Benjamin M. Blumberg

Sequence comparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains

The large (L) protein subunit of unsegmented negative-strand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagreement with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis.

Sendai virus contains overlapping genes expressed from a single mRNA

The mRNA coding for the Sendai virus P and C proteins was located on the viral genome using cloned DNA and the relevant regions of the DNA were sequenced. The nucleotide sequence revealed two overlapping open reading frames that could code for proteins of 568 and 204 amino acids. Primer extension and S1 nuclease mapping studies detected only a single 1.894 kb mRNA from this region. Hybrid arrest of translation studies using restriction fragments verified the overlapping nature of these genes. Sequence homologies at the beginning of three Sendai virus cistrons suggest that these genes may have arisen by duplication from a common ancestor, possibly an influenza-like virus gene.

Sequence determination of the Sendai virus HN gene and its comparison to the influenza virus glycoproteins

The nucleotide sequence of the Sendai virus (SV) HN (hemagglutinin-neuraminidase) gene was determined. The deduced primary structure of the protein showed only one hydrophobic domain likely to represent the transmembrane region, but at its N terminus. Since the SV F protein is anchored in the membrane at its C terminus, the two SV glycoproteins are thus membrane-anchored in opposite orientations, similar to the two influenza virus (FLU) glycoproteins. Amino acid sequence comparisons of the SV HN and the FLU HA and NA proteins revealed homologies between 100 amino acids of the hemagglutinin region of the FLU HA protein and the C terminus of the SV HN, and between 200 amino acids of the neuraminidase region of the FLU NA and the central region of SV HN. Alignment of the neuraminidase, hemagglutinin, and fusion regions shared by these glycoproteins suggest the structure of a possible ancestral gene.

Up-regulated p75NTR neurotrophin receptor on glial cells in MS plaques.

Objective: To investigate the expression of the neurotrophin receptor p75NTR on glial cells within MS plaques. Background: In recent studies on the pathogenesis of MS white matter plaques, we found large populations of inflammatory and resident glial cells, including oligodendrocytes undergoing cell death, and identified increased expression of Fas receptor and ligand death pathway signaling molecules on the same glial cell types. In another study, the p75NTR was shown to induce apoptotic death of maturing oligodendrocytes when exposed to NGF in vitro. Methods: We used immunohistochemistry and in situ reverse-transcription PCR to detect p75NTR expression on inflammatory and resident glial cells in MS plaques and used TUNEL staining for fragmented DNA to detect cell death. Results: Up-regulated p75NTR messenger RNA and protein were demonstrated in both oligodendrocytes and microglia/macrophages in MS plaques but not in control white matter. However, only a fraction of p75NTR expressing oligodendrocytes was also stained by TUNEL. Conclusions: Glial cell expression of p75NTR receptor is up-regulated during MS plaque formation. The exact role of this receptor in glial cell death and/or survival in MS remains to be elucidated.

An analytical review of defective infections of vesicular stomatitis virus.

Introduction. Following the discovery of interference in influenza infections by incomplete forms of the virus (von Magnus, 1951), Cooper & Bellett (1959) found a similar phenomenon in vesicular stomatitis virus (VSV) infections, and showed that it was due to transmissible components, defective interfering (DI) particles (Hackett, 1964; Huang & Wagner, 1966), which are usually generated when VSV is passaged at a high multiplicity of infection (m.o.i.). DI particles can replicate only during co-infection with standard or non-defective (ND) helper virus, and in such mixed virus infections the replication of the helper virus is greatly repressed. Bellett & Cooper (1959) observed a reciprocal exponential relationship between the m.o.i. of DI particles in the inoculum and the yield of infectious virion progeny, and deduced that co-infection of a cell with a single DI particle is sufficient to repress the helper virus in one passage (compare Sekellick & Marcus, 1980). DI particles enjoy a replicative advantage over ND virions, and once generated they quickly become the dominant viral species during high m.o.i. passage.

Comparison of Sendai virus and influenza virus surface glycoproteins

Nasopharyngeal secretions (NPS) collected by a mucus extractor from more than 1000 children with respiratory tract infections (including otitis) and aged from newborn to 10 years were investigated for the presence of viral antigens. The antigens were detected by an enzyme immunoassay (EIA) as originally described by Sarkkinen and coworkers (J. din. Microbiol. 13 (1981) 2158) Antigens of the following viruses could be found: RSV, Adeno, Parainfluenza 1, 2, 3, Influenza A and B. The study period was one year; an etiological diagnosis could be achieved within 24 h in each instance. Viral antigens could be demonstrated in 25 % from all NPS samples studied. RSV infection was diagnosed most frequently; RSV antigens being present in about 46% from all positive specimens, followed by Parainfluenza 3 (28 %) and Adenovirus (13 %). Clinically a pronounced association was found between RSV infection and lower respiratory tract disease in particular in infants less than 1 year old. A croup syndrome was often observed during Parainfluenza 3 infection, whereas tonsillitis and pharyngitis most frequently were due to Adenovirus infection.