Biomedical subjects

C W Naeve

Publications and source records attributed to C W Naeve.

42 records · Page 3Linked to original sources

PubMed1983

Sequence of the hemagglutinin gene from influenza virus A/Seal/Mass/1/80.

A double-strand DNA copy of the influenza virus A/Seal/Mass/1/80 (H7N7) [seal] hemagglutinin (HA) gene was cloned into the plasmid pAT153/PvuII/8 and sequenced to deduce the primary amino acid sequence. The gene is 1731 nucleotides long and codes for a protein of 560 amino acids with a nonglycosylated molecular weight of 62098 Da. The deduced amino acid sequence displays similarities to all other sequenced hemagglutinins by retaining six of seven potential glycosylation sites, showing conversation in the number and position of cysteine residues, conservation in the fusion and anchor peptides, and conservation in the putative receptor site of the molecule. However, three features of the primary amino acid sequence could be distinguished from the H7 amino acid sequence of A/fowl plague/Rostock/34 (FPV), another avian H7 influenza virus which does not produce disease in mammals. First, the seal HA sequence has three fewer amino acids in the connecting peptide region of the HA than FPV. This lack of multiple basic amino acids in the connecting peptide is similar to that found in avirulent H7 avian strains and to mammalian serotypes H1, H2, and H3. Second, the seal HA has gained four additional proline residues, all in HA1, as compared to FPV. These residues may alter the tertiary structure of the HA and ultimately contribute to the biological features of this virus. Third, the seal HA has lost a potential carbohydrate attachment site at residue 149 which lies at the tip of the HA structure. The loss of this carbohydrate could alter the seal HAs interaction with host cell receptors.

Amino Acid Sequence↗

PubMed1983

Analysis of antigenic variation in equine 2 influenza A viruses.

Influenza outbreaks involving viruses of the H3N8 subtype (equine 2) often occur in vaccinated horses. For this reason, a series of influenza viruses of the H3N8 subtype were examined to determine if antigenic variation could be detected in isolates during the period 1963-81. Antigenic analyses with post-infection ferret sera and monoclonal antibodies showed that the haemagglutinins of recent isolates were antigenically distinguishable from the prototype A/eq/Miami/1/63 and that antigenically distinguishable groups of equine 2 viruses co-circulate in the horse population. Based on these studies, it is recommended that a recent equine strain, A/equine/Fontainebleu/1/79 or A/equine/Kentucky/1/81, serve as an additional prototype strain for this subtype.Antigenic variation in equine 2 viruses may be of epidemiological significance, yet the overall conservation of these strains makes it unlikely that vaccine failures can be attributed solely to antigenic changes in these viruses. A sufficiently potent vaccine, containing a current representative of the most prevalent equine 2 strain, may improve the protection afforded by equine vaccines.

Animals↗

PubMed1981

Phosphorylation of vesicular stomatitis virus proteins as a possible contributing factor in virion uncoating.

The relationship between the in vitro phosphorylation of vesicular stomatitis virus (VSV) proteins and virion uncoating was examined. Activation of the VSV virion kinase with low concentrations of melittin, the active peptide component of bee venom, in the presence of gamma-[32P] ATP resulted in the phosphorylation of virion proteins. Following the in vitro phosphorylation of VSV proteins in the presence of melittin and deoxyadenosine triphosphate, the virion envelope was disrupted based on the accessibility of the internal ribonucleoprotein core (RNP) to the heavy metal stain, uranyl acetate, as determined by electron microscopic observation. The RNP structure was not observed in unphosphorylated virions treated with melittin and uranyl acetate. Phosphorylated virions treated with uranyl acetate subsequently lost the capacity for transcription whereas unphosphorylated virions treated with the stain retained transcriptase activity. These observations suggest that phosphorylation of VSV proteins may contribute to virion uncoating by disrupting the virus envelope.

DNA-Directed RNA Polymerases↗

PubMed1980

Comparison o;f vesicular stomatitis virus intracellular and virion ribonucleoproteins.

Vesicular stomatitis virus ribonucleoproteins (RNP) obtained by a detergent treatment of purified virus (vRNP) or from infected HeLa cell cytoplasm (icRNP) were examined by sedimentation in sucrose or Renografin gradients in the presence or absence of EDTA. It was shown that vRNP and icRNP sediment at the same rate in sucrose and Renografin in the absence of EDTA; however, icRNP sedimented more slowly in the presence of EDTA than did vRNP. Polyacrylamide gel electrophoresis of the proteins of vRNA and icRNP recovered from EDTA-containing gradients demonstrated that both RNP structures contained L, N, and NS proteins in the same proportion. Electron microscopy of both RNP structures, in the absence of EDTA, demonstrated that both exist as helical structures approximately 20 by 700 nm. However, in the presence of EDTA the icRNP was completely uncoiled with a mean length of 4,095 nm, whereas vRNP was hardly affected. The addition of excess Mg(2+) or Mn(2+) to uncoiled icRNP preparations partially restored the coiled configuration. These observations suggest that the change in sedimentation of icRNP in the presence of EDTA is due to a change from a coiled to an uncoiled conformation, that icRNP and vRNP are not structurally identical, and that icRNP must undergo a conformational change during maturation of VSV from the 20-by-700-nm intracellular form to the 50-by-175-nm form found in intact virus. The icRNP containing L, N, and NS proteins (icRNP(L,N,NS)) and icRNP containing only N protein (icRNP(N)), prepared by centrifugation of icRNP(L,N,NS) in CsCl to remove L and NS, were compared by cosedimentation in sucrose gradients. There was a decrease in sedimentation rate of icRNP(N) due to loss of L and NS. This sedimentation difference was also apparent in the presence of EDTA; however, both icRNP(L,N,NS) and icRNP(N) sedimented at a much slower rate in the presence of EDTA, and by electron microscopy both were completely uncoiled. These observations suggest that N protein alone is responsible for the 20-by-700-nm coiled structure and that the divalent cation interactions disrupted by EDTA are N-N or N-RNA interactions. These results are discussed with regard to vesicular stomatitis virus maturation.

Cations, Divalent↗

PubMed1980

Electron microscopy of vesicular stomatitis virus replicative ribonucleoproteins.

The objective of this investigation was to examine by electron microscopy the replicative ribonucleoprotein (RNP) structures synthesized in vesicular stomatitis virus-infected HeLa cells. Pulse-labeled in vivo products of vesicular stomatitis virus replication and transcription can be separated by centrifugation in Renografin gradients. Transcription complexes are dissociated, allowing nascent messenger RNPs to remain at the top of the gradient, whereas RNPs biochemically consistent with replication complexes sediment to the middle of the gradient. Examination of these structures by electron microscopy revealed that all exist as coiled or helical RNPs having dimensions of approximately 20 by 700 nm. These structures can be further subdivided into three major morphological classes: (i) linear forms (20 by 769 +/- 158 nm), which have both ends free; (ii) circular forms (20 by 679 +/- 95 nm), which appear to have both ends joined; and (iii) complex forms, which include those structures which are branched replicative complexes as well as those which are random. To distinguish random complexes and possible transcriptive complex contaminants from replicative complexes, it was necessary to uncoil the RNP structures with EDTA so that length measurements could be made relating the nascent strand length to its position on the template. After EDTA treatment, the linear RNPs uncoiled (10 by 4,035 +/- 3,802 nm), and the circular morphology virtually disappeared. However, a new form appeared which was one-half the length and double the width (20 by 2,103 +/- 306 nm) of full-length RNPs and contained a loop at one end and two free ends at the other (alpha-form RNP). The distribution and length analysis of these structures, plus and minus EDTA, suggest that the alpha-form RNPs arise by EDTA-induced uncoiling of circular forms held together at the ends. Close scrutiny of uncoiled complex RNPs revealed no single-strand RNP templates with single-strand nascents. However, several complexes were observed which appeared to contain alpha-form templates with single-strand nascent RNPs. Length measurements suggest these complexes are neither random nor transcriptive, but are replicative. These experiments suggest that replication may, in part, occur on circular coiled RNP templates.

Edetic Acid↗

PubMed1978

Identification of Saint Louis encephalitis virus mRNA.

Saint Louis encephalitis (SLE) virus-specific RNA was recovered from infected HeLa cells by sodium dodecyl sulfate (SDS)-phenol-chloroform extraction, and the molecular species were resolved by SDS-sucrose gradient centrifugation and agarose gel electrophoresis. Sucrose gradient centrifugation revealed the presence of a 45S species, minor 20 to 30S heterogeneous species, and an 8 to 10 S RNA species in the cytoplasmic extract. Analysis of the same samples by electrophoresis on agarose gels, under both nondenaturing and denaturing conditions, revealed only two virus-specific RNA molecules, the 45S genome-sized RNA and an 8 to 10S species. Varying the gel concentration to facilitate analysis of nucleic acids with molecular weights ranging from 25,000 to 25 X 10(6) failed to reveal additional RNA species, although low levels of a putative double-stranded replicative form could conceivably have escaped detection. From our observations it appears that the heterogeneous RNA species and presumably the 20S RNase-resistant species reported in other investigations of flavivirus RNA are degradation products or conformers of the 45S molecule. Polysomes from SLE virus-infected cells were prepared and separated from contaminating nucleocapsid by centrifugation on discontinuous sucrose gradients. RNA extracted from these polysome preparations was analyzed by sucrose gradient centrifugation and agarose gel electrophoresis. The 45S SLE virus genome-size molecule was found to be the only RNA species associated with the polysomes. This molecule was sensitive to RNase digestion and was released from polysomes by EDTA and puromycin treatment. These findings provide direct evidence that the 45 S SLE virus RNA serves as the messenger during virus replication, in contrast to the 26S RNA species which functions as the predominant messenger during alphavirus replication.

Encephalitis Virus, St. Louis↗