Influenza C and D Viruses Package Eight Organized Ribonucleoprotein Complexes.

Influenza A and B viruses have eight-segmented, single-stranded, negative-sense RNA genomes, whereas influenza C and D viruses have seven-segmented genomes. Each genomic RNA segment exists in the form of a ribonucleoprotein complex (RNP) in association with nucleoproteins and an RNA-dependent RNA polymerase in virions. Influenza D virus was recently isolated from swine and cattle, but its morphology is not fully studied. Here, we examined the morphological characteristics of D/bovine/Yamagata/10710/2016 (D/Yamagata) and C/Ann Arbor/50 (C/AA), focusing on RNPs packaged within the virions. By scanning transmission electron microscopic tomography, we found that more than 70% of D/Yamagata and C/AA virions packaged eight RNPs arranged in the "1+7" pattern as observed in influenza A and B viruses, even though type C and D virus genomes are segmented into only seven segments. These results imply that influenza viruses generally package eight RNPs arranged in the "1+7" pattern regardless of the number of RNA segments in their genome.IMPORTANCE The genomes of influenza A and B viruses are segmented into eight segments of negative-sense RNA, and those of influenza C and D viruses are segmented into seven segments. For progeny virions to be infectious, each virion needs to package all of their genomic segments. Several studies support the conclusion that influenza A and B viruses selectively package eight distinct genomic RNA segments; however, the packaging of influenza C and D viruses, which possess seven segmented genomes, is less understood. By using electron microscopy, we showed that influenza C and D viruses package eight RNA segments just as influenza A and B viruses do. These results suggest that influenza viruses prefer to package eight RNA segments within virions independent of the number of genome segments.

Morphology of influenza B/Lee/40 determined by cryo-electron microscopy.

Cryo-electron microscopy projection image analysis and tomography is used to describe the overall architecture of influenza B/Lee/40. Algebraic reconstruction techniques with utilization of volume elements (blobs) are employed to reconstruct tomograms of this pleomorphic virus and distinguish viral surface spikes. The purpose of this research is to examine the architecture of influenza type B virions by cryo-electron tomography and projection image analysis. The aims are to explore the degree of ribonucleoprotein disorder in irregular shaped virions; and to quantify the number and distribution of glycoprotein surface spikes (hemagglutinin and neuraminidase) on influenza B. Projection image analysis of virion morphology shows that the majority (∼83%) of virions are spherical with an average diameter of 134±19 nm. The aspherical virions are larger (average diameter = 155±47 nm), exhibit disruption of the ribonucleoproteins, and show a partial loss of surface protein spikes. A count of glycoprotein spikes indicates that a typical 130 nm diameter type B virion contains ∼460 surface spikes. Configuration of the ribonucleoproteins and surface glycoprotein spikes are visualized in tomogram reconstructions and EM densities visualize extensions of the spikes into the matrix. The importance of the viral matrix in organization of virus structure through interaction with the ribonucleoproteins and the anchoring of the glycoprotein spikes to the matrix is demonstrated.

Disassembly of influenza C viruses, distinct from that of influenza A and B viruses requires neutral-alkaline pH.

Influenza C (Flu C) viruses comprise an internal ribonucleoprotein (RNP) and an outer lipoprotein envelope with surface spike glycoproteins and the M1 protein matrix. The lipoprotein envelope and spike glycoproteins are solubilized by nonionic detergent in a pH-independent manner. In contrast, disassembly of the M1 protein matrix appears to depend on pH. Treatment of Flu C viruses with nonionic detergent in neutral or alkaline medium (pH 9.0-7.4) results in disintegration of the virion M1 matrix and leads to a significant release of RNP free of the M1 protein. In acidic medium (pH 6.0-5.0), the M1 matrix is not removed and the viral core-like complex of RNP along with the M1 matrix cover is released. Since Flu A and B viruses were characterized by acid-dependent disassembly of the virion M1 matrix, Flu C viruses seem to resemble the paramyxoviruses, which also show a neutral-alkaline pH dependence on matrix disintegration. These observations suggest that uncoating mechanisms of influenza C viruses and paramyxoviruses in target cells may be similar.

Mutations in the cytoplasmic tail of influenza A virus neuraminidase affect incorporation into virions.

The significance of the conserved cytoplasmic tail sequence of influenza A virus neuraminidase (NA) was analyzed by the recently developed reverse genetics technique (W. Luytjes, M. Krystal, M. Enami, J. D. Parvin, and P. Palese, Cell 59:1107-1113, 1989). A chimeric influenza virus A/WSN/33 NA containing the influenza B virus cytoplasmic tail rescued influenza A virus infectivity. The transfectant virus had less NA incorporated into virions than A/WSN/33, indicating that the cytoplasmic tail of influenza virus NA plays a role in incorporation of NA into virions. However, these results also suggest that the influenza A virus and influenza B virus cytoplasmic tail sequences share common features that lead to the production of infectious virus. Transfectant virus was obtained with all cytoplasmic tail mutants generated by site-directed mutagenesis of the influenza A virus tail, except for the mutant resulting from substitution of the conserved proline residue, presumably because of its contribution to the secondary structure of the tail. No virus was rescued when the cytoplasmic tail was deleted, indicating that the cytoplasmic tail is essential for production of the virus. The virulence of the transfectant viruses in mice was directly proportional to the amount of NA incorporated. The importance of the NA cytoplasmic tail in virus assembly and virulence has implications for use in developing antiviral strategies.

Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.

Electron microscopic evidence for the association of M2 protein with the influenza virion.

Immunogold electron microscopy revealed that site-specific antibodies elicited by a synthetic peptide representing the N-terminal sequence (residues 2-10) of influenza virus M2 protein were capable of binding to the surface of virions. Antibody binding was observed with two human influenza virus strains but not with an avian virus strain which has amino acid substitutions in the appropriate sequence of M2. These results provide direct evidence for the presence of M2 in the influenza virion.

Polylactosaminoglycan modification of a small integral membrane glycoprotein, influenza B virus NB.

The structure of the carbohydrate components of NB, the small integral membrane glycoprotein of influenza B virus, was investigated. The carbohydrate chains of NB are processed from the high-mannose form (NB18) to a heterogeneous form of much higher molecular weight, designated NBp. Selection of this carbohydrate-containing form of NB with Datura stramonium lectin, its susceptibility to digestion by endo-beta-galactosidase, and determination of the size of NBp glycopeptides by gel filtration chromatography suggested that the increase in molecular weight is due to processing to polylactosaminoglycan. Investigation of the polypeptides produced by influenza B/Lee/40 virus infection of several cell types and another strain of influenza B virus suggested that the signal for modification to polylactosaminoglycan is contained in NB. Expression of mutants of NB lacking either one or both of the normal N-terminal sites of asparagine-linked glycosylation indicated that both carbohydrate chains are modified to contain polylactosaminoglycan. NBp and a small amount of unprocessed NB18 are expressed at the infected-cell surface, as determined by digestion of the surfaces of intact cells with various endoglycosidases. Unglycosylated NB, expressed either in influenza B virus-infected cells treated with tunicamycin or in cells expressing the NB mutant lacking both N-linked glycosylation sites, was expressed at the cell surface, indicating that NB does not require carbohydrate addition for transport.

Analysis of the mechanism of influenza B virus inactivation by guinea pig serum.

Normal guinea pig serum lacking detectable antiviral antibody inactivated influenza B virus via the classical complement pathway. This virus inactivation appeared to result from the steric hindrance of HA activity by the association with the virus of serum proteins presumed to be complement components. Trypsin digestion of the associated proteins fully restored the HA activity but not infectivity. It was found that the virus underwent minor disruption of the envelope and degradation of M1 protein and genomic RNA.

Electron microscopy of influenza virus. A comparison of negatively stained and ice-embedded particles.

An electron microscopical study was made of the influenza virus, type B/Hong Kong, in the unstained, frozen, hydrated state after quench-freezing in cooled liquid ethane. The results are compared with data from negatively stained specimens. It is shown that cryo-electron microscopy confirms and extends the data obtained by conventional methods. In particular, the virus is shown to be circular in projection with no indication of icosahedral symmetry, the lipid membrane is clearly resolved as a bi-layer and it is demonstrated that the distribution of material within the bi-layer is non-uniform, with a shell of more electron dense material surrounding a less dense central region. Neuraminidase spikes are not clearly distinguished from haemaglutinin spikes. The diameter of the complete B/Hong Kong virus was estimated from cryo-micrographs as 1270(+/- 70) A. Some preliminary data for influenza virus type A/X31 are presented.

[Characteristics of the glycoproteins of vaccinal strains of the influenza viruses solubilized by octyl-beta-D-glucopyranoside]. / Kharakteristika glikoproteidov vaktsinnykh shtammov virusov grippa, soliubilizirovannykh oktil-beta-D-gliukoprianozidom.

The use of a nonionic detergent, octyl-beta-d-glucopyranoside (OG), under optimal conditions is highly effective in selective solubilization of membrane proteins (glycoproteins) of influenza A and B viruses, including the strains currently recommended for polytype inactivated influenza vaccines. The yield of hemagglutinin was 84-90% of the initial level in the virion concentrate. The antigenic stability of hemagglutinin of the A/H3 subtype upon storage for over 2 years and the A/H1 subtype for about 1 year (the observation period) was demonstrated. According to the results of electron microscopic studies, the glycoproteins solubilized with OG are present before removal of the detergent in the form of isolated subunits, "rosettes", and their agglomerates, and after removal of the detergent in the form of virosomes (clumps of subunits on liposomes) more heterogeneous in size (20 to 130 nm) and shape than the intact virus. The process of virosome formation is reversible and depends on the presence of the detergent. The resulting glycoproteins of influenza virus vaccine strains solubilized with the OG detergent possess the characteristics which make this agent promising for use in the manufacture of polytype subunit vaccine.

Changes in the morphology of influenza particles induced at low pH.

At low pH influenza virus causes membrane fusion. This phenomenon is thought to reflect a part of the infection mechanism of the virus. To obtain more information on the effect of low pH on the virus, the change in morphology of influenza virus particles was studied by electron microscopy. Further, the degradation of haemagglutinin (HA) after trypsin digestion as a function of pH was studied by gel electrophoresis. The results showed that a threshold value existed below which both a change in morphology and an increase in trypsin sensitivity were observed. This threshold pH was found to be strain specific. A number of strains showed a heterogeneity in the particle population with respect to the threshold pH. The various subpopulations appeared to differ genetically. Virus particles with uncleaved precursor HA, HAo, were not effected by the low pH treatment.