SummaryThis study has identified a single amino acid change in the viral glycoprotein that profoundly affects the ability of lymphocytic choriomeningitis virus (LCMV) to persist in its natural host . Adult immunocompetent mice infected with a variant--of the Armstrong strain, spleen isolate clone 13 (svA/svA), harbor virus for several months and exhibit suppressed T cell responses . In contrast, adult mice infected with a reassortant virus (svA/wtA) that contains the L segment of the spleen variant and the S segment of the parental wt Armstrong, make potent LCMV specific CTL responses and clear the infection within 2-4 wk . These two viruses, spleen variant clone 13 and the reassortant svA/wtA, are identical in their noncoding regions and show no amino acid changes in any of their viral genes except for one substitution in the glycoprotein . The reassortant virus svA/wtA has a phenylalanine at amino acid residue 260 of the glycoprotein, whereas the spleen variant clone 13 has a leucine at this position . This study constitutes one of the first reports defining the genetic basis of viral persistence at the whole animal level, and identifying a single mutation that markedly increases the ability of a virus to persist in its natural host . S uccessful resolution of a viral infection depends upon a critical balance between the extent of viral spread and replication, and the magnitude of the host's immune response. We have been studying infection of mice with lymphocytic choriomeningitis virus (LCMV)t as a model system to understand the host and viral determinants that lead to viral clearance or persistence (1-4) . Infection of immunocompetent adult mice with the Armstrong strain of LCMV induces a potent antiviral T cellresponse and virus is eliminated within 2 wk . This clearance is mediated by CD8 * virus-specific cytotoxic T lymphocytes (4-7) . In contrast, infection of adult mice with a naturally selected isolate of Armstrong, spleen variant clone 13, results in a disseminated infection, with virus persisting for several months (1, 2). This chronic infection is associated with suppressed T cell responses and susceptibility to opportunistic infection (1, 2, 8) .The LCMV genome consists of two segments of singlestranded RNA, a large (L) segment of 7 .2 kb and a small (S) segment of 3 .4 kb (9-12) . The L RNA segment codes for a large protein, L (molecular mass 250 kD), that is be-1 Abbreviations used in this paper. L, large; LCMV, lymphocytic choriomeningitis virus; S, small; sv, spleen variants; wt, wild type.lieved to be the viral polymerase, and also contains a second open reading frame, designated Z, that encodes for a protein of "10-12 kD. The S segment codes for the three major structural proteins : the internal nucleocapsid protein (NP ; 63 kD) and the two surface glycoproteins GP-1 (43 kD) and GP-2 (36 kD) that are derived from a common precursor polypeptide, GP-C. After coinfwtion of cells with two different LCMV strains, recombinants are generated by reassortment of genome segments. This permits genetic a...
Following several model experiments, conditions were developed for optimal deglycosylation of tracheal mucin glycoproteins. Exposure of rigorously dried material to trifluoromethanesulfonic acid at 0 degree C for up to 8 h results in cleavage of essentially all fucose, galactose, and N-acetylglucosamine, about 80% of the N-acetylneuraminic acid (NeuNAc), and a variable amount of N-acetylgalactosamine (GalNAc), the sugar involved in linkage to protein. Residual N-acetylneuraminic acid is sialidase susceptible and apparently in disaccharide units, presumably NeuNAc2----GalNAc. The remaining N-acetylgalactosamine is mostly present as monosaccharides, and a few Gal beta 1----3GalNAc alpha units are also present; both are cleaved by appropriate enzymatic treatment. The saccharide-free proteins obtained from either human or canine mucin glycoproteins have molecular weights of about 100,000 and require chaotropic agents or detergents for effective solubilization.
Cottontail rabbit papillomavirus is the major animal model for cancer-associated papillomaviruses. Here we show that vaccination with the nonstructural proteins E1 and E2 induces the regression of virus-induced papillomas and that vaccination is equally effective when proteins are given with and without adjuvant. There was no correlation between antibody levels and regression, suggesting that tumor regression may be due to a cell-mediated response.
Cottontail rabbit papillomavirus (CRPV) is a highly oncogenic papillomavirus and has been successfully used as a model to develop protective vaccines against papillomaviruses. Papillomas induced by the virus may spontaneously regress, suggesting that CRPV can also serve as a model to develop therapeutic vaccines. As a first step toward this goal, we have analyzed immunologic and viral aspects associated with papilloma regression and have identified several features unique to regression. Immunohistochemical staining of biopsies from growing and regressing papillomas and from sites after complete regression showed infiltration of CD8 ؉ cells into the basal and suprabasal layers of the epidermis only during active regression. In situ hybridizations with mRNA-specific probes were strongly positive for E6 and E7 mRNAs during regression, but no late mRNA was present. Viral DNA was detected by in situ hybridization during regression but not after regression. However, analysis by PCR revealed persistence of viral DNA for several months at the majority of regression sites. The results suggest that stimulation of a strong CD8 ؉ response to virus-infected cells is important for an effective therapeutic vaccine and that special attention should be given to the suppression of latent infection.
Canine tracheal mucin glycoprotein was isolated from beagle dogs fitted with tracheal pouches. Following exclusion chromatography on Sepharose CL-4B, noncovalently associated proteins were further resolved by dissociative density gradient centrifugation in CsBr-guanidinium chloride, and the mucin was then extracted with chloroform-methanol. The delipidated high-density product obtained had a nominal molecular weight of about 10(6) and an overall composition characteristic for a mucin glycoprotein, viz., a high content of serine and threonine, about 80% carbohydrate by weight, the absence of mannose or uronic acid, measurable ester sulfate, and a Pronase-resistant domain of molecular weight (1.75-3.0) X 10(5) which contains essentially all of the saccharide residues. Noncovalently bound lipid amounted to 6-10% by weight and was primarily cholesterol and cholesteryl esters. Cleavage of disulfide bonds by performic acid oxidation resulted in the release of a protein (Mr 65,000) not otherwise resolved by sodium dodecyl sulfate gel electrophoresis or the purification scheme.
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