Oligomeric organization and strain-specific proteolytic modification of the virion M2 protein of influenza A H1N1 viruses.

The M2 protein of influenza A H1N1 strains PR8, WS, and WSN is present in homooligomeric forms in virions grown in the allantoic cavity of embryonated eggs. The bulk of the virion M2 is detected as tetramers and dimers. The oligomeric forms of PR8 virions differ from those of WS and WSN not only in apparent molecular weight (MW) but also in that they seem to be composed of two types of monomers differing in MW by approximately 1.5 kDa. Evidence from monoclonal antibody binding (or lack of it) and from in vitro trypsin digestion suggests that, in ovo, the external NH2 region of some PR8 M2 monomers is proteolytically trimmed, resulting in heterogeneous oligomers composed of cleaved and uncleaved monomers in different proportions.

Replication of H1N1 influenza viruses in cultured mouse embryo brain cells: virus strain and cell differentiation affect synthesis of proteins encoded in RNA segments 7 and 8 and efficiency of mRNA splicing.

The aims of these studies are (1) to determine whether, and by what mechanism(s), underexpression of M1 and/or NS1 protein restricts replication and cytopathogenicity in mouse brain cells of human influenza viruses which are closely related to the neurovirulent WSN variant but not selected for the neurovirulent phenotype; (2) to learn, ultimately, whether similarly restricted replication in natural infections might be enough to cause direct or indirect, immunologically mediated, neuropathology. On the basis of immunostaining, we have suggested that, in "aged" mouse embryo brain (MEB) cell cultures infected with A/PR/8/34 (PR8) or A/WS/33 (WS), M1 protein expression is restricted mainly in mature astrocytes (the dominant cell type in such cultures), but not in mature oligodendrocytes or neurons. Here we show that amounts of radiolabeled M1 protein in lysates of MEB cultures infected with PR8, WS, or WSN differ in proportion to previously reported single-cycle yields of trypsin-activated infectious virions. Low or undetectable cell-associated M1 does not reflect accelerated degradation, but tends to be accompanied by increased M2 protein (a product of spliced mRNA7). Radiolabeled NS1 is reduced, NS2 relatively increased, in "aged" MEB cultures infected at low m.o.i. with PR8, at high m.o.i. with WS as well, but not with WSN. In contrast, actively dividing and differentiating astrocyte-enriched or "young" MEB cultures tend to produce greatly increased amounts of NS2 even though NS1 may be at "normal" levels, both relative to those in similarly infected CEF cultures. We show, in extension of comparative studies by others on permissive and abortive FPV-infected cell systems, that virus strain-, cell type-, and perhaps differentiation-dependent variations in efficiency of mRNA 7 and 8 transcription and/or splicing are primary factors controlling variable expression of M and NS proteins in mouse brain cell cultures.

Role of hemagglutinin cleavage and expression of M1 protein in replication of A/WS/33, A/PR/8/34, and WSN influenza viruses in mouse brain.

The combined presence of WSN gene segments 6 (neuraminidase), 7 (M1 and M2), and 8 (NS1 and NS2) in reassortants of WSN with A/Aichi/2/68 (H3N2) has been found by others to be necessary for full expression of neurovirulence in mice. We are examining the expression of the analogous three gene segments in brains of mice after intracerebral infection with non-neuroadapted strains A/WS/33 (WS) (from which WSN was derived) and A/PR/8/34 (PR8). Our aim is to determine possible mechanisms by which one or more of the five gene products may restrict replication of these strains in mouse brain cells to a single cycle, yielding noninfectious hemagglutinating particles (incomplete growth cycle). We found that minority subsets of such particles did produce plaques, provided they were activated by trypsin (analogous to other abortive systems producing virions with uncleaved HA), a step obviated for some WSN virions by indirect promotion of hemagglutinin cleavage by the neuraminidase of that strain. The percentage of such potentially infectious virions, relative to total hemagglutinating particles, was significantly lower in WS- or PR8-infected than in WSN-infected brains, suggesting possible defects in synthesis or function of M1 protein in the former. Cells in immunostained sections and appropriate bands in Western blots (immunoblots) of viral proteins electrophoretically separated from lysates of PR8-infected brains reacted with antibody to nucleoprotein but not to M1 protein. Either method revealed the presence of both proteins in WSN-infected brains. In contrast, Western blot analyses of particles concentrated from PR8-, WS-, or WSN-infected brains by hemadsorption, elution, and pelleting did reveal NP and M1 bands with comparable relative peroxidase-antiperoxidase staining intensities. The findings suggest that availability of M1 protein is a factor influencing the extent or rate of assembly of potentially infectious (i.e., trypsin-activated) progeny virions in mouse brains and that in this respect the two non-neurovirulent strains differ from WSN quantitatively rather than qualitatively.

Effects of cell differentiation on replication of A/WS/33, WSN, and A/PR/8/34 influenza viruses in mouse brain cell cultures: biological and immunological characterization of products.

The responses of mouse embryo brain (MEB) cell cultures and of Madin-Darby canine kidney cells and chicken embryo fibroblasts to infection with A/PR/8/34 (PR8), A/WS/33 (WS), or the neurovirulent WSN variant were compared in terms of (i) single-cycle yields of hemagglutinating and associated neuraminidase (NA) activities and plaque-forming particles, the latter with or without trypsin activation [PFU(TR++) or PFU(TR--), respectively], and (ii) expression of nucleoprotein (NP), M1, and NS1 protein, determined for specific cell types by immunostaining, for whole culture lysates by Western blot analysis of NP and M1. Primary MEB cultures grown in serum-enriched medium were infected after 6 days (young), when none of the cells reacted specifically and exclusively with any of the nerve cell marker antibodies used, or after greater than or equal to 21 days (aged), when astrocytes (the predominant cell type), neurons, and oligodendrocytes were morphologically and immunologically mature. Secondary astrocyte-enriched cultures were used when they contained 90 to 99% of their cells as astrocytes at an early stage of differentiation. By all criteria, young MEB cultures were only marginally less permissive for each of the three viruses than were chicken embryo fibroblasts or Madin-Darby canine kidney cells. Aged MEB cultures, by comparison, produced undiminished NP, hemagglutinin, and neuraminidase, but yields of PFU(TR++) and expression of M1 protein (relative to NP) were reduced for all three viruses, most for PR8 and least for WSN; relative reduction of NS1 protein was demonstrable only in PR8-infected aged cultures. Immunostaining revealed low levels of M1 and NS1 expression only in astrocytes, not in oligodendrocytes and neurons. In PR8-infected mature astrocytes, NP accumulated in the nucleus; it persisted in some cells for at least 8 weeks after infection. The presence of NP did not seem to interfere with cell division. Secondary MEB cultures containing 90 to 99% immature astrocytes were less restricted than were aged primary cultures. Thus, it appears that reduced permissivity of nerve cell cultures, as measured in this study, is most closely correlated with advancing differentiation and maturity of astroglial cells. Assembled virions, including those that score as PFU(TR++) in restricted cultures (e.g., PR8-infected aged MEB), may be mainly products of mature oligodendrocytes and neurons.

Nomenclature of flavivirus-specified proteins.

Flavivirus structural proteins are designated E, C, and M, replacing the former nomenclature of V3 (envelope), V2 (core), and V1 (membrane-like) proteins, respectively. The nonstructural proteins, formerly NV1 through NV5, are designated by their apparent size in kilodaltons, prefixed by P (or GP if a glycoprotein), if they are stable unrelated end-products; the lower case p or gp is used for variants or uncharacterized products. A relationship to any structural protein is indicated by adding E, C, or M parenthetically to the designation of the appropriate nonstructural protein.

The significance and nature of defective interfering viruses.

Deletions in viral genomes appear to be a common occurrence in the replication of all DNA and RNA viruses which have been adequately studied. Such defective genomes can replicate in the presence in the same cell of a helper virus as long as the deletion does not involve the initiation site for genome replication. Coinfection of a cell with defective and "normal" infectious virus leads to reduction in the yield of the latter. The nature of DI viruses and genomes found in Sindbis virus-infected vertebrate cells during "undiluted passage" series is discussed. This procedure leads to the accumulation of progressively shorter viral RNA genomes with internal deletions. The enrichment is limited to genome lengths which are integral fractions (1/2, 1/3, 1/4, etc.) of the complete genome, and these are also found in viral particles released at the corresponding passage levels. It is believed that the selective accumulation of these fragments is governed by constraints of assembly which demand that one full genome equivalent be packaged in a released particle. In contrast to vertebrate cells, cultured mosquito cells do not seem to produce or "recognize" DI particles. Possible implications for the epidemiology of arthropod-transmitted alphaviruses are presented.

Sindbis virus replication in vertebrate and mosquito cells: an interpretation.

This paper summarizes recent comparative studies of Sindbis virus (SV) replication in cultured Aedes albopictus (A. albo) or (A. aegypti (A. aeg) and BHK21 or chick embryo (CEF) cells. 1. Viral growth kinetics and yields are similar in A. albo cells at 28 degress C and in vertebrate cells at 37 degrees C. A. albo exhibit no CPE and yield persistenetly infected cultures. 2. SV grown in A. albo cells lacks sialic acid but is antigenically and in terms of particle/PFU or particle/HAU ratios equivalent to SV derived from vertebrate cells. The contrast to VSV in the latter respect is discussed. 3. SV from persistently infected A. albo or A. aeg cells is temperature-sensitive, thermolabile, and produces small plaques. Partial characterization of these mutants, of RNA associated with their replication, and their high reversion rate to ts+ upon serial undiluted passage in GHK21 cells are presented. 4. Hostdependent differences in the generation of defective-interfering (DI) SV particles and of low molecular weight viral RNA species have been observed upon undiluted serial passages in BHK21 and CEF. In contrast, serial passage in A. albo cells appears not to produce DI particles or small RNA species nor do these cells "recognize" as such DI particles from BHK21 cells. 5. Possible implications of these observations fro the natural life cycle of arthropod-borne togaviruses are discussed.