Human MxA Protein Protects Mice Lacking a Functional Alpha/Beta Interferon System against La Crosse Virus and Other Lethal Viral Infections

The human MxA protein is part of the antiviral state induced by alpha/beta interferon (IFN-alpha/beta). MxA inhibits the multiplication of several RNA viruses in cell culture. However, its antiviral potential in vivo has not yet been fully explored. We have generated MxA-transgenic mice that lack a functional IFN system by crossing MxA-transgenic mice constitutively expressing MxA with genetically targeted (knockout) mice lacking the beta subunit of the IFN-alpha/beta receptor (IFNAR-1(-/-) mice). These mice are an ideal animal model to investigate the unique antiviral activity of human MxA in vivo, because they are unable to express other IFN-induced proteins. Here, we show that MxA confers resistance to Thogoto virus, La Crosse virus, and Semliki Forest virus. No Thogoto virus progeny was detectable in MxA-transgenic mice, indicating an efficient block of virus replication at the primary site of infection. In the case of La Crosse virus, MxA restricted invasion of the central nervous system. In contrast, Semliki Forest virus multiplication in the brain was detectable in both MxA-expressing and nonexpressing IFNAR-1(-/-) mice. However, viral titers were clearly reduced in MxA-transgenic mice. Our results demonstrate that MxA does not need the help of other IFN-induced proteins for activity but is a powerful antiviral agent on its own. Moreover, the results suggest that MxA may protect humans from potential fatal infections by La Crosse virus and other viral pathogens.     

Constitutive Expression of Interferon-Induced Human MxA Protein in Transgenic Tobacco Plants Does not Confer Resistance to a Variety of RNA Viruses

MxA is a key component in the interferon-induced antiviral defense in humans. After viral infections, MxA is rapidly induced and accumulates in the cytoplasm. The multiplication of many RNA viruses,including all bunyaviruses tested so far, is inhibited by MxA. These findings prompted us to express MxA in plants in an attempt to create resistance to tospoviruses. Here, we report the generation of transgenic tobacco plants that constitutively express MxA under the control of the 35S cauliflower mosaic virus promotor. Northern and western blot analysis confirmed the expression of MxA in several transgenic plant lines. MxA expression had no obvious detrimental effects on plant growth and fertility. However, challenge experiments with tomato spotted wilt virus, tomato chlorotic spot virus, and groundnut ringspot virus revealed no increased resistance of MxA-transgenic tobacco plants to tospovirus infections. Neither was the multiplicationof tobacco mosaic virus, cucumber mosaic virus and potato virus Y inhibited in MxA-transgenic plants. The results indicate that the expression of human MxA alone does not enhance virus resistance in planta.     

Pandemic Influenza A Viruses Escape from Restriction by Human MxA through Adaptive Mutations in the Nucleoprotein

The interferon-induced dynamin-like MxA GTPase restricts the replication of influenza A viruses. We identified adaptive mutations in the nucleoprotein (NP) of pandemic strains A/Brevig Mission/1/1918 (1918) and A/Hamburg/4/2009 (pH1N1) that confer MxA resistance. These resistance-associated amino acids in NP differ between the two strains but form a similar discrete surface-exposed cluster in the body domain of NP, indicating that MxA resistance evolved independently. The 1918 cluster was conserved in all descendent strains of seasonal influenza viruses. Introduction of this cluster into the NP of the MxA-sensitive influenza virus A/Thailand/1(KAN-1)/04 (H5N1) resulted in a gain of MxA resistance coupled with a decrease in viral replication fitness. Conversely, introduction of MxA-sensitive amino acids into pH1N1 NP enhanced viral growth in Mx-negative cells. We conclude that human MxA represents a barrier against zoonotic introduction of avian influenza viruses and that adaptive mutations in the viral NP should be carefully monitored.     

The viral nucleoprotein determines Mx sensitivity of influenza A viruses

Host restriction factors play a crucial role in preventing trans-species transmission of viral pathogens. In mammals, the interferon-induced Mx GTPases are powerful antiviral proteins restricting orthomyxoviruses. Hence, the human MxA GTPase may function as an efficient barrier against zoonotic introduction of influenza A viruses into the human population. Successful viruses are likely to acquire adaptive mutations allowing them to evade MxA restriction. We compared the 2009 pandemic influenza A virus [strain A/Hamburg/4/09 (pH1N1)] with a highly pathogenic avian H5N1 isolate [strain A/Thailand/1(KAN-1)/04] for their relative sensitivities to human MxA and murine Mx1. The H5N1 virus was highly sensitive to both Mx GTPases, whereas the pandemic H1N1 virus was almost insensitive. Substitutions of the viral polymerase subunits or the nucleoprotein (NP) in a polymerase reconstitution assay demonstrated that NP was the main determinant of Mx sensitivity. The NP of H5N1 conferred Mx sensitivity to the pandemic H1N1 polymerase, whereas the NP of pandemic H1N1 rendered the H5N1 polymerase insensitive. Reassortant viruses which expressed the NP of H5N1 in a pH1N1 genetic background and vice versa were generated. Congenic Mx1-positive mice survived intranasal infection with these reassortants if the challenge virus contained the avian NP. In contrast, they succumbed to infection if the NP of pH1N1 origin was present. These findings clearly indicate that the origin of NP determines Mx sensitivity and that human influenza viruses acquired adaptive mutations to evade MxA restriction. This also explains our previous observations that human and avian influenza A viruses differ in their sensitivities to Mx.     

Functional compensation of a detrimental amino acid substitution in a cytotoxic-T-lymphocyte epitope of influenza a viruses by comutations

Influenza A viruses accumulate amino acid substitutions in cytotoxic-T-lymphocyte (CTL) epitopes, allowing these viruses to escape from CTL immunity. The arginine-to-glycine substitution at position 384 of the viral nucleoprotein is associated with escape from CTLs. Introduction of the R384G substitution in the nucleoprotein gene segment of influenza virus A/Hong Kong/2/68 by site-directed mutagenesis was detrimental to viral fitness. Introduction of one of the comutations associated with R384G, E375G, partially restored viral fitness and nucleoprotein functionality. We hypothesized that influenza A viruses need to overcome functional constraints to accumulate mutations in CTL epitopes and escape from CTLs.     

Human MxA protein inhibits tick-borne Thogoto virus but not Dhori virus.

Thogoto and Dhori viruses are tick-borne orthomyxoviruses infecting humans and livestock in Africa, Asia, and Europe. Here, we show that human MxA protein is an efficient inhibitor of Thogoto virus but is inactive against Dhori virus. When expressed in the cytoplasm of stably transfected cell lines, MxA protein interfered with the accumulation of Thogoto viral RNA and proteins. Likewise, MxA(R645), a mutant MxA protein known to be active against influenza virus but inactive against vesicular stomatitis virus, was equally efficient in blocking Thogoto virus growth. Hence, a common antiviral mechanism that is distinct from the antiviral action against vesicular stomatitis virus may operate against both influenza virus and Thogoto virus. When moved to the nucleus with the help of a foreign nuclear transport signal, MxA(R645) remained active against Thogoto virus, indicating that a nuclear step of virus replication was inhibited. In contrast, Dhori virus was not affected by wild-type or mutant MxA protein, indicating substantial differences between these two tick-transmitted orthomyxoviruses. Human MxB protein had no antiviral activity against either virus.     

Influenza A virus strains differ in sensitivity to the antiviral action of Mx-GTPase

Interferon-mediated host responses are of great importance for controlling influenza A virus infections. It is well established that the interferon-induced Mx proteins possess powerful antiviral activities toward most influenza viruses. Here we analyzed a range of influenza A virus strains for their sensitivities to murine Mx1 and human MxA proteins and found remarkable differences. Virus strains of avian origin were highly sensitive to Mx1, whereas strains of human origin showed much weaker responses. Artificial reassortments of the viral components in a minireplicon system identified the viral nucleoprotein as the main target structure of Mx1. Interestingly, the recently reconstructed 1918 H1N1 "Spanish flu" virus was much less sensitive than the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 when tested in a minireplicon system. Importantly, the human 1918 virus-based minireplicon system was almost insensitive to inhibition by human MxA, whereas the avian influenza A virus H5N1-derived system was well controlled by MxA. These findings suggest that Mx proteins provide a formidable hurdle that hinders influenza A viruses of avian origin from crossing the species barrier to humans. They further imply that the observed insensitivity of the 1918 virus-based replicon to the antiviral activity of human MxA is a hitherto unrecognized characteristic of the "Spanish flu" virus that may contribute to the high virulence of this unusual pandemic strain.     

Human MxA Protein Confers Resistance to Semliki Forest Virus and Inhibits the Amplification of a Semliki Forest Virus-Based Replicon in the Absence of Viral Structural Proteins

Mx proteins form a small family of interferon (IFN)-induced GTPases with potent antiviral activity against various negative-strand RNA viruses. To examine the antiviral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiviral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of viral structural proteins, we took advantage of a recombinant vector based on the SFV replicon. The vector contains only the coding sequence for the viral nonstructural proteins and the bacterial LacZ gene, which was cloned in place of the viral structural genes. Upon transfection of vector-derived recombinant RNA, expression of the β-galactosidase reporter gene was strongly reduced in the presence of MxA. This finding indicates that viral components other than the structural proteins are the target of MxA action.     

Human MxA protein inhibits the replication of Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Nairovirus within the family Bunyaviridae and is the causative agent of severe hemorrhagic fever. Despite increasing knowledge about hemorrhagic fever viruses, the factors determining their pathogenicity are still poorly understood. The interferon-induced MxA protein has been shown to have an inhibitory effect on several members of the Bunyaviridae family, but the effect of MxA against CCHFV has not previously been studied. Here, we report that human MxA has antiviral activity against CCHFV. The yield of progeny virus in cells constitutively expressing MxA was reduced up to 1,000-fold compared with control cells, and accumulation of viral genomes was blocked. Confocal microscopy revealed that MxA colocalizes with the nucleocapsid protein (NP) of CCHFV in the perinuclear regions of infected cells. Furthermore, we found that MxA interacted with NP by using a coimmunoprecipitation assay. We also found that an amino acid substitution (E645R) within the C-terminal domain of MxA resulted in a loss of MxA antiviral activity and, concomitantly, in the capacity to interact with CCHFV NP. These results suggest that MxA, by interacting with a component of the nucleocapsid, prevents replication of CCHFV viral RNA and thereby inhibits the production of new infectious virus particles.     

The Human Interferon-Induced MxA Protein Inhibits Early Stages of Influenza A Virus Infection by Retaining the Incoming Viral Genome in the Cytoplasm

The induction of an interferon-induced antiviral state is a powerful cellular response against viral infection that limits viral spread. Here, we show that a preexisting antiviral state inhibits the replication of influenza A viruses in human A549 cells by preventing transport of the viral genome to the nucleus and that the interferon-induced MxA protein is necessary but not sufficient for this process. This represents a previously unreported antiviral function of MxA against influenza A virus infection.     

Role of Nucleotide Binding and GTPase Domain Dimerization in Dynamin-like Myxovirus Resistance Protein A for GTPase Activation and Antiviral Activity

Myxovirus resistance (Mx) GTPases are induced by interferon and inhibit multiple viruses, including influenza and human immunodeficiency viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action.     

Expression of human MxA protein in mosquito cells interferes with LaCrosse virus replication

Human MxA protein inhibits LaCrosse virus (LAC virus; family Bunyaviridae) replication in vertebrate cells and MxA-transgenic mice. LAC virus is transmitted to humans by Aedes triseriatus mosquitoes. In this report, we have shown that transfected mosquito cells expressing the human MxA cDNA are resistant to LAC virus but permissive for Sindbis virus (family Togaviridae) infection.     

Enhanced virus resistance of transgenic mice expressing the human MxA protein.

MxA is a GTPase that accumulates to high levels in the cytoplasm of interferon-treated human cells. Expression of MxA cDNA confers to transfected cell lines a high degree of resistance against several RNA viruses, including influenza, measles, vesicular stomatitis, and Thogoto viruses. We have now generated transgenic mice that express MxA cDNA in the brain and other organs under the control of a constitutive promoter. Embryonic fibroblasts derived from the transgenic mice were nonpermissive for Thogoto virus and showed reduced susceptibility for influenza A and vesicular stomatitis viruses. The transgenic animals survived challenges with high doses of Thogoto virus by the intracerebral or intraperitoneal route. Furthermore, the transgenic mice were more resistant than their nontransgenic littermates to intracerebral infections with influenza A and vesicular stomatitis viruses. These results demonstrate that MxA is a powerful antiviral agent in vivo, indicating that it may protect humans from the deleterious effects of infections with certain viral pathogens.     

植物抗病毒分子机制

在与植物病毒的长期斗争中,植物进化出多种抗病毒机制,其中RNA沉默和R基因介导的病毒抗性是最受人们关注的两种机制.一方面,RNA沉默是植物抵抗病毒侵染的重要手段.植物在病毒侵染过程中可形成病毒来源的双链RNA,经过DCL蛋白的切割、加工形成sRNA,与AGO蛋白结合形成RISC指导病毒RNA的沉默,用于清除病毒.相应地,病毒在与植物的竞争中进化出RNA沉默抑制子,抑制宿主RNA沉默系统以逃避宿主RNA沉默抗病毒反应,增强致病能力.另一方面,植物也进化出R基因介导植物对包括病毒在内的多类病原的抗性.R蛋白直接或间接识别病毒因子,通过一系列的信号转导途径激活植物防御反应,限制病毒的进一步侵染.对植物抗病毒的研究有助于人们对植物抗病分子基础的理解,有重要的科学意义和潜在应用价值.本文综述了植物抗病毒分子机制的重要进展.