N L McQueen

Determinants of pantropism of the F1-R mutant of Sendai virus: specific mutations involved are in the F and M genes.

SummaryMutations in the fusion, F, protein of Sendai virus resulting in increased cleavability by ubiquitous host protease(s), and mutations in the matrix, M, protein resulting in bipolar budding, are both important determinants for the systemic infection in mice caused by the protease activating pantropic mutant, F1-R. Several mutants of Sendai virus (BY, BF, and KD-M) with phenotypes of bipolar budding and/or increased cleavability of F protein were isolated. Genomic RNA sequence analysis of the F and M genes of the mutants revealed that several deduced amino acids in the F and M proteins were different from those of F1-R, T-5 (a revertant of F1-R), and wild-type viruses. The BF and KD-M mutants that budded bipolarly and were also activated by ubiquitous proteases were examined for replication in tissue culture cells and in mice. All of the mutants exhibited multiple-step replication in MDCK, MDBK, and LLC-MK2 cells without trypsin, but formed plaques only in MDCK cells. One of the mutants, designated KD-52M, was similar to F1-R in that it formed plaques in all three cell lines without addition of exogenous protease. However, none of the mutant viruses, including KD-52M, caused a systemic infection in mice. The mutated M protein of F1-R enhances the disruption of microtubles. However, none of the mutants with a bipolar budding phenotype (BY, BF, and KD-M), disrupted the microtubules to the same extent as F1-R. All of these mutants had mutations in the M protein that were different from those found in F1-R. Taken together, these results suggest that mutations at Ser115 to Pro in the F protein and at Asp 128 to Gly and Ile210 to Thr in the M protein of F1-R are the mutations specifically required for the systemic infection caused by F1-R.

Mutations in Sendai virus variant F1-R that correlate with plaque formation in the absence of trypsin

With the emergence of new viruses, such as the SARS virus and the avian influenza virus, the importance of investigations on the genetic basis of viral infections becomes clear. Sendai virus causes a localized respiratory tract infection in rodents, while a mutant, F1-R, causes a systemic infection. It has been suggested that two determinants are responsible for the systemic infection caused by F1-R [Okada et al (1998) Arch Virol 143:2343–2352]. The primary determinant of the pantropism is the enhanced proteolytic cleavability of the fusion (F) protein of F1-R, which allows the virus to undergo multiple rounds of replication in many different organs, whereas wild-type virus can only undergo multiple rounds of replication in the lungs. The enhanced cleavability of F1-R F was previously attributed to an amino acid change at F115 that is adjacent to the cleavage site at amino acid 116. Secondly, wild-type virus buds only from the apical domain of bronchial epithelium, releasing virus into the lumen of the respiratory tract, whereas F1-R buds from both apical and basolateral domains. Thus, virus is released into the basement membrane where it can easily gain access to the bloodstream for dissemination. The microtubule disruption is attributed to two amino acid differences in M protein. To confirm that the F and M gene mutations described above are solely responsible for the phenotypic differences seen in wld-type versus F1-R infections, reverse genetics was used to construct recombinant Sendai viruses with various combinations of the mutations found in the M and F genes of F1-R. Plaque assays were performed with or without trypsin addition. A recombinant virus containing all F1-R M and F mutations formed plaques in LLC-MK2 cells and underwent multiple cycles of replication without trypsin addition. To clarify which mutation(s) are necessary for plaque formation, plaque assays were done using other recombinant viruses. A virus with only the F115 change, which was previously thought to be the only change important for plaque formation of F 1-R F, did not confer upon the virus the ability to form plaques without the addition of trypsin. Another virus with the F115 and both M changes gave the same result. Therefore, more than one mutation in the F gene contributes to the ability of F1-R to form plaques without trypsin addition.

Molecular diagnostics — An upper division/graduate course

Abstract We have developed a course in Molecular Diagnostics suitable as an upper division/Master’s level elective class for Biology, Microbiology, and Biochemistry majors who have already been introduced to basic genetics and molecular biology. The course provides an intensive hands-on laboratory experience in current molecular techniques for disease diagnosis coupled with lecture on major biological topics and concepts underlying disease and disease processes. Its structure and content make the course appropriate not only for students who envision careers in molecular diagnosis, but also for those aspiring to the teaching, research, or medical professions.

EUKARYOTIC EXPRESSION OF CLONED cDNA CODING FOR INFLUENZA VIRAL GLYCOPROTEINS USING AN SV40 VECTOR: USE OF RECOMBINANT DNA MUTANTS TO STUDY STRUCTURE-FUNCTION RELATIONSHIPS

Publisher Summary Influenza virus has been widely used to study the biosynthesis, sorting, distribution, and orientation of membrane proteins. Influenza has three membrane associated proteins; hemagglutinin (HA), neuraminidase (NA), and matrix protein (M). HA and NA are integral membrane proteins located on the outer envelope, whereas M protein is associated with the inner lining of the viral membrane. As influenza contains these two types of integral membrane proteins, it is an ideal candidate for the study of the mechanism by which integral membrane proteins are transported from their site of synthesis, via the rough endoplasmic reticulum and golgi apparatus, to their final destination in eukaryotic cells. Co-translational and post-translational modifications along with specific structural features of a protein are presumably responsible for guiding the protein through the elaborate cellular machineries to its final destination. The conserved residues may, however, play an important role in sorting directional transport or interactions with other viral proteins such as M in the viral membrane. This chapter describes methods used to express HA and NA in CV1P cells by using SV40 as a vector. The cDNA is cloned into the late region of SV40 between Hpa II and Bam HI, replacing the late SV40 genes. CV1P cells are transfected with the recombinant virus and SVsal.32—a helper virus defective in the early genes.