Structural insight into HIV-1 restriction by MxB.
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ABSTRACT:
SUBMITTER: Fribourgh JL
PROVIDER: S-EPMC4252739 | biostudies-literature | 2014 Nov
REPOSITORIES: biostudies-literature
Publications
Structural insight into HIV-1 restriction by MxB.
Fribourgh Jennifer L JL Nguyen Henry C HC Matreyek Kenneth A KA Alvarez Frances Joan D FJD Summers Brady J BJ Dewdney Tamaria G TG Aiken Christopher C Zhang Peijun P Engelman Alan A Xiong Yong Y
Cell host & microbe 20141009 5
The myxovirus resistance (Mx) proteins are interferon-induced dynamin GTPases that can inhibit a variety of viruses. Recently, MxB, but not MxA, was shown to restrict HIV-1 by an unknown mechanism that likely occurs in close proximity to the host cell nucleus and involves the viral capsid. Here, we present the crystal structure of MxB and reveal determinants involved in HIV-1 restriction. MxB adopts an extended antiparallel dimer and dimerization, but not higher-ordered oligomerization, is criti ...[more]
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Project description:BackgroundInterferon-induced cellular proteins play important roles in the host response against viral infection. The Mx family of dynamin-like GTPases, which include MxA and MxB, target a wide variety of viruses. Despite considerable evidence demonstrating the breadth of antiviral activity of MxA, human MxB was only recently discovered to specifically inhibit lentiviruses. Here we assess both host and viral determinants that underlie MxB restriction of HIV-1 infection.ResultsHeterologous expression of MxB in human osteosarcoma cells potently inhibited HIV-1 infection (~12-fold), yet had little to no effect on divergent retroviruses. The anti-HIV effect manifested as a partial block in the formation of 2-long terminal repeat circle DNA and hence nuclear import, and we accordingly found evidence for an additional post-nuclear entry block. A large number of previously characterized capsid mutations, as well as mutations that abrogated integrase activity, counteracted MxB restriction. MxB expression suppressed integration into gene-enriched regions of chromosomes, similar to affects observed previously when cells were depleted for nuclear transport factors such as transportin 3. MxB activity did not require predicted GTPase active site residues or a series of unstructured loops within the stalk domain that confer functional oligomerization to related dynamin family proteins. In contrast, we observed an N-terminal stretch of residues in MxB to harbor key determinants. Protein localization conferred by a nuclear localization signal (NLS) within the N-terminal 25 residues, which was critical, was fully rescuable by a heterologous NLS. Consistent with this observation, a heterologous nuclear export sequence (NES) abolished full-length MxB activity. We additionally mapped sub-regions within amino acids 26-90 that contribute to MxB activity, finding sequences present within residues 27-50 particularly important.ConclusionsMxB inhibits HIV-1 by interfering with minimally two steps of infection, nuclear entry and post-nuclear trafficking and/or integration, without destabilizing the inherent catalytic activity of viral preintegration complexes. Putative MxB GTPase active site residues and stalk domain Loop 4 -- both previously shown to be necessary for MxA function -- were dispensable for MxB antiviral activity. Instead, we highlight subcellular localization and a yet-determined function(s) present in the unique MxB N-terminal region to be required for HIV-1 restriction.
| S-EPMC4213484 | biostudies-literature
Project description:UnlabelledThe alpha interferon (IFN-α)-inducible restriction factor myxovirus B (MxB) blocks HIV-1 infection after reverse transcription but prior to integration. MxB binds to the HIV-1 core, which is composed of capsid protein, and this interaction leads to inhibition of the uncoating process of HIV-1. Previous studies suggested that HIV-1 restriction by MxB requires binding to capsid. This work tests the hypothesis that MxB oligomerization is important for the ability of MxB to bind to the HIV-1 core. For this purpose, we modeled the structure of MxB using the published tertiary structure of MxA. The modeled structure of MxB guided our mutagenic studies and led to the discovery of several MxB variants that lose the capacity to oligomerize. In agreement with our hypothesis, MxB variants that lost the oligomerization capacity also lost the ability to bind to the HIV-1 core. MxB variants deficient for oligomerization were not able to block HIV-1 infection. Overall, our work showed that oligomerization is required for the ability of MxB to bind to the HIV-1 core and block HIV-1 infection.ImportanceMxB is a novel restriction factor that blocks infection of HIV-1. MxB is inducible by IFN-α, particularly in T cells. The current work studies the oligomerization determinants of MxB and carefully explores the contribution of oligomerization to capsid binding and restriction. This work takes advantage of the current structure of MxA and models the structure of MxB, which is used to guide structure-function studies. This work leads to the conclusion that MxB oligomerization is important for HIV-1 capsid binding and restriction.
| S-EPMC4337540 | biostudies-literature
Project description:Human dynamin-like, interferon-induced myxovirus resistance 2 (Mx2 or MxB) is a potent HIV-1 inhibitor. Antiviral activity requires both the amino-terminal region of MxB and protein oligomerization, each of which has eluded structural determination due to difficulties in protein preparation. We report that maltose binding protein-fused, full-length wild-type MxB purifies as oligomers and further self-assembles into helical arrays in physiological salt. Guanosine triphosphate (GTP), but not guanosine diphosphate, binding results in array disassembly, whereas subsequent GTP hydrolysis allows its reformation. Using cryo-electron microscopy (cryoEM), we determined the MxB assembly structure at 4.6 Å resolution, representing the first near-atomic resolution structure in the mammalian dynamin superfamily. The structure revealed previously described and novel MxB assembly interfaces. Mutational analyses demonstrated a critical role for one of the novel interfaces in HIV-1 restriction.
| S-EPMC5600524 | biostudies-literature
Project description: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 (MX2) 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.IMPORTANCE 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 MX2 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.
| S-EPMC6096802 | biostudies-literature
Project description:The type I interferon (IFN) system plays an important role in controlling herpesvirus infections, but it is unclear which IFN-mediated effectors interfere with herpesvirus replication. Here we report that human myxovirus resistance protein B (MxB, also designated Mx2) is a potent human herpesvirus restriction factor in the context of IFN. We demonstrate that ectopic MxB expression restricts a range of herpesviruses from the Alphaherpesvirinae and Gammaherpesvirinae, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), and Kaposi's sarcoma-associated herpesvirus (KSHV). MxB restriction of HSV-1 and HSV-2 requires GTPase function, in contrast to restriction of lentiviruses. MxB inhibits the delivery of incoming HSV-1 DNA to the nucleus and the appearance of empty capsids, but not the capsid delivery to the cytoplasm or tegument dissociation from the capsid. Our study identifies MxB as a potent pan-herpesvirus restriction factor which blocks the uncoating of viral DNA from the incoming viral capsid.
| S-EPMC5958057 | biostudies-literature
Project description:Viruses impose diverse and dynamic challenges on host defenses. Diversifying selection of codons and gene copy number variation are two hallmarks of genetic innovation in antiviral genes engaged in host-virus genetic conflicts. The myxovirus resistance (Mx) genes encode interferon-inducible GTPases that constitute a major arm of the cell-autonomous defense against viral infection. Unlike the broad antiviral activity of MxA, primate MxB was recently shown to specifically inhibit lentiviruses including HIV-1. We carried out detailed evolutionary analyses to investigate whether genetic conflict with lentiviruses has shaped MxB evolution in primates. We found strong evidence for diversifying selection in the MxB N-terminal tail, which contains molecular determinants of MxB anti-lentivirus specificity. However, we found no overlap between previously-mapped residues that dictate lentiviral restriction and those that have evolved under diversifying selection. Instead, our findings are consistent with MxB having a long-standing and important role in the interferon response to viral infection against a broader range of pathogens than is currently appreciated. Despite its critical role in host innate immunity, we also uncovered multiple functional losses of MxB during mammalian evolution, either by pseudogenization or by gene conversion from MxA genes. Thus, although the majority of mammalian genomes encode two Mx genes, this apparent stasis masks the dramatic effects that recombination and diversifying selection have played in shaping the evolutionary history of Mx genes. Discrepancies between our study and previous publications highlight the need to account for recombination in analyses of positive selection, as well as the importance of using sequence datasets with appropriate depth of divergence. Our study also illustrates that evolutionary analyses of antiviral gene families are critical towards understanding molecular principles that govern host-virus interactions and species-specific susceptibility to viral infection.
| S-EPMC4687636 | biostudies-literature
Project description:Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505-527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515-519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.
| S-EPMC7212661 | biostudies-literature
Project description:The interferon-inducible myxovirus resistance B (MxB) protein has been reported to inhibit HIV-1 and herpesviruses by blocking the nuclear import of viral DNA. Here, we report a new antiviral mechanism in which MxB restricts the nuclear import of HIV-1 regulatory protein Rev, and as a result, diminishes Rev-dependent expression of HIV-1 Gag protein. Specifically, MxB disrupts the interaction of Rev with the nuclear transport receptor, transportin 1 (TNPO1). Supporting this, the TNPO1-independent Rev variants become less restricted by MxB. In addition, HIV-1 can overcome this inhibition by MxB through increasing the expression of multiply spliced viral RNA and hence Rev protein. Therefore, MxB exerts its anti-HIV-1 function through interfering with the nuclear import of both viral DNA and viral Rev protein.
| S-EPMC7534208 | biostudies-literature
Project description:Tripartite motif protein isoform 5 alpha (TRIM5α) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5α recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5α induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5α PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5α PRY/SPRY, but not the human TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5α that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5α-mediated retroviral restriction.
| S-EPMC3494900 | biostudies-literature
Project description:BackgroundThe IFN-α-inducible restriction factor MxB blocks HIV-1 infection after reverse transcription but prior to integration. Genetic evidence suggested that capsid is the viral determinant for restriction by MxB. This work explores the ability of MxB to bind to the HIV-1 core, and the role of capsid-binding in restriction.ResultsWe showed that MxB binds to the HIV-1 core and that this interaction leads to inhibition of the uncoating process of HIV-1. These results identify MxB as an endogenously expressed protein with the ability to inhibit HIV-1 uncoating. In addition, we found that a benzimidazole-based compound known to have a binding pocket on the surface of the HIV-1 capsid prevents the binding of MxB to capsid. The use of this small-molecule identified the MxB binding region on the surface of the HIV-1 core. Domain mapping experiments revealed the following requirements for restriction: 1) MxB binding to the HIV-1 capsid, which requires the 20 N-terminal amino acids, and 2) oligomerization of MxB, which is mediated by the C-terminal domain provides the avidity for the interaction of MxB with the HIV-1 core.ConclusionsOverall our work establishes that MxB binds to the HIV-1 core and inhibits the uncoating process of HIV-1. Moreover, we demonstrated that HIV-1 restriction by MxB requires capsid binding and oligomerization.
| S-EPMC4145229 | biostudies-literature