicating that Cs-sensitive host cell cyclophilins other than CypA contribute to the activity of IFN-α-induced blocks. We propose that cellular interactions with incoming HIV-1 capsids help shield the virus from recognition by antiviral effector mechanisms. Thus, the CA protein is a fulcrum for the dynamic interplay between cell-encoded functions that inhibit or promote HIV-1 infection.IMPORTANCE HIV-1 is the causative agent of AIDS. During acute HIV-1 infection, numerous proinflammatory cytokines are produced, including type I interferons (IFNs). IFNs can limit HIV-1 replication by inducing the expression of a set of antiviral genes that inhibit HIV-1 at multiple steps in its life cycle, including the postentry steps of reverse transcription and nuclear import. This is observed in cultured cell systems, as well as in clinical trials in HIV-1-infected patients. The identities of the cellular antiviral factors, their viral targets, and the underpinning mechanisms are largely unknown. We show here that the HIV-1 Capsid protein plays a central role in protecting the virus from IFN-induced inhibitors that block early postentry steps of infection. We further show that host cell cyclophilins play an important role in regulating these processes, thus highlighting the complex interplay between antiviral effector mechanisms and viral survival.

BackgroundThe deoxynucleotide-triphosphate (dNTP) hydrolase sterile alpha motif domain and HD domain 1 (SAMHD1) is a nuclear protein that inhibits HIV-1 infection in myeloid cells as well as quiescent CD4 T-cells, by decreasing the intracellular dNTP concentration below a level that is required for efficient reverse transcription. The Vpx proteins of the SIVSMM/HIV-2 lineage of lentiviruses bind SAMHD1 and recruit an ubiquitin ligase, leading to polyubiquitination and proteasomal degradation.ResultsHere, we have investigated the importance of nuclear localization for SAMHD1′s antiviral function as well as its sensitivity to the Vpx protein of SIVMAC. Using GST pull down assays, as well as RNA silencing approaches, we show that SAMHD1 preferentially uses karyopherin α2 (KPNA2) and a classical N-terminal nuclear localization signal (14KRPR17) to enter the nucleus. Reduction of karyopherin β1 (KPNB1) or KPNA2 by RNAi also led to cytoplasmic re-distribution of SAMHD1. Using primary human monocyte-derived macrophages (MDM), a cell type in which SAMHD1 is naturally expressed to high levels, we demonstrate that nuclear localization is not required for its antiviral activity. Cytoplasmic SAMHD1 still binds to VpxMAC, is efficiently polyubiquitinated, but is not degraded. We also find that VpxMAC-induced SAMHD1 degradation was partially reversed by ubiquitin carrying the K48R or K11R substitution mutations, suggesting involvement of K48 and K11 linkages in SAMHD1 polyubiquitination. Using ubiquitin K-R mutants also revealed differences in the ubiquitin linkages between wild type and cytoplasmic forms of SAMHD1, suggesting a potential association with the resistance of cytoplasmic SAMHD1 to VpxMAC induced degradation.ConclusionsOur work extends published observations on SAMHD1 nuclear localization to a natural cell type for HIV-1 infection, identifies KPNA2/KPNB1 as cellular proteins important for SAMHD1 nuclear import, and indicates that components of the nuclear proteasomal degradation machinery are required for SAMHD1 degradation.

Human immunodeficiency virus type 1 (HIV-1) infection of dividing and nondividing cells involves regulatory interactions with the nuclear pore complex (NPC), followed by translocation to the nucleus and preferential integration into genomic areas in proximity to the inner nuclear membrane (INM). To identify host proteins that may contribute to these processes, we performed an overexpression screen of known membrane-associated NE proteins. We found that the integral transmembrane proteins SUN1/UNC84A and SUN2/UNC84B are potent or modest inhibitors of HIV-1 infection, respectively, and that suppression corresponds to defects in the accumulation of viral cDNA in the nucleus. While laboratory strains (HIV-1NL4.3 and HIV-1IIIB) are sensitive to SUN1-mediated inhibition, the transmitted founder viruses RHPA and ZM247 are largely resistant. Using chimeric viruses, we identified the HIV-1 capsid (CA) protein as a major determinant of sensitivity to SUN1, and in vitro-assembled capsid-nucleocapsid (CANC) nanotubes captured SUN1 and SUN2 from cell lysates. Finally, we generated SUN1−/− and SUN2−/− cells by using CRISPR/Cas9 and found that the loss of SUN1 had no effect on HIV-1 infectivity, whereas the loss of SUN2 had a modest suppressive effect. Taken together, these observations suggest that SUN1 and SUN2 may function redundantly to modulate postentry, nuclear-associated steps of HIV-1 infection.IMPORTANCE HIV-1 causes more than 1 million deaths per year. The life cycle of HIV-1 has been studied extensively, yet important steps that occur between viral capsid release into the cytoplasm and the expression of viral genes remain elusive. We propose here that the INM components SUN1 and SUN2, two members of the linker of nucleoskeleton and cytoskeleton (LINC) complex, may interact with incoming HIV-1 replication complexes and affect key steps of infection. While overexpression of these proteins reduces HIV-1 infection, disruption of the individual SUN2 and SUN1 genes leads to a mild reduction or no effect on infectivity, respectively. We speculate that SUN1/SUN2 may function redundantly in early HIV-1 infection steps and therefore influence HIV-1 replication and pathogenesis.