Animal cells harbour multiple innate effector mechanisms that inhibit virus replication. For the pathogenic retrovirus human immunodeficiency virus type 1 (HIV-1), these include widely expressed ...restriction factors, such as APOBEC3 proteins, TRIM5-α, BST2 (refs 4, 5) and SAMHD1 (refs 6, 7), as well as additional factors that are stimulated by type 1 interferon (IFN). Here we use both ectopic expression and gene-silencing experiments to define the human dynamin-like, IFN-induced myxovirus resistance 2 (MX2, also known as MXB) protein as a potent inhibitor of HIV-1 infection and as a key effector of IFN-α-mediated resistance to HIV-1 infection. MX2 suppresses infection by all HIV-1 strains tested, has equivalent or reduced effects on divergent simian immunodeficiency viruses, and does not inhibit other retroviruses such as murine leukaemia virus. The Capsid region of the viral Gag protein dictates susceptibility to MX2, and the block to infection occurs at a late post-entry step, with both the nuclear accumulation and chromosomal integration of nascent viral complementary DNA suppressed. Finally, human MX1 (also known as MXA), a closely related protein that has long been recognized as a broadly acting inhibitor of RNA and DNA viruses, including the orthomyxovirus influenza A virus, does not affect HIV-1, whereas MX2 is ineffective against influenza virus. MX2 is therefore a cell-autonomous, anti-HIV-1 resistance factor whose purposeful mobilization may represent a new therapeutic approach for the treatment of HIV/AIDS.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Herpesvirus infections are highly prevalent in the human population and persist for life. They are often acquired subclinically but potentially progress to life-threatening diseases in ...immunocompromised individuals. The interferon system is indispensable for the control of herpesviral replication. However, the responsible antiviral effector mechanisms are not well characterized. The type I interferon-induced, human myxovirus resistance 2 (
) gene product MxB, a dynamin-like large GTPase, has recently been identified as a potent inhibitor of HIV-1. We now show that MxB also interferes with an early step of herpesvirus replication, affecting alpha-, beta-, and gammaherpesviruses before or at the time of immediate early gene expression. Defined MxB mutants influencing GTP binding and hydrolysis revealed that the effector mechanism against herpesviruses is thoroughly different from that against HIV-1. Overall, our findings demonstrate that MxB serves as a broadly acting intracellular restriction factor that controls the establishment of not only retrovirus but also herpesvirus infection of all three subfamilies.
Human herpesviruses pose a constant threat to human health. Reactivation of persisting herpesvirus infections, particularly in immunocompromised individuals and the elderly, can cause severe diseases, such as zoster, pneumonia, encephalitis, or cancer. The interferon system is relevant for the control of herpesvirus replication as exemplified by fatal disease outcomes in patients with primary immunodeficiencies. Here, we describe the interferon-induced, human
gene product MxB as an efficient restriction factor of alpha-, beta-, and gammaherpesviruses. MxB has previously been described as an inhibitor of HIV-1. Importantly, our mutational analyses of MxB reveal an antiviral mechanism of herpesvirus restriction distinct from that against HIV-1. Thus, the dynamin-like MxB GTPase serves as a broadly acting intracellular restriction factor that controls retrovirus as well as herpesvirus infections.
Persistent infection with the hepatitis C virus (HCV) is a major risk factor for the development of liver cirrhosis and hepatocellular carcinoma. With an estimated about 3% of the world population ...infected with this virus, the lack of a prophylactic vaccine and a selective therapy, chronic hepatitis C currently is a main indication for liver transplantation. The establishment of cell-based replication and virus production systems has led to first insights into the functions of HCV proteins. However, the role of nonstructural protein 5A (NS5A) in the viral replication cycle is so far not known. NS5A is a membrane-associated RNA-binding protein assumed to be involved in HCV RNA replication. Its numerous interactions with the host cell suggest that NS5A is also an important determinant for pathogenesis and persistence. In this study we show that NS5A is a key factor for the assembly of infectious HCV particles. We specifically identify the C-terminal domain III as the primary determinant in NS5A for particle formation. We show that both core and NS5A colocalize on the surface of lipid droplets, a proposed site for HCV particle assembly. Deletions in domain III of NS5A disrupting this colocalization abrogate infectious particle formation and lead to an enhanced accumulation of core protein on the surface of lipid droplets. Finally, we show that mutations in NS5A causing an assembly defect can be rescued by trans-complementation. These data provide novel insights into the production of infectious HCV and identify NS5A as a major determinant for HCV assembly. Since domain III of NS5A is one of the most variable regions in the HCV genome, the results suggest that viral isolates may differ in their level of virion production and thus in their level of fitness and pathogenesis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The HIV-1 genome enters cells inside a shell comprised of capsid (CA) protein. Variation in CA sequence alters HIV-1 infectivity and escape from host restriction factors. However, apart from the ...Cyclophilin A-binding loop, CA has no known interfaces with which to interact with cellular cofactors. Here we describe a novel protein-protein interface in the N-terminal domain of HIV-1 CA, determined by X-ray crystallography, which mediates both viral restriction and host cofactor dependence. The interface is highly conserved across lentiviruses and is accessible in the context of a hexameric lattice. Mutation of the interface prevents binding to and restriction by CPSF6-358, a truncated cytosolic form of the RNA processing factor, cleavage and polyadenylation specific factor 6 (CPSF6). Furthermore, mutations that prevent CPSF6 binding also relieve dependence on nuclear entry cofactors TNPO3 and RanBP2. These results suggest that the HIV-1 capsid mediates direct host cofactor interactions to facilitate viral infection.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 ...was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recently, we demonstrated that sterile α motif and HD domain containing protein 1 (SAMHD1) is a major barrier in acute myelogenous leukemia (AML) cells to the cytotoxicity of cytarabine (ara-C), the ...most important drug in AML treatment. Ara-C is intracellularly converted by the canonical dNTP synthesis pathway to ara-CTP, which serves as a substrate but not an allosteric activator of SAMHD1. Using an AML mouse model, we show here that wild type but not catalytically inactive SAMHD1 reduces ara-C treatment efficacy in vivo. Expanding the clinically relevant substrates of SAMHD1, we demonstrate that THP-1 CRISPR/Cas9 cells lacking a functional SAMHD1 gene showed increased sensitivity to the antimetabolites nelarabine, fludarabine, decitabine, vidarabine, clofarabine, and trifluridine. Within this Extra View, we discuss and build upon both these and our previously reported findings, and propose SAMHD1 is likely active against a variety of nucleoside analog antimetabolites present in anti-cancer chemotherapies. Thus, SAMHD1 may constitute a promising target to improve a wide range of therapies for both hematological and non-haematological malignancies.
The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G) and APOBEC3F (A3F), act as potent ...human immunodeficiency virus type-1 (HIV-1) restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC) region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC) fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rational design in combination with a screening process was used to develop affinity polymers for a specific binding site on the surface of immunoglobulin G (IgG) proteins. The concept starts with ...the identification of critical amino acid residues on the protein interface and their topological arrangement. Appropriate binding monomers were subsequently synthesized. Together with a sugar monomer (2–5 equiv) for water solubility and a dansyl monomer (0.5 equiv) as a fluorescent label, they were subjected in aqueous solution to linear radical copolymerization in various compositions (e.g., azobisisobutyronitrile (AIBN), homogeneous water/DMF mixtures). After ultrafiltration and lyophilization, colorless dry water‐soluble powders were obtained. NMR spectroscopic and gel permeation chromatography (GPC) characterization indicated molecular weights between 30 and 500 kD and confirmed retention of monomer composition as well as the absence of monomers. In a competitive enzyme‐linked immunosorbent assay (ELISA) screen of the polymer libraries (20–50 members), few copolymers qualified as strong and selective binders for the protein A binding site on the Fc fragment of the antibody. Their monomer composition precisely reflected the critical amino acids found at the interface. The simple combination of a charged and a nonpolar binding monomer sufficed for selective submicromolar IgG recognition by the synthetic polymer. Affinities were confirmed by fluorescence titrations; they increased with decreasing salt load but remained largely unaltered at lowered pH. Other proteins, including those of similar size and isoelectric point (pI), were bound 10–1000 times less tightly. This example indicates that interaction domains in other proteins may also be targeted by synthetic polymers if their comonomer composition reflects the nature and arrangement of amino acid residues on the protein surface.
Some like it hot: Designed affinity polymers recognize the hot spot on a protein surface by virtue of their carefully selected binding monomers. Here, it is the protein A binding site on the Fc fragment of immunoglobulin G proteins (see figure), which is essential for medicinal antibody purification.
The recently identified restriction factor tetherin/BST-2/CD317 is an interferon-inducible trans-membrane protein that restricts HIV-1 particle release in the absence of the HIV-1 countermeasure ...viral protein U (Vpu). It is known that Tantalus monkey CV1 cells can be rendered non-permissive to HIV-1 release upon stimulation with type 1 interferon, despite the presence of Vpu, suggesting species-specific sensitivity of tetherin proteins to viral countermeasures such as Vpu. Here we demonstrate that Tantalus monkey tetherin restricts HIV-1 by nearly two orders of magnitude, but in contrast to human tetherin the Tantalus protein is insensitive to HIV-1 Vpu. We have investigated tetherin's sensitivity to Vpu using positive selection analyses, seeking evidence for evolutionary conflict between tetherin and viral countermeasures. We provide evidence that tetherin has undergone positive selection during primate evolution. Mutation of a single amino acid (showing evidence of positive selection) in the trans-membrane cap of human tetherin to that in Tantalus monkey (T45I) substantially impacts on sensitivity to HIV-1 Vpu, but not on antiviral activity. Finally, we provide evidence that cellular steady state levels of tetherin are substantially reduced by Vpu, and that the T45I mutation abrogates this effect. This study provides evidence that tetherin is important in protecting mammals against viral infection, and that the HIV-1 Vpu-mediated countermeasure is specifically adapted to act against human tetherin. It also emphasizes the power of selection analyses to illuminate the molecular details of host-virus interactions. This work suggests that tetherin binding agents might protect it from viral encoded countermeasures and thus make powerful antivirals.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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