To analyze the pathogenesis of the neurotropic murine coronavirus JHMV, we used monoclonal antibodies to the E2 viral glycoprotein to select antigenic variant viruses. Monoclonal antibodies J.7.2 and J.2.2 were shown to bind to topographically distinct regions of the E2 molecule, and the variants selected with the two antibodies demonstrated very different disease pictures in mice. Variants selected with J.7.2 were, like the parental virus, highly virulent and caused an acute encephalitic illness. By contrast, J.2.2-selected variants predominantly caused a subacute paralytic disease clinically and extensive demyelination histologically. Antigenic differences among the variants and parental virus were readily demonstrable with anti-E2 monoclonal antibodies. However, no differences between the viruses could be shown in binding studies with monoclonal antibodies directed against either E1 or N, the other two JHMV structural proteins. Since only J.2.2 selected demyelinating variants with reduced neurovirulence, it is likely that this monoclonal antibody recognizes a subregion of the E2 molecule that is particularly important in JHMV pathogenesis.

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Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies.

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.

https://www.mdpi.com/1999-4915/6/7/2826/pdf

Membranous Replication Factories Induced by Plus-Strand RNA Viruses

Newly synthesized Dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization

Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer.

Publisher Summary The vast majority of monoclonal antibodies to flaviviruses produced so far are specific for the E glycoprotein; therefore, this chapter deals with the characteristics of epitopes on this immunologically dominant protein. In addition, recent studies with NV3-specific monoclonal antibodies provide evidence that this nonstructural glycoprotein may play an important role in mounting a protective immune response. These new aspects have been emphasized. The analysis of flavivirus structural glycoproteins with monoclonal antibodies revealed a serological complexity that goes far beyond that established by polyclonal sera. At the epitope level, antigenic relationships are strongly determined by the assay system used, and differing patterns of cross-reactivities are obtained when different functional tests are applied. For the tick-borne encephalitis (TBE) virus system, it has been shown that increased binding is due to up to six-fold enhancement of antibody avidity. The structural characteristics of monoclonal antibody-defined epitopes have been determined for TBE virus. Epitopes in both major domains appear to be strongly conformational dependent, with the difference that domain A is very labile and easily denatured, whereas domain B is strongly stabilized by disulfide bridges. For all flaviviruses analyzed with monoclonal antibodies, the dissociation of different functional activities is apparent at the epitope level. All types of different qualitative and quantitative combinations can be observed.

Epitope mapping of flavivirus glycoproteins.

Enzymatic markers and electron microscopy were utilized to determine the cellular origin of the membrane types isolated from type 2 dengue virus-infected BHK cells by discontinuous sucrose gradient centrifugation. The results showed an apparent separation of plasma membrane, smooth and rough endoplasmic reticulum with increasing density. Virus-induced protein and RNA synthesis, as indicated by the incorporation of radiolabled precursors, was localized on the rough endoplasmic reticulum. Glycosylation, measured by the incorporation of radiolabeled glucosamine into membrane-associated proteins, was most active in the bands of intermediate and smooth endoplasmic reticulum. Polyacrylamide gel electrophoresis of isolated membrane bands, radiolabeled in the presence of actinomycin D, after pulse inhibition by cycloheximide, revealed seven virus-specific proteins associated with all membrane fractions. Viral structural protein V-3, and nonstructural proteins NV-3 and NV-2, increased with decreasing density, whereas NV-5 and NV-4 remained constant. The viral capsid protein V-2 was depleted in the intermediate and smooth endoplasmic reticulum, suggesting that these membranes may serve as the sites for viral maturation. NV-3 was the most prominent virus-specified protein found in the plasma membrane.

Dengue virus-induced modifications of host cell membranes.

The membranes isolated from type 2 dengue virus-infected BHK-21/15 cells contain three glycosylated virus-specified proteins; one corresponds to the virion envelope glycoprotein, V-3, whereas the other two are nonstructural virus-specified proteins, NV-2 and NV-3 A combination of two nonionic detergents, Nonidet P-40 and Triton X-305, solubilized greater than or equal to 80% of the membrane-bound protein and the majority of the type 2 dengue virus complement-fixing antigens The soluble material was adsorbed by concanavalin A-Sepharose in the presence of the nonionic detergents, which were subsequently removed by washing with deoxycholate-containing buffer Finally, the bound glycoprotein was eluted by the addition of alpha-methyl glucopyranoside V-3 was the only virus-specified protein in the alpha-methyl glucopyranoside eluate The V-3-containing fraction did not cross-react with antisera against other selected Flaviviruses in the complement fixation tests The V-3 contained in the isolated fraction differed from the parent membrane-bound V-3 in two interesting, and as yet unexplained, ways: (i) it exhibited hemagglutinating activity similar to that of the infectious virus, but (ii) it did not block the action of neutralizing antibody

Isolation of the dengue virus envelope glycoprotein from membranes of infected cells by concanavalin A affinity chromatography.

Membranes from type 2 dengue virus (DEN-2) infected BHK cells, when used as immunogen, elicit antibodies detectable by complement fixation, hemagglutination inhibition and plaque reduction neutralization tests with suckling-mouse-brain-derived DEN-antigens. Membrane fractions from infected cells separated by sucrose density gradient centrifugation were employed as antigen in serological tests with anti-DEN-2 infected suckling mouse brain hyperimmune mouse serum and hyperimmune mouse ascitic fluid. Antigen (s) capable of fixing complement were associated with all membrane fractions, including the plasma membrane, whereas antigen capable of reacting with antihemagglutinin was largely confined to the two membrane bands consisting of (1) a mixture of rough and smooth endoplasmic reticulum (2) predominantly rough endoplasmic reticulum.

S A Stohlman

Papers

Dengue virus-induced modifications of host cell membranes.

Isolation of the dengue virus envelope glycoprotein from membranes of infected cells by concanavalin A affinity chromatography.

Dengue viral antigens in host cell membranes.