Heterogeneity of an influenza virus population in the manifestation of biological functions (author's transl)]

In a population of influenza virus, virions have different degree of the functional capacities to produce infectious virus, to induce neuraminidase production and to interfere with infectious virus. Fractions of virus were found which had a poor capacity for production of infectious progeny and a relatively high induction of neuraminidase synthesis as well as induction of heterogenous light fractions of RNA in cells. In the population of defective virus a fraction of particles possessing marked interfering activity in the absence of infectivity was found.     

Characterisation of an avian influenza virus nucleoprotein expressed inE. coli and in insect cells

The nucleoprotein (NP) gene from influenza virus A/Shearwater/Australia/72 has been expressed intracellularly in both E. coli and insect cells. E. coli-derived NP was identified by Western blot analysis as a 56 kDa protein which co-migrates with virion-derived NP. This protein was purified by immunoaffinity chromatography and a nitrocellulose binding assay showed that NP formed complexes with positive- and negative-sense influenza neuraminidase RNA transcribed in vitro. ELISA and Western blot analysis revealed that recombinant NP of 56 kDa was produced in high yields in insect cells using a baculovirus vector. Immunofluorescence microscopy revealed that NP was localised to the nucleus of infected insect cells.     

Translational regulation of influenza virus mRNAs

cDNAs for genome RNAs of influenza virus A/PR/8/34 were cloned, and portions containing the ATG for initiation codon of translation were inserted into the 5 leader sequence of the chloramphenicol acetyltransferase (CAT) gene in a pSV2cat vector. When transfected cells were super-infected with influenza virus, the CAT activity was found to vary in a time-dependent fashion: A construct containing a cDNA segment for the nonstructural (NS) protein directed the highest activity during the early stage of infection, while a construct containing a cDNA segment for the neuraminidase (NA) directed the highest activity during the late stage of infection. This time-dependent variation in the CAT activity is in good agreement with that of the synthesis rate of respective viral proteins in infected cells. We propose that the translational efficiency of viral mRNA is subjected to temporal control following viral infection, although viral protein synthesis itself is regulated primarily at the level of mRNA synthesis.     

Intracellular localization of influenza C virus NS2 protein (NEP) in infected cells and its incorporation into virions

RNA segment 7 of influenza C virus encodes two non-structural (NS) proteins, NS1 and NS2. The influenza C virus NS2 protein has been proposed to possess nuclear export activity like that of influenza A and B virus NS2 proteins (NEP). In the present study, we investigated the kinetics and localization of the NS2 protein in influenza C virus-infected cells, and analysed whether NS2 is present in virions. Immunofluorescent staining analysis of the infected cells indicated that NS2 was localized in the nucleus immediately after synthesis and predominantly in the cytoplasm in the later stages of infection. Confocal microscopy revealed that a part of the NS2 protein was colocalized with nucleoprotein NP/vRNP in the cytoplasm and on the cell membrane in the late stages of infection. The NS2 protein was detected in influenza C virions purified by gradient centrifugations and/or affinity chromatography. Trypsin treatment demonstrated that the NS2 protein was present inside the viral envelope. Furthermore, glycerol gradient analysis of detergent-solubilized virions revealed that the NS2 protein cosedimented with vRNPs. These data suggest that the influenza C virus NS2 protein is incorporated into virions, where it associates with vRNP.     

Polypeptide composition of incomplete influenza virus grown in MDBK cells.

The polypeptide composition of incomplete WSN influenza virions grown in MDBK cells is altered relative to a control grown at low m.o.i. The relative amount of viral glycoproteins in the third and fourth serial undiluted passages were increased to approximately twice the level in control virus, while the NP and M proteins were decreased. Neuraminidase activity was also increased, suggesting that the level of NA (neuraminidase) glycoprotein is also higher in incomplete virions. The increase in neuraminidase activity was already evident in the first undiluted passage, where no substantial increase in overall glycoprotein content was demonstrated. Similar alterations in protein composition were seen in several different preparations exhibiting different morphological aberrations and in incomplete particles that were of normal morphology. These results permitted the following conclusions regarding incomplete WSN virus grown in MDBK cells: (1) Neuraminidase and HA (hemagglutinin) glycoprotein levels are regulated independently in the incomplete virion; (2) the level of these proteins in the virion can be influenced by growth conditions; (3) the glycoproteins do not play a primary role in determining particle morphology.     

甲型流感病毒核蛋白基因的克隆表达及纯化

目的　将甲型流感病毒核蛋白(NP)基因克隆到原核表达载体进行可溶性融合表达,制备纯化的病毒核蛋白,为制备甲型流感病毒单克隆抗体提供材料。方法　提取甲型流感病毒RNA ,设计引物,RT PCR扩增NP基因,利用基因工程的手段,将甲型流感病毒的NP基因在大肠埃希菌中进行融合表达,并将表达产物进行亲和层析。结果　成功构建了甲型流感病毒NP基因原核表达载体,经亲和层析制备了较高纯度的目的表达产物。结论　通过合理控制发酵时间、生长温度和诱导物浓度,制备了较为理想的可溶性甲型流感病毒核蛋白。     

Avian and human influenza a virus strains possess different intracellular nucleoprotein oligomerization efficiency

We have previously shown (Prokudina-Kantorovich EN and Semenova NP, Virology 223, 51-56, 1996) that the nucleoprotein (NP) of influenza A virus forms in infected cells oligomers which in the presence of SDS and 2-mercaptoethanol (ME) as reducing agent are stable at room temperature (RT) and dissociate at 100 degrees C. Here we report that the efficiency of intracellular NP oligomerization depends on the host origin of influenza A virus strain. Thus, in the cells infected with avian influenza A virus strains the viral NP was almost completely oligomerized and only traces of monomeric NP were detected by polyacrylamide gel electrophoresis (PAGE) in unboiled samples. However, in the cells infected with human influenza A virus strains, besides oligomeric NP also a significant amount of non-oligomerized monomeric NP was detected in unboiled samples. In purified virions of avian and human strains the same difference in NP monomers/oligomers ratio was detected as in the infected cells. A reassortant having all internal protein genes from a human strain and the glycoprotein genes from an avian strain revealed the same intracellular pattern of NP monomers/oligomers ratio as its parental human virus. These findings suggest that the type of NP oligomerization is controlled by the NP gene. The possible connection between the accumulation of protease-sensitive monomeric NP in cells infected with a human influenza strain and the parallel accumulation of cleaved NP in these cells is discussed.     

Influenza virus proteins. Incorporation of newly synthesized viral proteins into virions

Incorporation of individual viral proteins (P1, P2, P3, NP, NA, HA, and M) into virions was studied by the "pulse-chase" method followed by electrophoretic analysis of the proteins in chick fibroblast cultures infected with influenza WSN virus. Viral proteins were found to incorporate into virions rather rapidly and for a long time after synthesis, with the exception of M protein incorporation of which into the virus markedly decreased by 4 hours after synthesis. Nucleocapsid proteins (NP, P1, P2, and P3) incorporated into the virus asynchronously: by 17 hours postinoculation, in NP protein prevailed the molecules synthesized after 6 hours of infection, in P2 protein the molecules synthesized in the first hours of infection, and P1 and P3 proteins were represented by a uniform set of molecules synthesized at various periods after inoculation. The evidence on heterogeneity of P2 and M proteins in the virus has been obtained. Possible mechanisms regulating the selection of molecules of individual viral proteins in formation of virus particles are discussed.     

The fourth genus in the Orthomyxoviridae: sequence analyses of two Thogoto virus polymerase proteins and comparison with influenza viruses

The tick-borne Thogoto virus (THOV) is the type species of a newly recognized fourth genus, Thogotovirus, in the family Orthomyxoviridae. Because of the distant relationship of THOV with the influenza viruses, determination of its genomic information can potentially be used to identify important domains in influenza virus proteins. We have determined the complete nucleotide sequence of the second longest RNA segment of THOV. The molecule comprises 2212 nucleotides with a single large open reading frame encoding a protein of 710 amino acids, estimated M_r 81 284. The protein shares 77% amino acid similarity with the PB1-like protein of Dhori virus, a related tick-borne virus, and 50–53% with the PB1 polymerase proteins of influenza virus A, B and C. All the motifs characteristic of RNA-dependent polymerases were identified including the SSDD motif common to all RNA-dependant RNA polymerases, indicating that the THOV protein is functionally analogous to the influenza virus PB1 proteins and involved in chain elongation. We also report the corrected sequence of the third longest RNA segment of THOV, encoding a protein which shares 44–47% amino acid similarity with the PA-like polymerase proteins of influenza virus A, B and C. The biological significance of conserved domains in these orthomyxovirid proteins is discussed.     

Characterization of the host cell entry of filamentous influenza virus

Summary. Most laboratory-adapted strains of influenza virus exist as spheres of approximately 100 nm in diameter, which are well established to enter cells by endocytosis in a pH-dependent manner. However, influenza virus isolated from the lungs of infected individuals is believed to exist as predominantly filamentous particles, up to several micrometers in length. Here, we have attempted an initial characterization of the entry of purified influenza virus filaments into host cells – in comparison to more commonly studied spherical forms of the virus. We demonstrate that the internalization of filamentous influenza virus particles is delayed, relative to spherical particles, and that this delay is a result of morphological rather than strain differences. The filamentous influenza particles appear to retain their dependence on low-pH for entry, as demonstrated by a vacuolar-ATPase inhibitor, and viral trafficking to late endosomes, as demonstrated by the requirement for protein kinase C function. However, our data suggest that the endocytic uptake of the filamentous virus particles may be dynamin-independent, unlike spherical virions. Overall, these data provide a view of the entry of influenza virus in its filamentous morphology, demonstrating potential differences between the endocytosis of spherical virions in vitro and filamentous virions in vivo.     

Effects of hexose starvation and the role of sialic acid in influenza virus release

We previously reported that growth of influenza virus in the presence of cytochalasin B (CB), a drug that disrupts microfilaments and blocks hexose transport, yields particles with glycoproteins that are heterogeneous and unlabeled by [3H]glucosamine. When the virus was grown in glucose-free medium, we observed reduced virus titers similar to those produced by CB. In contrast, treatment of cells with cytochalasin D (CD) and dihydrocytochalasin B (H2CB), drugs which are known to inhibit microfilament function without affecting hexose transport, did not cause a reduction in virus titers or a change in the electrophoretic mobility of viral glycoproteins. Partial inhibition of glycosylation of viral glycoproteins resulting from either CB-induced inhibition of hexose transport or from glucose starvation resulted in the formation of aggregates of virions on cell surfaces. These aggregates can be dissociated by exogenous neuraminidase. Under these conditions the virions contained a functional hemagglutinin glycoprotein (HA) but an inactive neuraminidase glycoprotein (NA) which was not able to cleave sialic acid, the HA receptor, from viral glycoproteins, or from cellular glycoproteins and glycolipids. Neuraminidase treatment of membrane fractions of CB-treated cells did not cause a shift in the electrophoretic mobility of HA or in the gel elution profile of HA glycopeptides obtained after extensive pronase digestion from HA synthesized in glucose-free medium. These findings suggest that sialic acid is not present on labeled glycoproteins in either of these preparations. We obtained evidence that the sialic acid to which HA binds when NA is inactive is on glycoproteins and glycolipids of cellular origin. Our results support the idea that even when NA is functional, sialylated cellular components impede influenza virus release.     

Flocculation of influenza virus by a neuraminidase inhibitor, neuraminin, produced by Streptomyces sp.

Flocculation of virions by a viral neuraminidase inhibitor, neuraminin, was examined by low speed centrifugation and electron microscopy. The inhibitor could flocculate virions of influenza viruses but not those of Newcastle disease viruses. Virion-free neuraminidase of influenza virus was also flocculated by this inhibitor. The flocculation was temperature dependent and proceeded rapidly over a wide pH range. The amount of the inhibitor required for complete flocculation of virions was similar to that for the maximum inhibition of neuraminidase. Viral neuraminin receptors were stable to various enzymological, physical, and chemical treatments which caused a loss or reduction of the activity of neuraminidase.     

Ultrastructure and some biological properties of influenza A virus. III. The role of influenza A virus lipids in interferon induction

Experiments were carried out with A/USSR/053/74/H3N2 influenza virus. To remove lipids from virus particles, the enzyme phospholipase C, butanol-ether and the detergent cytylpyridinum chloride were used. Mild treatment of the influenza virus with phospholipase C resulted in a slight decrease in the activity of both surface antigens of the virions as well as in a suppression of their infectivity. Such virus is capable of IFN induction in mouse. Long-term (25 h) treatment of the virus with phospholipase C caused gross destruction of virions. Thus, despite the preserved neuraminidase activity the preparation was no longer capable of IFN induction. The treatment with butanol-ether and the detergent cytylpyridinum chloride brought similar results--fully preserved hemagglutination and neuraminidase activity and a complete loss of IFN induction.     

Human monoclonal single chain antibodies (HuScFv) that bind to the polymerase proteins of influenza A virus

Current anti-influenza drugs target the viral neuraminidase or inhibit the function of the ion channel M2 protein. Not only is the supply of these drugs unlikely to meet the demand during a large influenza epidemic/ pandemic, but also has an emergence of drug resistant influenza virus variants been documented. Thus a new effective drug or antiviral alternative is required. The influenza virus RNA polymerase complex consists of nucleoproteins (NP) that bind to three polymerase subunits: two basic polymerases, PB1 and PB2, and an acidic polymerase (PA). These proteins play a pivotal role in the virus life cycle; thus they are potential targets for the development of new anti-influenza agents. In this study, we produced human monoclonal antibodies that bound to the influenza A polymerase proteins by using a human antibody phage display library. Complementary DNA was prepared from the total RNA of a highly pathogenic avian influenza (HPAI) virus: A/duck/Thailand/144/2005(H5N1). The cDNA synthesized from the total virus RNA was used as template for the amplification of the gene segments encoding the N-terminal halves of the PB1, PB2 and PA polymerase proteins which encompassed the biologically active portions of the respective proteins. The cDNA amplicons were individually cloned into appropriate vectors and the recombinant vectors were introduced into Escherichia coli bacteria. Transformed E. coli clones were selected, and induced to express the recombinant proteins. Individually purified proteins were used as antigens in bio-panning to select the phage clones displaying specific human monoclonal single chain variable fragments (HuScFv) from a human antibody phage display library constructed from Thai blood donors in our laboratory. The purified HuScFv that bound specifically to the recombinant polymerase proteins were prepared. The inhibitory effects on the biological functions of the respective polymerase proteins should be tested. We envisage the use of the HuScFv in their cell penetrating version (transbodies) as an alternative influenza therapeutic to current anti-virus drugs.     

Polypeptides specified by the influenza virus genome: I. Evidence for eight distinct gene products specified by fowl plague virus

The structural polypeptides of fowl plague virus (influenza A) and those synthesized in fowl plague virus-infected chick embryo fibroblasts have been analyzed by high resolution polyacrylamide gel electrophoresis. We detected eight distinct virus gene products: three polymerase-associated polypeptides (P₁, P₂, P₃), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), membrane polypeptide (M), and a nonstructural polypeptide (NS). The molecular weights of these polypeptides correlate closely with the molecular weights of the eight genome RNA species found in fowl plague virus.The three high molecular weight polypeptides, P₁, P₂, and P₃, were detected both in virions and infected cells, and their separate identity established by a two-dimensional tryptic peptide mapping procedure. An active RNA polymerase enzyme complex isolated from virions and virus-infected cells contained all three P proteins together with the NP protein. The nonstructural polypeptide (NS), together with the P proteins and the NP, appeared early in the infectious cycle, while the M protein and HA protein appeared later in infection. The NS and M polypeptides, which have similar molecular weights, were separated on SDS-polyacrylamide gels and shown to be distinct by tryptic peptide mapping.     

Avian cells expressing the Newcastle disease virus hemagglutinin-neuraminidase protein are resistant to Newcastle disease virus infection

The cDNA derived from the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene was inserted into a replication-competent Schmidt-Ruppin Rous sarcoma virus-derived vector. Chick embryo cells transfected with this vector expressed HN-sized protein which could be precipitated with anti-HN antibody. These cells adsorbed avian red blood cells and the cell surfaces exhibited neuraminidase activity while cells transfected with an antisense version of the gene were negative for hemadsorption and neuraminidase. The cells transfected with the retroviral vector containing the HN gene were resistant to infection by NDV and influenza virus, viruses which bind to sialic acid containing receptors, but sensitive to vesicular stomatitis virus (VSV). Cells transfected with the antisense version of the HN gene were sensitive to NDV, influenza virus, and VSV infection. Thus the HN protein-expressing cells are likely resistant to NDV and influenza virus due to the destruction of the cellular receptors by the neuraminidase of the HN protein. The expression of the influenza virus HA protein using the same retrovirus vector has been reported previously (L. A. Hunt, D. W. Brown, H. L. Robinson, C. W. Naeve, and R. G. Webster, 1988, J. Virol. 62, 3014-3019). Cells infected with this vector were sensitive to infection with influenza virus, NDV, and VSV. Thus expression of a viral surface protein does not necessarily confer resistance of the cell to the homologous virus.     

The selective inactivation of influenza virus haemagglutinin by pyridine

Summary Pyridine has been shown to selectively inactivate the haemagglutinating activity of influenza virus. V-antigen and neuraminidase activity were hardly affected and the virions maintained their basic integrity, although some change in morphology was evident. The pyridine treated virus particles obtained were thus unlike the products of virus degradation reported with other solvents or biological agents. Production of multivalent neuraminidase components with no haemagglutinating activity provided the first unequivocal demonstration that neuraminidase does not contribute to the haemagglutinating activity of native virions. The pyridine treated virus retained its antigenic ability although its antigenicity was less than that of the normal virus.     

Use of FITC-labeled influenza virus and flow cytometry to assess binding and internalization of virus by monocytes-macrophages and lymphocytes

Summary The binding of influenza virus to the surface of cells and the internalization of virus particles by all or a subset of cells are key points in the pathogenesis of viral infection. The current studies established a method for discrimination of surface-bound from internalized influenza virus. Fluorescein isothiocyanate (FITC) was attached to the viral hemagglutinin and neuraminidase proteins; the fluorescent virus retained infectivity. A flow cytometric technique was then adapted for study of virus-cell interactions, with addition of ethidium bromide to quench green fluorescence associated with FITC-labeled virus that was cell-bound but remained external. Ethidium bromide was excluded by intact cell membranes, and internalized virions retained green fluorescence. Cells could be examined by fluorescence microscopy or flow cytometry, with flow cytometry allowing rapid, kinetic assessment of large numbers of cells and subsets of virus-exposed cells. The data showed that, whereas a majority of both monocytes-macrophages and lymphocytes bound influenza virus, a large percentage of monocytes-macrophages but only a very small percentage of lymphocytes internalized the virus. This procedure provides a simple and effective method to distinguish surface-bound from internalized influenza virus, and allows precise kinetic analyses on large numbers of cells.