Nuclear import and export of influenza virus nucleoprotein

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

Structure and function of influenza virus NS1 and NS2 proteins

This review discusses the structure and function of the influenza virus NS1 and NS2 proteins. The NS1 is a phosphoprotein and has two nuclear localization signals. In the nucleus, the NS1 interferes with the splicing as well as the nuclear export of cellular mRNAs. In the later time of the infection, the NS1 is present in the cytoplasm and associates with the polysomes. The NS1 binds to the 5'UTR of some viral mRNAs and stimulates translation. The NS2 is a phosphoprotein and binds to the nucleoporin yRip1 and Rab/hRip as well as the M1 protein which associates with the vRNPs. Therefore, the NS2 protein plays an important role in the nuclear export of the vRNPs. Improved technique to genetically manipulate influenza virus allowed us to rescue NS1 and NS2 mutants which are useful for further study.     

Association of influenza virus NP and M1 proteins with cellular cytoskeletal elements in influenza virus-infected cells

We have investigated the association of the influenza virus matrix (M1) and nucleoprotein (NP) with the host cell cytoskeletal elements in influenza virus-infected MDCK and MDBK cells. At 6.5 h postinfection, the newly synthesized M1 was Triton X-100 (TX-100) extractable but became resistant to TX-100 extraction during the chase with a t1/2 of 20 min. NP, on the other hand, acquired TX-100 resistance immediately after synthesis. Significant fractions of both M1 and NP remained resistant to differential detergent (Triton X-114, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS], octylglucoside) extraction, suggesting that M1 and NP were interacting with the cytoskeletal elements. However, the high-molecular-weight form of the viral transmembrane protein hemagglutinin (HA), which had undergone complex glycosylation, also became resistant to TX-100 extraction but was sensitive to octylglucoside detergent extraction, indicating that HA, unlike M1 or NP, was interacting with TX-100-insoluble lipids and not with cytoskeletal elements. Morphological analysis with cytoskeletal disrupting agents demonstrated that M1 and NP were associated with microfilaments in virus-infected cells. However, M1, expressed alone in MDCK or HeLa cells from cloned cDNA or coexpressed with NP, did not become resistant to TX-100 extraction even after a long chase. NP, on the other hand, became TX-100 insoluble as in the virus-infected cells. M1 also did not acquire TX-100 insolubility in ts 56 (a temperature-sensitive mutant with a defect in NP protein)-infected cells at the nonpermissive temperature. Furthermore, early in the infectious cycle in WSN-infected cells, M1 acquired TX-100 resistance very slowly after a long chase and did not acquire TX-100 resistance at all when chased in the presence of cycloheximide. On the other hand, late in the infectious cycle, M1 acquired TX-100 resistance when chased in either the presence or absence of cycloheximide. Taken together, these results demonstrate that M1 and NP interact with host microfilaments in virus-infected cells and that M1 requires other viral proteins or subviral components (possibly viral ribonucleoprotein) for interaction with host cytoskeletal components. The implication of these results for viral morphogenesis is discussed.     

Evaluation of a competitive ELISA for detection of antibodies against avian influenza virus nucleoprotein

BACKGROUND: Growth hormone (GH) has been shown to promote wound healing and to improve protein metabolism in burned patients. Through immunomodulation, GH has also protected rats infected with Salmonella typhimurium and mice infected with Escherichia coli. In spite of advances in the management of patient care for those with thermal injuries, high mortality rates of burned patients as a result of infections are of special concern. An improvement in the resistance of burned patients to certain infections will make the beneficial role of GH very clear. In this study, therefore, the immunomodulating effects of recombinant human GH (rhGH) in thermally injured mice exposed to opportunistic herpesvirus infections were investigated. METHODS: (1) Burned mice, exposed to herpes simplex virus type 1 (HSV-1), were treated subcutaneously with rhGH (4 mg/kg) and observed for 21 days to determine the protective antiviral effect of rhGH. (2) Because of reports describing a lack of interferon-gamma (IFN-gamma) responsiveness in burned mice, the IFN-gamma-producing ability of the splenic mononuclear cells (SMNC) from burned mice treated with rhGH was examined. (3) Because the generation of burn-associated suppressor macrophages that can inhibit the IFN-gamma production by SMNC has been previously described, the suppressor cell activities of macrophages from burned mice treated with rhGH were examined. RESULTS: After exposure to lethal amounts of HSV-1, mice treated with rhGH displayed a reduced mortality rate compared with control mice treated with saline. SMNC from burned mice treated with rhGH produced IFN-gamma, whereas this cytokine was not produced by SMNC from burned mice treated with saline. Also, an inhibition of the generation of burn-associated suppressor macrophages was displayed in burned mice treated with rhGH. CONCLUSION: Exogenous administration of rhGH caused an improvement in the resistance of burned mice to HSV-1 infection. In burned mice treated with rhGH, the impaired IFN-gamma responsiveness was restored and the generation of burn-associated suppressor macrophages was inhibited. IFN-gamma, a typical antiviral cytokine induced by rhGH through the regulation of the suppressor macrophage generation, may therefore play a role in the protection of burned mice infected with a lethal amount of HSV-1.     

Structure of the haemagglutinin-esterase-fusion glycoprotein of influenza C virus

The spike glycoproteins of the lipid-enveloped orthomyxoviruses and paramyxoviruses have three functions: to recognize the receptor on the cell surface, to mediate viral fusion with the cell membrane, and to destroy the receptor. In influenza C virus, a single glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, possesses all three functions. In influenza A and B, the first two activities are mediated by haemagglutinin and the third by a second glycoprotein, neuraminidase. Here we report the crystal structure of the HEF envelope glycoprotein of influenza C virus. We have identified the receptor-binding site and the receptor-destroying enzyme (9-O-acetylesterase) sites, by using receptor analogues. The receptor-binding domain is structurally similar to the sialic acid-binding domain of influenza A haemagglutinin, but binds 9-O-acetylsialic acid. The esterase domain has a structure similar to the esterase from Streptomyces scabies and a brain acetylhydrolase. The receptor domain is inserted into a surface loop of the esterase domain and the esterase domain is inserted into a surface loop of the stem. The stem domain is similar to that of influenza A haemagglutinin, except that the triple-stranded, alpha-helical bundle diverges at both of its ends, and the amino terminus of HEF2, the fusion peptide, is partially exposed. The segregation of HEF's three functions into structurally distinct domains suggests that the entire stem region, including sequences at the amino and carboxy termini of HEF1 which precede the post-translational cleavage site between HEF1 and HEF2, forms an independent fusion domain which is probably derived from an ancestral membrane fusion protein.     

Enhanced protection against influenza virus of mice immunized as newborns with a mixture of plasmids expressing hemagglutinin and nucleoprotein

Effective immunization of neonates is an important goal for vaccinology. We show that inoculation of newborn mice with a mixture of plasmids expressing influenza hemagglutinin (HA) and nucleoprotein (NP) genes leads to an enhanced protection subsequent to the lethal challenge with two distinct strains. In sharp contrast, neonatal injection with UV-inactivated influenza virus strain WSN, failed to induce protection against the homologous challenge. Our results show that while plasmid immunization of neonates elicits a protective immunity, the immunization with inactivated virus does not.     

GABA(A) receptor function in the cerebral cortex of alcohol-naive P and NP rats

In our previous paper (Biochim. Biophys. Acta 1379 (1998) 257-263), we demonstrated that bicarbonate promotes a cleavage of lactone ring of dehydroascorbate (DHA) on the basis of in vitro experiments. In the present study, we examined the degradation of DHA in blood circulation in vivo by using a high-performance liquid chromatographic method for the determination of ascorbate (AsA), DHA and 2,3-diketogulonate (2,3-DKG), which required no pretreatment of biological fluids. When DHA was intravenously administered to rats, a rapid disappearance of DHA (t1/2 < 1 min) and a concomitant appearance of 2,3-DKG in blood circulation were observed. Approximately 90% of the administered DHA were excreted into urine as resulting 2,3-DKG (55%) and AsA (31%), respectively. Furthermore, we elucidated that rat plasma lacks an enzyme having an aldonolactonase-like activity. The result of the present study suggests that this DHA disappearance is a function of both a chemical degradation to 2,3-DKG and a reduction to AsA.     

Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745-1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+ T cells and cytokine-secreting CD4+ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.     

The structure and function of the HE protein of influenza C virus

The hemagglutinin-esterase (HE) glycoprotein is an integral membrane protein and the major surface antigen of influenza C virion. It displays three biological activities: receptor-binding activity for N-acetyl-9-O-acetylneuraminic acid, fusion with the host cell membrane, and receptor-destroying activity which is a neuraminate-O-acetylesterase. This protein undergoes four kinds of posttranslational processing: proteolytic cleavage, N-glycosylation, fatty acid acylation, and phosphorylation. We summarize here the data concerning the sites on the HE molecule responsible for the biological activities including antigenic epitopes as well as those concerning the role of posttranslational modifications in virus replication.     

Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity

The C terminus of the influenza virus hemagglutinin (HA) contains three cysteine residues that are highly conserved among HA subtypes, two in the cytoplasmic tail and one in the transmembrane domain. All of these C-terminal cysteine residues are modified by the covalent addition of palmitic acid through a thio-ether linkage. To investigate the role of HA palmitylation in virus assembly, we used reverse genetics technique to introduce substitutions and deletions that affected the three conserved cysteine residues into the H3 subtype HA. The rescued viruses contained the HA of subtype H3 (A/Udorn/72) in a subtype H1 helper virus (A/WSN/33) background. Rescued viruses which do not contain a site for palmitylation (by residue substitution or substitution combined with deletion of the cytoplasmic tail) were obtained. Rescued virions had a normal polypeptide composition. Analysis of the kinetics of HA low-pH-induced fusion of the mutants showed no major change from that of virus with wild-type (wt) HA. The PFU/HA ratio of the rescued viruses grown in eggs ranged from that of virus with wt HA to 16-fold lower levels, whereas the PFU/HA ratio of the rescued viruses grown in MDCK cells varied only 2-fold from that of virus with wt HA. However, except for one rescued mutant virus (CAC), the mutant viruses were attenuated in mice, as indicated by a > or = 400-fold increase in the 50% lethal dose. Interestingly, except for one mutant virus (CAC), all of the rescued mutant viruses were restricted for replication in the upper respiratory tract but much less restricted in the lungs. Thus, the HA cytoplasmic tail may play a very important role in the generation of virus that can replicate in multiple cell types.     

Structure and function of human IgA Fc receptors (Fc alpha R)

Receptors for the Fc region of IgA are expressed by many human cell types, especially phagocytes located in mucosal areas, where IgA is the prevalent antibody isotype. Binding of IgA-opsonized particles (e.g., bacteria, viruses) to Fc alpha R may trigger a plethora of cell-mediated immune effector functions designed to rid the body of the foreign invader. The IgA receptor present on myeloid cells such as neutrophils, eosinophils, and monocytes (Fc alpha RI or CD89) is a transmembrane glycoprotein that binds both IgA isotypes with similar affinity. Genetic characterization showed Fc alpha RI to be a more distantly related member of the Ig receptor gene family. Recently, Fc alpha RI was found to associate with the FcR gamma-chain signaling molecule through a unique charge-based mechanism. Fc alpha RI is, thus, connected to the intracellular machinery via the ITAM signaling motifs located within the cytoplasmic tail of FcR gamma-chain. Evidence exists in support of receptors for IgA (distinct from Fc alpha RI) on human T and B cells. IgA Fc receptors may, therefore, play a role in both the induction and control of an efficient (mucosal) immune response.     

Modulation of RecA nucleoprotein function by the mutagenic UmuD'C protein complex

The RecA, UmuC, and UmuD' proteins are essential for error-prone, replicative bypass of DNA lesions. Normally, RecA protein mediates homologous pairing of DNA. We show that purified Umu(D')2C blocks this recombination function. Biosensor measurements establish that the mutagenic complex binds to the RecA nucleoprotein filament with a stoichiometry of one Umu(D')2C complex for every two RecA monomers. Furthermore, Umu(D')2C competitively inhibits LexA repressor cleavage but not ATPase activity, implying that Umu(D')2C binds in or proximal to the helical groove of the RecA nucleoprotein filament. This binding reduces joint molecule formation and even more severely impedes DNA heteroduplex formation by RecA protein, ultimately blocking all DNA pairing activity and thereby abridging participation in recombination function. Thus, Umu(D')2C restricts the activities of the RecA nucleoprotein filament and presumably, in this manner, recruits it for mutagenic repair function. This modulation by Umu(D')2C is envisioned as a key event in the transition from a normal mode of genomic maintenance by "error-free" recombinational repair, to one of "error-prone" DNA replication.     

Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus

The biochemical properties of a second protein (CM2) encoded by RNA segment 6 of influenza C virus were investigated. Three forms of CM2 with different electrophoretic mobilities (CM2(0), CM2a, and CM2b) were detected in infected cells by immunoprecipitation with antiserum to the glutathione S-transferase (GST)-CM2 fusion protein. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with endoglycosidase H or peptide-N-glycosidase F suggested that a mannose-rich oligosaccharide core is added to unglycosylated CM2(0) (Mr, approximately 16,000) to form CM2a (Mr, approximately 18,000) and that the processing of the carbohydrate chain from the high-mannose type to the complex type converts CM2a into CM2b, which is heterogeneous in electrophoretic mobility (Mr, approximately 22,000 to 30,000). Labeling of infected cells with [3H]palmitic acid showed that CM2 is fatty acylated. The fatty acid bond was sensitive to treatment with hydroxylamine and mercaptoethanol, which indicates a labile thioester-type linkage. The CM2 protein was also found to form disulfide-linked dimers and tetramers on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Trypsin treatment of infected cell surfaces as well as of microsome vesicles from infected cells followed by immunoprecipitation with antiserum to the GST fusion protein containing the 56 C-terminal amino acid residues of CM2 suggested that this C-terminal domain is intracellular and exposed to the cytoplasms of microsomes. Furthermore, evidence that a small amount of CM2 is incorporated into progeny virus particles was obtained by Western blot analysis. These results, altogether, suggest that CM2 is an integral membrane protein with biochemical properties similar to those of influenza A virus M2 and influenza B virus NB proteins.