- Volume 18, Issue 1, 1973

Selenocystamine dihydrochloride (0.003 mm) inhibited particle-associated RNA-dependent RNA polymerase activity of influenza B/LEE/40, influenza A/WSN/34 (H0 N1), A/RI-5+ (H2 N2), recombinant A/RI-5+ (N2)-A/WSN/34 (H0) and recombinant A/KOREA/68 (H2)-A/BEL (N1) in vitro. The structurally related compounds L-cystine, cystamine and N,N′-diacetyl selenocystamine were less active and inhibited influenza B/LEE particle-associated RNA-dependent RNA polymerase activity by 50% at concentrations of >0.3, 0.3 and 0.018 mm, respectively. Particle-associated RNA-dependent RNA polymerase activity was inhibited immediately by addition of selenocystamine. The inhibitory activity of 0.01 mm-selenocystamine was reversed completely by the addition of 0.06 mm-β-mercaptoethanol and partially by dialysis of a virus-selenocystamine mixture. High concentrations (3 mm) of the compound had no detectable effect on influenza virus haemagglutinin or neuraminidase activities, but virus infectivity was reduced significantly after incubation with 0.03 mm-selenocystamine.

The growth of varicella-zoster virus in human diploid cells was studied. Newly synthesized varicella-zoster virus appeared to be transferred from the perinuclear cisterna to the Golgi region of the cell, probably by a discontinuous route involving endoplasmic reticulum. In the Golgi, varicella-zoster virus and acid phosphatase, a lysosomal marker enzyme, were packaged together into small vesicles. These small vesicles apparently coalesced to form larger vacuoles. Virus particles were probably transported through cytoplasm in these vacuoles with lysosomal enzymes, and particles seemed to be released by exocytosis. Extracellular virus was pleomorphic. Rupture of lysosomes into cell cytoplasm in varicella-zoster virus infected cells was not detected. It is postulated that the presence of lysosomal enzymes and varicella-zoster virus in the same cytoplasmic vacuoles may result in inactivation of varicella-zoster virus during egress of the virus from the cell. This may account for the ‘cell-associated’ character of varicella-zoster virus infection in tissue culture.

The possibility that the haemagglutinin of encephalomyocarditis (EMC) virus might be a glycoprotein was investigated by looking for sugars and amino sugars in purified EMC virus particles grown in Krebs ascitestumour cells in vitro in the presence of isotopically labelled glucosamine, acetyl glucosamine, galactosamine, mannosamine or fucose. With either isotopically labelled glucosamine or galactosamine the virus became significantly radioactive, whereas radioactivity was low in virusgrown in the presence of mannosamine, or fucose, possibly because the latter was poorly taken up by the cells; acetyl glucosamine was completely excluded by Krebs cells, so its ability to be incorporated into virus could not be tested.

Radioactivity derived from glucosamine remained firmly attached to the purified virus through both rate-zonal and equilibrium density centrifugation and chromatography on calcium phosphate. This radioactivity did not appear due to simple adsorption of either glucosamine or (after synthesis) acetyl glucosamine, or to contamination of the preparation with infected host cell membranes. Estimates of the number of glucosamine molecules incorporated into the virus had values which varied with the position of the isotope in the glucosamine added, suggesting that it was not incorporated unchanged.

Polyacrylamide gel electrophoresis of disrupted whole virus showed radio-activity derived from glucosamine to be present in all virus polypeptides, indicating that glucosamine was perhaps metabolized, amongst other things, to amino acids. Disruption of the virus with phenol revealed that 70 % of the radioactivity was in the virus RNA, mostly in ribose, the remaining 30% being in virus protein, presumably as amino acids. The total amount of radioactivity that could be in the form of glucosamine did not allow for more than 0.6 glucosamine residues per virus particle. From this it was concluded that glucosamine is not a virus constituent per se, that the EMC virus particle does not contain glycoprotein and, consequently, that the virus haemagglutinin is not a glycoprotein.