Structure of an avian influenza A virus NS1 protein effector domain

Influenza A virus NS1 protein is a multifunctional virulence factor. Here, we report a crystal structure for the NS1 effector domain of avian influenza virus A/Duck/Albany/76. Comparison of this structure with that reported for a human strain shows both proteins share a common monomer conformation, albeit with subtle differences. Strikingly, our data reveal a novel helix-helix dimeric interface between monomers of the avian NS1 protein, which is also found in the human NS1 crystal lattice. We re-evaluate the current model of NS1 dimeric assembly, and provide biochemical evidence to show tryptophan-187 (a residue located at the helix-helix interface) is essential for dimerization of this effector domain.     

The polybasic region is not essential for membrane binding of the matrix protein M1 of influenza virus

The matrix protein M1, the organizer of assembly of influenza virus, interacts with other virus components and with cellular membranes. It has been proposed that M1 binding to lipids is mediated by its polybasic region, but this could hitherto not been investigated in vivo since M1 accumulates in the nucleus of transfected cells. We have equipped M1 with nuclear export signals and showed that the constructs are bound to cellular membranes. Exchange of the complete polybasic region and of further hydrophobic amino acids in its vicinity did not prevent association of M1 with membranes. We therefore suppose that M1 probably interacts with membranes via multiple binding sites.     

Combined results from solution studies on intact influenza virus M1 protein and from a new crystal form of its N-terminal domain show that M1 is an elongated monomer

The amino-terminal domain of influenza A virus matrix protein (residues 1-164) was crystallized at pH 7 into a new crystal form in space group P1. This packing of the protein implies that M1(1-164) was monomeric in solution when it crystallized. Otherwise, the structure of the M1 fragment in the pH 7 crystals was the same as the monomers in crystals formed at pH 4 where crystal packing resulted in dimer formation [B. Sha and M. Luo, 1997, Nature Struct. Biol. 4, 239-244]. Analysis of intact M1 protein, the N-terminal domain, and the remaining C-terminal fragment (residues 165-252) in solution also showed that the N-terminal domain was monomeric with the same dimensions as determined from the crystal structure. Intact M1 protein was also monomeric but with an elongated shape due to the presence of the C-terminal part. Circular dichroism showed that the C-terminal part of M1 contained helical structure. A model for soluble M1 is presented, based on the assumption that the C-terminal domain is spherical, in which the N- and C-terminal domains are connected by a linker sequence which is available for proteolytic attack.     

Characterisation of interaction between NS3 and NS5B protein of classical swine fever virus by deletion of terminal sequences of NS5B

The NS3-NS5B interaction of classical swine fever virus (CSFV) is important for viral replication. For characterisation of the interaction between the NS3 and NS5B, a series of NS5B mutants with deletion of N-, C-terminal amino acids and quadruple alanine substitution mutations were produced. GST pull-down assays and immunoprecipitation analyses showed that NS5B and some NS5B mutants have NS3 binding activity. Further experimental data indicated that CSFV NS5B might contain two NS3 binding sites, one covering amino acids 63-99 located at the N-terminal end, another covering amino acids 611-642 at the C-terminal end. Assays for RNA-dependent RNA polymerase (RdRp) activity revealed that CSFV NS3 is able to enhance the RdRp activity of NS5B and some NS5B mutants in vitro. The enhancement might be obtained by NS3 binding to the two terminal sequences of NS5B, which could be attractive targets for drug development against CSFV.     

H5N1禽流感病毒NS1蛋白与干扰素诱导蛋白10表达的相关性研究

目的 研究高致病性禽流感(HPAI)H5N1病毒NS1蛋白对干扰素诱导蛋白10(IP-10)的影响.方法 分别将禽流感病毒A/Anhui/1/2005(H5N1)的NS1基因、插入80-84位缺失氨基酸的NS1突变基因及流感病毒A/Puerto Rico/8/1934(H1N1)的NS1基因克隆至真核表达载体pEGFP-N1,转染人支气管上皮细胞BEAS-2B,流式细胞仪检测转染细胞内IP-10的表达情况.结果 与pEGFP-N1对照组相比,三种NS1蛋白均能下调BEAS-2B细胞IP-10的表达(P<0.01),但三者之间下调程度差异无统计学意义(P>0.01).结论 A/Anhui/1/2005(H5N1)禽流感病毒单一NS1蛋白能够抑制BEAS-2B细胞IP-10表达,但这并不能完全阐明其与病毒致病性之间的关系.     

Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein

During replication, hepatitis C virus (HCV) NS4B protein rearranges intracellular membranes to form foci, or the web, the putative site for HCV replication. To understand the role of the C-terminal domain (CTD) in NS4B function, mutations were introduced into NS4B alone or in the context of HCV polyprotein. First, we show that the CTD is required for NS4B-induced web structure, but it is not sufficient to form the web nor is it required for NS4B membrane association. Interestingly, all the mutations introduced into the CTD impeded HCV genome replication, but only two resulted in a disruption of NS4B foci. Further, we found that NS4B interacts with NS3 and NS5A, and that mutations causing NS4B mislocalization have a similar effect on these proteins. Finally, we show that the redistribution of Rab5 to NS4B foci requires an intact CTD, suggesting that Rab5 facilitates NS4B foci formation through interaction with the CTD.     

Expression and characterisation of the influenza A virus non-structural protein NS1 in yeast

Summary The influenza A virus non-structural protein NS₁ was produced using a copper-inducible expression system in the yeastSaccharomyces cerevisiae. The protein produced had a molecular weight of 26 kDa by SDS-PAGE and was reactive with anti-NS₁ antisera. The recombinant NS₁ protein was targetted to the nucleolus/nuclear envelope fraction of the yeast cell nucleus, showing that its localisation signals remain functional in yeast. In addition, immune-electron microscopy detected cytoplasmic inclusions reminiscent of those seen in cells infected with some influenza strains. The NS₁ protein was shown to be capable of in vivo self-interaction which probably forms the basis of its propensity to form inclusions. Expression of the protein was found to be toxic to yeast cells expressing it, supporting a role for the protein in the shutdown of influenza virus-infected cells. Deletion mapping of NS₁ pointed to 2 regions of the molecule being important for this toxicity: a basic C-terminal stretch which has been shown to act as a nuclear localisation signal, and an N-terminal region implicated in RNA binding.     

Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication

Robust hepatitis B virus (HBV) replication is stimulated by the regulatory HBx protein. HBx binds the cellular protein DDB1; however, the importance of this interaction for HBV replication remains unknown. We tested whether HBx binding to DDB1 was required for HBV replication using a plasmid based replication assay in HepG2 cells. Three DDB1 binding-deficient HBx point mutants (HBx⁶⁹, HBx^90/91, HBx^R96E) failed to restore wildtype levels of replication from an HBx-deficient plasmid, which established the importance of the HBx-DDB1 interaction for maximal HBV replication. Analysis of overlapping HBx truncation mutants revealed that both the HBx-DDB1 binding domain and the carboxyl region are required for maximal HBV replication both in vitro and in vivo, suggesting the HBx-DDB1 interaction recruits regulatory functions critical for replication. Finally we demonstrate that HBx localizes to the Cul4A-DDB1 complex, and discuss the possible implications for models of HBV replication.     

Structure of a knockout mutant of influenza virus M1 protein that has altered activities in membrane binding, oligomerisation and binding to NEP (NS2)

The influenza virus M1 (matrix) protein forms the connection between the membrane component of the virus and its replication component eight ribonucleoprotein particles (RNPs). For this activity, M1 self-polymerises in the infected cell in order to pull glycoprotein containing membrane segments together. Later in the process of infection, M1 enters the nucleus and is active in the nuclear export process of newly made RNPs for virus particle assembly. The N-terminal domain (residues 1-164) of M1 carries the nuclear localisation sequence (NLS) motif and is important for membrane binding, self-polymerisation and nuclear export of RNPs. An NLS-mutant M1 has been used in functional studies in order to implicate the positive charges in the NLS in these three activities. In this paper, the crystal structure of the N-terminal domain of this NLS-mutant is determined and is found to be the same as that of the wild-type protein, clearly indicating that it is the absence of the positively charged residues of the NLS that causes the knock-out phenotype rather than a change in the overall structure of the mutant protein.     

Biological functions of the NS1 protein of an influenza B virus mutant which has a long carboxyl terminal deletion

Summary To clarify the function of the NS gene of a highly cytolytic mutant of influenza virus B/Yamagata/1/73 which expresses an NS₁ protein with a long carboxyl terminal deletion (clone 201), we prepared a single gene reassortant (201 L-77) and a control reassortant (YL-20) in which all the genes were of wild type influenza virus B/Lee/40 origin except NS gene which was derived from either clone 201 or wild type B/Yamagata. Comparative studies have revealed that 201 L-77 destructed infected cells more severely and much earlier after infection than did YL-20, although both produced comparable amount of infectious virus. The highly cytolytic reassortant 201 L-77 produced a small plaque, while the weakly cytolytic reassortant YL-20 produced a large plaque in MDCK cells. There was little difference between the two reassortants in the time course and the amount of synthesis of viral proteins within the infected cells. However, the mode of synthesis of viral RNA (vRNA) by 201 L-77 was greatly altered compared with YL-20.     

Loss of function of the influenza A virus NS1 protein promotes apoptosis but this is not due to a failure to activate phosphatidylinositol 3-kinase (PI3K)

A panel of influenza A viruses encoding mutant NS1 proteins was created in which a number of NS1 functions, including interactions with dsRNA, PI3K, CPSF30 and PKR, were inhibited. Surprisingly, given previous reports that NS1 activates PI3K to prevent apoptosis, the mutant viruses rUd-Y89F and rUd-P164/7A that fail to activate PI3K did not induce any more apoptosis than wild-type virus in MRC-5 and A549 cells, even though these cells are highly sensitive to inducers of apoptosis. Induction of cell death by the apoptogenic rUd-184-8(P) virus could not be prevented by serum-mediated activation of PI3K/Akt. Neither infection of MRC-5 or A549 cells with wild-type virus nor constitutive expression of NS1 prevented cell death caused by apoptosis inducers, suggesting that NS1 is not directly anti-apoptotic. Our data suggest that the loss of a functionally intact NS1 protein promotes apoptosis, but this is not due to an inability to activate PI3K.     

Acute GB virus B infection of marmosets is accompanied by mutations in the NS5A protein

GBV-B, a member of the Flaviviridae family of viruses, is the virus most closely related to HCV, and GBV-B infection in tamarin monkeys might represent a valuable surrogate animal model of HCV infection. In the current study, GBV-B was successfully transmitted to two marmosets (Callithrix jaccus). The infection resulted in viremia of 14- and 17-week duration, respectively, and was accompanied by elevation of isocitrate dehydrogenase activity. These data confirm that marmosets might represent an attractive model for GBV-B infection. The sequence of GBV-B NS5A, which was previously reported to have one of the highest mutation rates during infection in tamarins, was determined for viruses recovered from the inoculum and from marmoset blood samples obtained at weeks 1, 8, and 14 post inoculation in one marmoset and at weeks 2, 8, and 17 post inoculation in the other marmoset. In both animals, we detected four substitutions (R1945K, K2052G, F2196L, and G2268E), in the virus recovered immediately before viral clearance. Interestingly, two of these mutations (F2196L and G2268E) were described recently for viruses recovered from persistently infected tamarins. Appearance of these mutations presumably reflects a mechanism of immune escape rather than adaptation of the virus to a new host.     

Restoration of replication-defective dengue type 1 virus bearing mutations in the N-terminal cytoplasmic portion of NS4A by additional mutations in NS4B

Flavivirus NS4A has an N-terminal hydrophilic cytoplasmic portion; however, the role of this portion remains poorly understood. In this study, we show that a recombinant dengue type 1 virus (DENV-1) in which a subportion (amino acids 27–34) of the N-terminal portion of NS4A is replaced by the corresponding region from Japanese encephalitis virus (JEV) is defective in replication. Using the defective mutant clone NS4A(27–34^JEV), we recovered suppressor mutant viruses that carry various non-synonymous mutations. Site-directed mutational analysis indicated that a single non-synonymous mutation in NS4B that is found in the suppressor viruses is sufficient to restore NS4A(27–34^JEV). Recombinant DENV-1 with single mutations in NS4B had increased growth properties as compared to the wild-type virus and NS4A(27–34^JEV) virus bearing the same NS4B mutation. Collectively, our results suggest that the NS4B mutation enhanced the growth of DENV-1, irrespective of the sequence of the 27–34 subportion NS4A.     

Experimental basis of enhancing the immunogenicity of influenza B virus by genetic recombination

Redistribution of the immunogenicity marker in the course of genetic recombination of influenza B virus was studied in animal experiments on virus strains differing in their ability to induce antibody formation following a single peroral or intraperitoneal immunization. Immunogenicity of influenza virus could be enhanced by recombination of a strain possessing a low activity with a highly immunogenic homotypic strain. Efficiency of the transfer of the immunogenicity marker depended on the properties of viruses used for recombination. Strains at a low passage level proved to be more prospective "donors of immunogenicity" than hyperattenuated thermosensitive viruses which were unsuitable for this purpose. There was no complete correlation between hemagglutinating activities of influenza B viruses and of the recombinant.     

Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus

Vaccination programs for the control of avian influenza (AI) in poultry have limitations due to the problem of differentiating between vaccinated and virus-infected birds. We have used NS1, the conserved nonstructural protein of influenza A virus, as a differential diagnostic marker for influenza virus infection. Experimentally infected poultry were evaluated for the ability to induce antibodies reactive to NS1 recombinant protein produced in Escherichia coli or to chemically synthesized NS1 peptides. Immune sera were obtained from chickens and turkeys inoculated with live AI virus, inactivated purified vaccines, or inactivated commercial vaccines. Seroconversion to positivity for antibodies to the NS1 protein was achieved in birds experimentally infected with multiple subtypes of influenza A virus, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In contrast, animals inoculated with inactivated gradient-purified vaccines had no seroconversion to positivity for antibodies to the NS1 protein, and animals vaccinated with commercial vaccines had low, but detectable, levels of NS1 antibodies. The use of a second ELISA with diluted sera identified a diagnostic test that results in seropositivity for antibodies to the NS1 protein only in infected birds. For the field application phase of this study, serum samples were collected from vaccinated and infected poultry, diluted, and screened for anti-NS1 antibodies. Field sera from poultry that received commercial AI vaccines were found to possess antibodies against AI virus, as measured by the standard agar gel precipitin (AGP) test, but they were negative by the NS1 ELISA. Conversely, diluted field sera from AI-infected poultry were positive for both AGP and NS1 antibodies. These results demonstrate the potential benefit of a simple, specific ELISA for anti-NS1 antibodies that may have diagnostic value for the poultry industries.     

CDK/ERK-mediated phosphorylation of the human influenza A virus NS1 protein at threonine-215

Posttranslational modification of viral proteins by cellular enzymes is a feature of many virus replication strategies. Here, we report that during infection the multifunctional human influenza A virus NS1 protein is phosphorylated at threonine-215. Substitution of alanine for threonine at this position reduced early viral propagation, an effect apparently unrelated to NS1 antagonizing host interferon responses or activating phosphoinositide 3-kinase signaling. In vitro, a subset of cellular proline-directed kinases, including cyclin dependent kinases (CDKs) and extracellular signal-regulated kinases (ERKs), potently phosphorylated NS1 protein at threonine-215. Our data suggest that CDK/ERK-mediated phosphorylation of NS1 at threonine-215 is important for efficient virus replication.