RESUMO
IMPORTANCE: Herpesviruses present a major global disease burden. Understanding the host cell mechanisms that block viral infections, as well as how viruses can evolve to counteract these host defenses, is critically important for understanding viral disease pathogenesis. This study reveals that the major human variant of the antiviral protein myxovirus resistance protein B (MxB) inhibits the human pathogen herpes simplex virus (HSV-1), whereas a minor human variant and orthologous MxB genes from even closely related primates do not. Thus, in contrast to the many antagonistic virus-host interactions in which the virus is successful in thwarting the host's defense systems, here the human gene appears to be at least temporarily winning at this interface of the primate-herpesvirus evolutionary arms race. Our findings further show that a polymorphism at amino acid 83 in a small fraction of the human population is sufficient to abrogate MxB's ability to inhibit HSV-1, which could have important implications for human susceptibility to HSV-1 pathogenesis.
Assuntos
Herpesvirus Humano 1 , Interações entre Hospedeiro e Microrganismos , Proteínas de Resistência a Myxovirus , Polimorfismo Genético , Animais , Humanos , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 1/fisiologia , Interações entre Hospedeiro e Microrganismos/genética , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Primatas/genética , Primatas/virologia , Especificidade da EspécieRESUMO
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.
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Capsídeo/metabolismo , Núcleo Celular/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Multimerização Proteica , Transporte Ativo do Núcleo Celular , Núcleo Celular/genética , Núcleo Celular/virologia , Células HEK293 , Infecções por HIV/genética , HIV-1/genética , Células HeLa , Humanos , Proteínas de Resistência a Myxovirus/genética , Sinais de Exportação Nuclear , Prolina , Ligação ProteicaRESUMO
Mx proteins are interferon (IFN) type I (α/ß)- and type III (λ)-induced effector proteins with intrinsic antiviral activity. Mammalian Mx proteins show different subcellular localizations and distinct yet partially overlapping viral specificities. However, the precise mechanism(s) of antiviral action are still unresolved. Human MxA accumulates in the cytoplasm and inhibits a wide variety of RNA and DNA viruses, among them influenza A virus (IAV). In contrast, MxB, the second human Mx protein, localizes via its amino (N) terminus to the outer nuclear membrane at or near nuclear pores and inhibits the nuclear import of incoming human immunodeficiency viruses (HIV) and herpesviruses, but not that of IAV. Here, we evaluated whether the antiviral specificity of MxB is determined by its subcellular localization. For this purpose, we redirected MxB to the nucleus or cytoplasm by either attaching a nuclear localization signal to its N terminus or by exchanging the N terminus of MxB with that of MxA. Interestingly, ectopic expression of these MxB variants in the nucleus or in the cytoplasm rendered the host cells resistant to IAV, revealing that the capacity of MxB to block IAV replication critically depends on the site where the protein accumulates in the infected cell. Furthermore, coimmunoprecipitation (co-IP) assays demonstrated that MxB physically interacted with the nucleoprotein (NP) of IAV. Taken together, the data indicate that the subcellular localization of the MxB protein plays a pivotal role in determining its antiviral specificity.IMPORTANCE The interferon system plays a pivotal role in the defense against viral infections. The dynamin-related Mx proteins form a small family of interferon-induced effector proteins with distinct antiviral specificities and subcellular localizations. So far, it is not clear whether the different virus specificities of Mx proteins are the result of distinct mechanisms of action or are due rather to their different subcellular localization. We show here that the human MxB protein, normally localized to the outer membrane of the cell nucleus, acquires antiviral activity against IAV when redirected to the nucleus or cytoplasm, subcellular sites where other members of the Mx protein family efficiently interfere with IAV replication. Our findings thus strongly suggest that Mx proteins act primarily through a common mechanism and that their viral specificity is at least in part determined by their individual subcellular localization.
Assuntos
Antivirais/metabolismo , Vírus da Influenza A/efeitos dos fármacos , Proteínas de Resistência a Myxovirus/metabolismo , Transporte Ativo do Núcleo Celular , Antivirais/farmacologia , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células HEK293 , Células HeLa , Humanos , Vírus da Influenza A/metabolismo , Interferons/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/farmacologia , Nucleoproteínas , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: The human myxovirus resistance 2 (Mx2/MxB) protein was originally found to regulate cytoplasmic-nuclear transport but was recently reported to restrict HIV-1 replication by binding to HIV-1 capsid (CA), preventing uncoating, the nuclear import of pre-integration complex (PIC) and viral DNA integration. This work explores the mechanisms of MxB-mediated HIV-1 inhibition. RESULTS: We demonstrated that MxB represses NUP358-mediated PIC nuclear import and HIV-1 replication. Moreover, MxB's effects on PIC nuclear import and HIV-1 replication depend critically on cofactor cleavage and polyadenylation specificity factor subunit 6 (CPSF6). MxB binds nucleoporin NUP358, blocks NUP358-CA interaction, thereby impeding the nuclear import of HIV-1 PIC with CPSF6 binding to PIC. More intriguingly, CPSF6's role in nuclear import depends on MxB, being a facilitator of HIV-1 nuclear import on its own, but becoming an inhibitor when MxB is present. CONCLUSIONS: Our work establishes that MxB impedes the NUP358-mediated HIV-1 nuclear import and viral replication cooperatively with CPSF6.
Assuntos
Núcleo Celular/metabolismo , HIV-1/fisiologia , Chaperonas Moleculares/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Transporte Ativo do Núcleo Celular , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Humanos , Indóis/farmacologia , Chaperonas Moleculares/genética , Proteínas de Resistência a Myxovirus/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Ligação Proteica , Replicação Viral , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Type I interferon (IFN) inhibits viruses by inducing the expression of antiviral proteins. The IFN-induced myxovirus resistance B (MxB) protein has been reported to inhibit a limited number of viruses, including HIV-1 and herpesviruses, but its antiviral coverage remains to be explored further. Here we show that MxB interferes with RNA replication of hepatitis C virus (HCV) and significantly inhibits viral replication in a cyclophilin A (CypA)-dependent manner. Our data further show that MxB interacts with the HCV protein NS5A, thereby impairing NS5A interaction with CypA and NS5A localization to the endoplasmic reticulum, two events essential for HCV RNA replication. Interestingly, we found that MxB significantly inhibits two additional CypA-dependent viruses of the Flaviviridae family, namely, Japanese encephalitis virus and dengue virus, suggesting a potential link between virus dependence on CypA and virus susceptibility to MxB inhibition. Collectively, these data have identified MxB as a key factor behind IFN-mediated suppression of HCV infection, and they suggest that other CypA-dependent viruses may also be subjected to MxB restriction.IMPORTANCE Viruses of the Flaviviridae family cause major illness and death around the world and thus pose a great threat to human health. Here we show that IFN-inducible MxB restricts several members of the Flaviviridae, including HCV, Japanese encephalitis virus, and dengue virus. This finding not only suggests an active role of MxB in combating these major pathogenic human viruses but also significantly expands the antiviral spectrum of MxB. Our study further strengthens the link between virus dependence on CypA and susceptibility to MxB restriction and also suggests that MxB may employ a common mechanism to inhibit different viruses. Elucidating the antiviral functions of MxB advances our understanding of IFN-mediated host antiviral defense and may open new avenues to the development of novel antiviral therapeutics.
Assuntos
Ciclofilina A/farmacologia , Hepacivirus/fisiologia , Interferons/farmacologia , Proteínas de Resistência a Myxovirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Ciclosporina/farmacologia , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Hepacivirus/efeitos dos fármacos , Humanos , Proteínas de Resistência a Myxovirus/genética , Ligação Proteica/efeitos dos fármacos , Células VeroRESUMO
Interferons limit viral replication by inducing intracellular restriction factors, such as the GTPase MxB (also designated MX2), which inhibits HIV-1 and, as recently shown, herpesviruses. Inhibition of these viruses occurs at ill-defined steps after viral entry and requires formation of MxB dimers or oligomers, but GTP hydrolysis is needed only for blocking herpesviruses. Together with previous findings on related MxA, the new research on MxB highlights the mechanistic diversity by which MX proteins interfere with viral replication.
Assuntos
HIV-1/fisiologia , Herpesviridae/fisiologia , Interferons/farmacologia , Proteínas de Resistência a Myxovirus/metabolismo , HIV-1/efeitos dos fármacos , Herpesviridae/efeitos dos fármacos , Humanos , Modelos Moleculares , Proteínas de Resistência a Myxovirus/química , Conformação Proteica , Multimerização Proteica , Regulação para Cima , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Type I interferon inhibits viruses through inducing the expression of antiviral proteins, including the myxovirus resistance (Mx) proteins. Compared to the human MxA protein, which inhibits a wide range of viruses, the MxB protein has been reported to specifically inhibit primate lentiviruses, including HIV-1, and herpesviruses. Further, the role of endogenous MxB in alpha interferon-mediated inhibition of HIV-1 infection was questioned by a recent study showing that MxB knockout did not increase the level of infection by HIV-1 which carried the G protein of vesicular stomatitis virus (VSV), allowing infection of CD4-negative HT1080 cells. In order to further examine the anti-HIV-1 activity of endogenous MxB, we have used CRISPR/Cas9 to deplete MxB in different cell lines and observed a substantial restoration of HIV-1 infection in the presence of alpha interferon treatment. However, this rescue effect of MxB knockout became much less pronounced when infection was performed with HIV-1 carrying the VSV G protein. Interestingly, a CRISPR/Cas9 knockout screen of alpha interferon-stimulated genes in U87-MG cells revealed that the genes for interferon-induced transmembrane protein 2 (IFITM2) and IFITM3 inhibited VSV G-pseudotyped HIV-1 much more strongly than the rest of the genes tested, including the gene for MxB. Therefore, our results demonstrate the importance of MxB in alpha interferon-mediated inhibition of HIV-1 infection, which, however, can be underestimated if infection is performed with VSV G protein-pseudotyped HIV-1, due to the high sensitivity of VSV G-mediated infection to inhibition by IFITM proteins.IMPORTANCE The results of this study reconcile the controversial reports regarding the anti-HIV-1 function of alpha interferon-induced MxB protein. In addition to the different cell types that may have contributed to the different observations, our data also suggest that VSV G protein-pseudotyped HIV-1 is much less inhibited by alpha interferon-induced MxB than HIV-1 itself is. Our results clearly demonstrate an important contribution of MxB to alpha interferon-mediated inhibition of HIV-1 in CD4+ T cells, which calls for using HIV-1 target cells and wild-type virus to test the relevance of the anti-HIV-1 activity of endogenous MxB and other restriction factors.
Assuntos
Antivirais/metabolismo , Infecções por HIV/imunologia , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Fatores Imunológicos/metabolismo , Interferon-alfa/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Modelos Biológicos , Proteínas de Resistência a Myxovirus/genéticaRESUMO
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.
Assuntos
Infecções por Herpesviridae/prevenção & controle , Herpesviridae/fisiologia , Mutação , Proteínas de Resistência a Myxovirus/genética , Replicação Viral/genética , Células A549 , Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Imunidade Inata , Interferons , Proteínas de Resistência a Myxovirus/imunologia , Replicação Viral/imunologiaRESUMO
Interferon (IFN) is a key component of the innate immune response to exogenous pathogens. Interferon increases the mRNA levels of interferon-stimulated genes (ISGs) in vivo, which is thought to account for its antiviral activity. Recent studies have indicated that human myxovirus resistance protein 2 (Mx2 or MxB), one of these ISGs, contributes to the inhibition of HIV-1 replication by interferon. MxB may bind to HIV-1 relatively late in the post-entry phase, and it leads to a reduced level of integrated viral DNA, thereby restricting HIV-1 infection. The N-terminal 91-aa domain of MxB and the assembly of MxB mediated by the Stalk domain have also been shown to be indispensible for MxB's anti-viral functions, but the mechanism involved has remained elusive. Here, we report the crystal structure (2.9Å) of the human MxB Stalk domain. MxB Stalk shows one dimer in the asymmetric unit. Each monomer contains a four-helix bundle. Interestingly, analyses of MxB dimer interfaces show that the majority of residues involved in the interface are not conserved between MxB and MxA, contributing to the building of a more stable MxB dimer. MxA and MxB Stalk domains share 46.7% sequence identity, and the structure of the MxA Stalk domain and the overall structure of MxB Stalk have a similar conformation. Our results indicate that although human Mx proteins share common structural characteristics, their dimerization strategies are unique, contributing to their unique contributions to viral restriction.
Assuntos
Dinaminas/química , Proteínas de Resistência a Myxovirus/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , HIV-1/fisiologia , Humanos , Interferons/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Replicação ViralRESUMO
Type I interferon (IFN) inhibits a wide spectrum of viruses through stimulating the expression of antiviral proteins. As an IFN-induced protein, myxovirus resistance B (MXB) protein was reported to inhibit multiple highly pathogenic human viruses. It remains to be determined whether MXB employs a common mechanism to restrict different viruses. Here, we find that IFN alters the subcellular localization of hundreds of host proteins, and this IFN effect is partially lost upon MXB depletion. The results of our mechanistic study reveal that MXB recognizes vimentin (VIM) and recruits protein kinase B (AKT) to phosphorylate VIM at amino acid S38, which leads to reorganization of the VIM network and impairment of intracellular trafficking of virus protein complexes, hence causing a restriction of virus infection. These results highlight a new function of MXB in modulating VIM-mediated trafficking, which may lead towards a novel broad-spectrum antiviral strategy to control a large group of viruses that depend on VIM for successful replication.
RESUMO
HIV-1 replication is tightly regulated in host cells, and various restriction factors have important roles in inhibiting viral replication. SAMHD1, a well-known restriction factor, suppresses HIV-1 replication by hydrolyzing intracellular dNTPs, thereby limiting the synthesis of viral cDNA in quiescent cells. In this study, we revealed an additional and distinct mechanism of SAMHD1 inhibition during the postviral cDNA synthesis stage. Using immunoprecipitation and mass spectrometry analysis, we demonstrated the interaction between SAMHD1 and MX2/MxB, an interferon-induced antiviral factor that inhibits HIV-1 cDNA nuclear import. The disruption of endogenous MX2 expression significantly weakened the ability of SAMHD1 to inhibit HIV-1. The crucial region within SAMHD1 that binds to MX2 has been identified. Notably, we found that SAMHD1 can act as a sensor that recognizes and binds to the incoming HIV-1 core, subsequently delivering it to the molecular trap formed by MX2, thereby blocking the nuclear entry of the HIV-1 core structure. SAMHD1 mutants unable to recognize the HIV-1 core showed a substantial decrease in antiviral activity. Certain mutations in HIV-1 capsids confer resistance to MX2 inhibition while maintaining susceptibility to suppression by the SAMHD1-MX2 axis. Overall, our study identifies an intriguing antiviral pattern wherein two distinct restriction factors, SAMHD1 and MX2, collaborate to establish an alternative mechanism deviating from their actions. These findings provide valuable insight into the complex immune defense networks against exogenous viral infections and have implications for the development of targeted anti-HIV therapeutics. IMPORTANCE: In contrast to most restriction factors that directly bind to viral components to exert their antiviral effects, SAMHD1, the only known deoxynucleotide triphosphate (dNTP) hydrolase in eukaryotes, indirectly inhibits viral replication in quiescent cells by reducing the pool of dNTP substrates available for viral cDNA synthesis. Our study provides a novel perspective on the antiviral functions of SAMHD1. In addition to its role in dNTP hydrolysis, SAMHD1 cooperates with MX2 to inhibit HIV-1 nuclear import. In this process, SAMHD1 acts as a sensor for incoming HIV-1 cores, detecting and binding to them, before subsequently delivering the complex to the molecular trap formed by MX2, thereby immobilizing the virus. This study not only reveals a new antiviral pathway for SAMHD1 but also identifies a unique collaboration and interaction between two distinct restriction factors, establishing a novel line of defense against HIV-1 infection, which challenges the traditional view of restriction factors acting independently. Overall, our findings further indicate the intricate complexity of the host immune defense network and provide potential targets for promoting host antiviral immune defense.
Assuntos
Infecções por HIV , HIV-1 , Proteínas de Resistência a Myxovirus , Proteína 1 com Domínio SAM e Domínio HD , Replicação Viral , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética , Humanos , HIV-1/fisiologia , HIV-1/genética , Proteínas de Resistência a Myxovirus/metabolismo , Proteínas de Resistência a Myxovirus/genética , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Infecções por HIV/genética , DNA Viral/metabolismo , DNA Viral/genética , Células HEK293 , Interações Hospedeiro-Patógeno , Ligação ProteicaRESUMO
Introduction: Hepatitis C virus (HCV) infection was the primary reason causing critical hepatic Q7 diseases. Although direct-acting antiviral agents (DAA) were widely used in clinics, anti-drug mutation, the outcome of patients with different viral subtypes, and recurrence suggested that HCV pathogenic mechanism should be studied further. HCV infection, replication, and outcome were influenced by the IFNL4 and itsdownstream genes (MxA and MxB). However, whether genetic polymorphisms of these genes played necessary roles required verification in the Yunnan population. Methods and Results: After analyzing the genotypes and allele frequencies of seven single nucleotide polymorphisms (SNP), we found the association between the genotype and allele frequencies of rs11322783 in the IFNL4 gene and HCV infection in Yunnan population. Furthermore, the genetic polymorphisms of the MxA and MxB genescould influence liver function of HCV patients. The indirect bilirubin (IBIL) and albumin (ALB) levels showed significant differences among HCV patients, who carried various genotypes. The IBIL levels were associated with genotypes of rs17000900 (P= 0.025) and rs2071430 (P= 0.037) in the MxA gene, and ALB levels were associated with genotypes of rs2838029 (P= 0.010) in the MxB gene. Similarly, the genotypes of SNPs also showed significant difference in patients infected with subtype 3a (P=0.035) and 2a (P=0.034). However, no association was identified between expression level and SNPs of the MxA and MxB genes. Furthermore, HCV subtype 3b was found to be the predominantly epidemic strain in Yunnan Province. Conclusion: In conclusion, the association between biochemical indices/HCV subtypes and SNPs in the MxA and MxB genes was identified in Yunnan HCV population.
Assuntos
Hepatite C Crônica , Hepatite C , Proteínas de Resistência a Myxovirus , Humanos , Antivirais/uso terapêutico , China/epidemiologia , Genótipo , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Interleucinas/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Resistência a Myxovirus/genéticaRESUMO
Hepatitis B virus (HBV) infection causes Hepatitis B, which is one of the most common causes of hepatocellular carcinoma (HCC). The single nucleotide polymorphisms (SNPs) of the host immune genes could impact HBV infection, viral clearance, and treatment effect. However, the contradictory roles of several studies suggest further analysis of various populations. The whole blood and biochemical indexes of 448 HBV patients and matched controls were collected from the Yunnan population to investigate the genetic roles of IFNL4 and the downstream genes (MxA and MxB). The genotypes, alleles, and haplotypes frequencies of the seven SNPs (rs11322783, rs117648444, rs2071430, rs17000900, rs9982944, rs408825, and rs2838029) from the HBV patients and controls were analyzed. However, no association was identified between the SNPs and HBV infection. Then, biochemical index levels were evaluated among the HBV patients with different genotypes of the seven SNPs. The results indicated that the liver function index levels (including alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), and albumin (ALB)) were influenced by the genotypes of the SNPs in HBV patients. Moreover, when the HBV patients were divided into HBsAg-positive and -negative groups, the association between the SNP genotypes and the biochemical indexes still existed. In addition, although the genetic polymorphisms in the IFNL4, MxA, and MxB genes were not significantly associated with HBV infection in the Yunnan population, these genes could indirectly influence disease progression by associating with the biochemical index levels of Yunnan HBV patients.
Assuntos
Carcinoma Hepatocelular , Hepatite B Crônica , Interleucinas , Neoplasias Hepáticas , Proteínas de Resistência a Myxovirus , Humanos , Bilirrubina , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , China/epidemiologia , Predisposição Genética para Doença/genética , Vírus da Hepatite B , Hepatite B Crônica/genética , Interleucinas/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Polimorfismo de Nucleotídeo Único , Proteínas de Resistência a Myxovirus/genéticaRESUMO
The human myxovirus resistance B (MxB) protein is an interferon-induced restriction factor that fights a wide range of viruses. We previously demonstrated that MxB binds to hepatitis C virus (HCV)-encoded non-structural protein 5A (NS5A) and inhibits HCV infection by impairing the formation of cyclophilin A (CypA)-NS5A complex. However, the molecular details about how the presence of MxB diminishes the binding of NS5A to CypA remain uncovered. In this study, through molecular dynamic simulations and biochemical assays, we characterized that MxB binds to NS5A domain I through its N-terminal and GTPase domains. Specifically, amino acids (aa.) 189-191 and aa. 330-334 within MxB, together with NS5A residues aa. 71-73, are crucial for MxB-NS5A interaction. Furthermore, we predicted the CypA:NS5A and CypA:NS5A:MxB complexes and calculated the per-residue energy decomposition for identified key residues of the CypA-NS5A interface. A 28% decrease in CypA-NS5A binding affinity was observed in the presence of MxB, suggesting a weakened CypA-NS5A association upon binding of MxB to NS5A, which may contribute to the MxB-mediated inhibitory effect on the formation of CypA-NS5A complex. This work provides information for the antiviral mechanism of MxB and may facilitate the discovery of new strategies to combat CypA-dependent viruses.
RESUMO
A restriction factor induced by interferons blocks the replication of herpesviruses by disassembling the capsid proteins surrounding their genome.
Assuntos
Proteínas do Capsídeo , Herpesviridae , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Herpesviridae/genética , InterferonsRESUMO
Host proteins sense viral products and induce defence mechanisms, particularly in immune cells. Using cell-free assays and quantitative mass spectrometry, we determined the interactome of capsid-host protein complexes of herpes simplex virus and identified the large dynamin-like GTPase myxovirus resistance protein B (MxB) as an interferon-inducible protein interacting with capsids. Electron microscopy analyses showed that cytosols containing MxB had the remarkable capability to disassemble the icosahedral capsids of herpes simplex viruses and varicella zoster virus into flat sheets of connected triangular faces. In contrast, capsids remained intact in cytosols with MxB mutants unable to hydrolyse GTP or to dimerize. Our data suggest that MxB senses herpesviral capsids, mediates their disassembly, and thereby restricts the efficiency of nuclear targeting of incoming capsids and/or the assembly of progeny capsids. The resulting premature release of viral genomes from capsids may enhance the activation of DNA sensors, and thereby amplify the innate immune responses.
Assuntos
Capsídeo , Herpesviridae , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Interferons/metabolismo , SimplexvirusRESUMO
Interferon exerts its antiviral activity by stimulating the expression of antiviral proteins. These interferon stimulate genes (ISGs) often target a group of viruses with unique molecular mechanisms. One such ISG is myxovirus resistance B (MxB) that has been reported to inhibit human immunodeficiency virus type 1 (HIV-1) by targeting viral capsid and impairing nuclear import of viral DNA. The antiviral specificity of MxB is determined by its N-terminal 25 amino acids sequence which has the nuclear localization activity, therefore functions as a nuclear localization signal (NLS). In this study, we report that the bipartite NLS, but not the classic NLS, the PY-NLS, nor the arginine-rich NLS, when used to replace the N-terminal sequence of MxB, drastically suppress HIV-1 gene expression and virus production, thus creates a new anti-HIV-1 mechanism. MxB preserves its anti-HIV-1 activity when its N-terminal sequence is replaced by the arginine-rich NLS. Interestingly, the arginine-rich NLS allows MxB to inhibit HIV-1 CA mutants that are otherwise resistant to wild type MxB, which suggests sequence specific targeting of viral capsid. Together, these data implicate that it is not the nuclear import function itself, but rather the sequence and the mechanism of action of the NLS which define the antiviral property of MxB.
RESUMO
The human myxovirus resistance 2 (Mx2/MxB) protein, a member of interferon (IFN)-inducible dynamin-like large GTPases, restricts a number of virus infections. Inhibition of these viruses occurs at poorly-defined steps after viral entry and has a common requirement for MxB oligomerization. However, the GTPase activity is essential for the anti-viral effects of MxB against herpesviruses and HBV but not HIV-1. To understand the role of MxB GTPase activity, including GTP binding and GTP hydrolysis, in restriction of HIV-1 infection, we genetically separated these two functions and evaluated their contributions to restriction. We found that both the GTP binding and hydrolysis function of MxB involved in the restriction of HIV-1 replication. The GTPase activity of MxB contributed to its nuclear location, interaction with nucleoporins (NUPs) and HIV-1 capsids. Furthermore, MxB disrupted the association between NUPs and HIV-1 cores dependently upon its GTPase activity. The function of GTPase activity was therefore multi-faceted, led to fundamentally distinct mechanisms employed by wild-type MxB and GTPase activity defective MxB mutations to restrict HIV-1 replication.
Assuntos
Infecções por HIV , HIV-1 , Capsídeo/metabolismo , HIV-1/metabolismo , Humanos , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Complexo de Proteínas Formadoras de Poros NuclearesRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Proteínas de Resistência a Myxovirus/metabolismo , beta Carioferinas/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Transporte Ativo do Núcleo Celular , Regulação Viral da Expressão Gênica , Produtos do Gene gag/metabolismo , Variação Genética , Células HEK293 , Infecções por HIV/virologia , HIV-1/genética , Células HeLa , Humanos , Internalização do Vírus , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genéticaRESUMO
The type one interferon induced restriction factor Myxovirus resistance B (MxB) restricts HIV-1 nuclear entry evidenced by inhibition of 2-LTR but not linear forms of viral DNA. The HIV-1 capsid is the key determinant of MxB sensitivity and cofactor binding defective HIV-1 capsid mutants P90A (defective for cyclophilin A and Nup358 recruitment) and N74D (defective for CPSF6 recruitment) have reduced dependency on nuclear transport associated cofactors, altered integration targeting preferences and are not restricted by MxB expression. This has suggested that nuclear import mechanism may determine MxB sensitivity. Here we have use genetics to separate HIV-1 nuclear import cofactor dependence from MxB sensitivity. We provide evidence that MxB sensitivity depends on HIV-1 capsid conformation, rather than cofactor recruitment. We show that depleting CPSF6 to change nuclear import pathway does not impact MxB sensitivity, but mutants that recapitulate the effect of Cyclophilin A binding on capsid conformation and dynamics strongly impact MxB sensitivity. We demonstrate that HIV-1 primary isolates have different MxB sensitivities due to cytotoxic T lymphocyte (CTL) selected differences in Gag sequence but similar cofactor dependencies. Overall our work demonstrates a complex relationship between cyclophilin dependence and MxB sensitivity likely driven by CTL escape. We propose that cyclophilin binding provides conformational flexibility to HIV-1 capsid facilitating simultaneous evasion of capsid-targeting restriction factors including TRIM5 as well as MxB.