Robert Anderson

In vivo and in vitro models of demyelinating diseases: Tropism of the JHM strain of murine hepatitis virus for cells of glial origin

Abstract Infection of mice with the neurotropic JHM strain of murine hepatitis virus causes demyelinating lesions resulting from an infection of the oligodendroglia. This was most evident in mice inoculated intraperitoneally with JHM. Such CNS lesions were not observed in mice inoculated intraperitoneally with the MHV3 strain. An in vitro system is described in which the rat glial RN2 cell line functions as a discriminating host for the JHM virus. Shortly after inoculation, this virus establishes a persistent infection in which there is a cyclical rise and fall in titer with an accompanying cytopathology. Furthermore, this host cell confers a thermal lability which the virus does not demonstrate in the fully permissive host cell, L-2. By comparison, infection of RN2 cells with the prototype MHV3 is aborted immediately. In the persistent infection of RN2 cells with measles virus, Hallé strain, the cell again confers a temperature sensitivity which the virus does not possess when replicating in Vero cells. This appears to be the first instance in which a cloned cell line of glial origin determines the outcome of the infectious process, discriminating in favor of a neurotropic variant which possesses a tropism for the glia in vivo. Systems such as the one described here may now offer a specific screening procedure for selecting, identifying and characterizing the nature of neurotropic viruses.

In vivo and in vitro models of demyelinating diseases II. Persistence and host-regulated thermosensitivity in cells of neural derivation infected with mouse hepatitis and measles viruses

Abstract Following inoculation of continuous cell lines of neural and other derivations, persistent infections are established with facility by mouse hepatitis and measles viruses. This occurs equally with the prototype MHV3 and its neurotropic variant JHM as well as with the Edmonston vaccine and SSPE Hallé measles variants. In almost every instance that the infection becomes persistent at 32.5°, virus replication is found to be thermosensitive at 39.5°; however, progeny virus derived from such infections at 32.5° is itself thermostable when replicating in the indicator, fully permissive cell lines. The new data, therefore, reveal the existence of a host-conferred interrelationship between persistence and virus restriction at elevated temperature. They indicate that the two agents with neurotropic potential, when they become established as pathogens in the nervous system, could be under close host cell regulation involving as yet unknown mechanisms.

Translational Control in Murine Hepatitis Virus Infection

High multiplicity infection of mouse fibroblast L-2 cells with mouse hepatitis virus (MHV) resulted, within 6 h, in a decline in total protein synthesis to about 7% of that observed in uninfected cells. The amount of intracellular total translatable RNA, however, increased approximately threefold, as a result of the accumulation of virus-encoded mRNAs. MHV-infected cells could be superinfected with vesicular stomatitis virus, demonstrating that MHV infection did not irreversibly alter the cellular translational machinery to the exclusion of non-MHV mRNAs. Comparative polysome analysis from MHV-infected and uninfected L-2 cells showed that MHV infection resulted in an increase in single 80S ribosomes and in a shift from longer to shorter polysomes. These observations suggest first, that MHV infection inhibits total protein synthesis at a very early stage, as evidenced by the increase in 80S ribosomes, and, second, that the increased number of viral mRNAs produced after infection compete with cellular mRNAs for cellular ribosomes. In vitro translation of RNA extracted from MHV-infected and mock-infected cells suggested that levels of cellular mRNAs were decreased after infection. This suggestion was confirmed by demonstrating the loss of cellular actin mRNA, using a radiolabelled cDNA probe, as a consequence of MHV infection.

Intracellular murine hepatitis virus-specific RNAs contain common sequences

Abstract A major polyadenylated viral RNA of approximately 0.8 × 106 daltons was isolated from murine hepatitis virus (A59)-infected cells by preparative polyacrylamide gel electrophoresis in formamide. This RNA was shown to encode the viral nucleocapsid protein by direct in vitro translation in a cell-free, reticulocyte-derived system. Single stranded 32P-labeled complementary DNA was prepared from this RNA and was demonstrated to be virus specific. Using this complementary DNA in a Northern blotting procedure, we were able to identify six major virus-specific intracellular RNA species with estimated molecular weights of 0.8, 1.1, 1.4, 1.6, 3, and 4 × 106 daltons. All of these RNA species were polyadenylated. Our results support the idea that coronavirus-infected cells contain multiple intracellular polyadenylated RNAs which share common sequences.

Attenuation of murine coronavirus infection by ammonium chloride

Abstract Ammonium chloride at a concentration of 20 mM delayed by 4–5 hr the production of virus progeny in mouse L-2 cells infected at high multiplicity with mouse hepatitis virus (MHV). This delay was seen in the production of both intracellular and extracellular virus. However, the final titers were similar to those produced by MHV-infected cells maintained in normal medium. The manifestation of virus-induced cell fusion was similarly found to be delayed, but not otherwise decreased in severity, when ammonium chloride was present in the culture medium. Ammonium chloride caused similar delays in production of virus-specific, positive-sense RNAs and of viral polypeptides. The relative proportions and apparent molecular weights of viral RNAs and polypeptides were similar to those found in MHV-infected cells cultured in normal medium. In vitro translation of endogenously produced viral RNAs in cell extracts, prepared from MHV-infected cells, was not inhibited by ammonium chloride. Thus, ammonium chloride has no specific, inhibitory effect on viral protein synthesis. Ammonium chloride did not reduce the number of virus-infected cells in culture, as monitored by infectious center assay. Analysis of early events in MHV infection showed that ammonium chloride did not affect adsorption or internalization of MHV by L-2 cells. However, the subsequent eclipse phase, as monitored by decline in infectivity of internalized virus inoculum proceeded less efficiently in the presence of ammonium chloride. On the basis of the known inhibitory effects of ammonium chloride on lysosomal/endosomal functions, the results suggest an endosomal mechanism of MHV uncoating. Thus the primary effect of ammonium chloride on MHV infection of L-2 cells is to attenuate virus uncoating, thereby chronologically displacing all subsequent virus-encoded functions.

Fusion resistance and decreased infectability as major host cell determinants of coronavirus persistence

Abstract Mouse hepatitis virus persists in cultures of a subline (designated LM-K) of mouse LM cells but produces a lytic infection in L-2 cells. Persistence in the LM-K cells was not accompanied by production of is mutants or of soluble anti-MHV factors. Infectious center assay demonstrated an approximately 500-fold lower level of infectibility by MHV of the LM-K cells as compared to L-2 cells. On an infected cell basis, production levels of infectious progeny and viral RNA were comparable between the two cell lines. The extent of virus-induced cell-cell fusion, however, was markedly reduced in the LM-K cells. Cell-mixing experiments showed that both infected L-2 and LM-K cells have the capacity of fusing with neighboring uninfected L-2 cells but not with uninfected LM-K cells. This suggests that the decreased level of fusion observed in the LM-K infection is due not to absence of viral fusion protein at the cell surface, but rather to an inherent resistance of the LM-K cell membrane to MHV-induced fusion. It is believed that such fusion resistance in LM-K cells moderates virus dissemination throughout the culture, thereby contributing to a state of virus persistence.

Cellular Synthesis and Modification of Murine Hepatitis Virus Polypeptides

Mouse L fibroblasts infected with mouse hepatitis virus, MHV3, and radiolabelled with 35S-methionine, contained, in addition to the three major structural polypeptides, p24, p56 and p180, two additional ones, p22 and p50. Of these total five polypeptides, only three, p22, p56 and p180, were labelled in infected cells during a 2 min 35S-methionine pulse and are, therefore, presumed to be immediate translation products. Pulse-chases and chymotryptic peptide mapping experiments showed apparent precursor-product relationships between p56 and p50 and between p22 and p24. Protein synthesis in infected cells was synchronized at the initiation stage by pre-exposure to hypertonic medium. Using a 0.5 min pulse-10 min chase sequence, to limit incorporation of 35S-methionine to stretches of approx. 100 amino acids adjacent to translational initiation sites, it was found that all three polypeptides, p22, and p56 and p180 contained radiolabel. It is thus apparent that translation of the three major structural proteins (or precursors) is initiated independently rather than at a single site as in the cases of other positive-strand RNA viruses such as polio or Semliki Forest virus.

RNA and polypeptide homology among murine coronaviruses

Abstract Using a 32P complementary DNA (cDNA) prepared against the A59 nucleocapsid protein messenger RNA, we have investigated the extent of homology between A59 and four other strains of murine hepatitis virus (MHV). Analysis by hybridization kinetics of the annealing between A59 [32P]cDNA and infected cell RNA from the other four MHV strains demonstrated 70–80% homology. By gel transfer analysis, the A59 [32P]cDNA was able to detect subgenomic-size virus-specific RNAs in cells infected with all of the five MHV strains. A similar pattern of seven viral RNAs was detected in cells infected with A59, MHV-1, MHV-3, and JHM. In contrast, cells infected with MHV-S contained seven virus-specific RNAs, of which only the two smallest species comigrated with RNAs from the other four strains. The results suggest that as previously shown with A59 (S. Cheley, R. Anderson, M. J. Cupples, E. C. M. Lee Chan, and V. L. Morris (1981) Virology, 112, 596–604), all MHV strains tested encode a nested set of subgenomic RNAs. Analysis of [35S]methionine-labeled viral proteins by SDS-polyacrylamide gel electrophoresis revealed that each strain of MHV specified four major viral polypeptides with apparent molecular weights very similar to those previously reported for the E2, N, El, and PEI polypeptides of A59. The strong degree of interstrain homology among the five MHV strains investigated was confirmed by comparative chymotryptic peptide mapping of the viral N proteins. A majority of the chymotryptic peptides from each of the [35Sknethionine-labeled N proteins was found to coelute by high-performance liquid chromotography. Moreover, this technique of peptide mapping indicated a particularly strong relatedness between MHV-1 and MHV-S and among MHV-3, JHM, and A59.

Regulation of humoral immune responses by a bone marrow-derived glycolipid-like molecule

Abstract Bone marrow cells have been shown to nonspecifically suppress primary in vitro antibody responses. This suppression appears to be mediated by a low-molecular-weight soluble factor, B-SF which was released from a fraction of cells of similar size to the suppressor as obtained by velocity sedimentation. Like the suppressor cell, B-SF was also shown to be effective very early in the immune response. It was produced by all strains of mice tested and functioned across strain barriers. Characterization of the active suppressor molecule showed it to be a highly heat-stable, nonsialic acid-containing glycolipid of 1000 to 35000 daltons in molecular weight. Recovery of the purified suppressor from thin-layer chromatography plates was achieved indicating that the major glycolipid component visualized on TLC is likely the active suppressor molecule. The characteristics of this suppressor may show it to be a fundamental immune regulatory mechanism.

Comparison of polypeptides of two strains of murine hepatitis virus

Abstract Viral polypeptides labeled with [ 35 S]methionine in mouse L fibroblasts infected with either of two strains (JHM or MHV3) of mouse hepatitis virus (MHV) were analyzed by polyacrylamide gel electrophoresis (PAGE). Four major polypeptides of apparent molecular weights, 180,000 (p′180′), 56,000 (p′56′), 24,000 (p′24′), and 22,000 (p′22′) were detected after a 15-min pulse of methionine. During a 2-hr chase performed late in infection (5.5 hr PI) a polypeptide of molecular weight 50,000 (p′50) was observed which appears to be derived from p′56′ by proteolytic processing. Both p′56′ and p′50′ were enriched in the amino acid arginine. Of the five major viral polypeptides only p′180′ could be labeled by growing infected cells in the presence of [ 14 C]glucosamine; moreover, it was the only polypeptide whose mobility of PAGE was altered after labeling infected cells with [ 35 S]methionine in the presence of glycosylation-inhibiting drugs. Despite the extreme similarity in polypeptide patterns specified by JHM and MHV3, the apparent molecular weights of each of the five major JHM polypeptides were consistently lower (by about 500–1000) daltons than the corresponding ones of MHV3.