Relationship between the loss of neutralizing antibody binding and fusion activity of the F protein of human respiratory syncytial virus

To elucidate the relationship between resistance to HRSV neutralizing antibodies directed against the F protein and the fusion activity of the F protein, a recombinant approach was used to generate a panel of mutations in the major antigenic sites of the F protein. These mutant proteins were assayed for neutralizing mAb binding (ch101F, palivizumab, and MAb19), level of expression, post-translational processing, cell surface expression, and fusion activity. Functional analysis of the fusion activity of the panel of mutations revealed that the fusion activity of the F protein is tolerant to multiple changes in the site II and IV/V/VI region in contrast with the somewhat limited spectrum of changes in the F protein identified from the isolation of HRSV neutralizing antibody virus escape mutants. This finding suggests that aspects other than fusion activity may limit the spectrum of changes tolerated within the F protein that are selected for by neutralizing antibodies

Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function

The mature F protein of all known isolates of human respiratory syncytial virus (HRSV) contains fifteen absolutely conserved cysteine (C) residues that are highly conserved among the F proteins of other pneumoviruses as well as the paramyxoviruses. To explore the contribution of the cysteines in the extracellular domain to the fusion activity of HRSV F protein, each cysteine was changed to serine. Mutation of cysteines 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 abolished or greatly reduced cell surface expression suggesting these residues are critical for proper protein folding and transport to the cell surface. As expected, the fusion activity of these mutations was greatly reduced or abolished. Mutation of cysteine residues 212, 382, and 422 had little to no effect upon cell surface expression or fusion activity at 32°C, 37°C, or 39.5°C. Mutation of C37 and C69 in the F2 subunit either abolished or reduced cell surface expression by 75% respectively. None of the mutations displayed a temperature sensitive phenotype

Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations

BACKGROUND: The thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. METHODS: A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. RESULTS: Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAA(r)5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture. CONCLUSIONS: This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2

A Case-Control Study of Hantavirus Pulmonary Syndrome during an Outbreak in the Southwestern United States

In May 1993, an outbreak of hantavirus pulmonary syndrome( HPS) occurred in the south-western United States. A case-control study determined risk factors for HPS. Seventeen case-patients were compared with 3 groups of controls: members of case-patient households( household controls), members of neighboring households( near controls), and members of randomly selected households ≥ 24 km away ( far controls). Investigators trapped more small rodents at case households than at near ( P = .03) or far control households( P = .02). After the number of small rodents was controlled for,case-patients were more likely than household controls to hand plow (odds ratio [OR], 12.3; 95% confidence interval [ CI], 1.1-143.0) or to clean feed storage areas (OR, 33.4; 95% CI, 1.7-666.0). Case-patients were more likely than near controls to plant( OR, 6.2; 95% CI, 1.1-34.0) and more likely than far controls to clean animal sheds( OR, 11.9;95% CI, 1.4-103.0). Peridomestic cleaning, agricultural activities, and an increased number of small rodents at the household were associated with HPS

A Case-Control Study of Hantavirus Pulmonary Syndrome during an Outbreak in the Southwestern United States

In May 1993, an outbreak of hantavirus pulmonary syndrome( HPS) occurred in the south-western United States. A case-control study determined risk factors for HPS. Seventeen case-patients were compared with 3 groups of controls: members of case-patient households( household controls), members of neighboring households( near controls), and members of randomly selected households ≥ 24 km away ( far controls). Investigators trapped more small rodents at case households than at near ( P = .03) or far control households( P = .02). After the number of small rodents was controlled for,case-patients were more likely than household controls to hand plow (odds ratio [OR], 12.3; 95% confidence interval [ CI], 1.1-143.0) or to clean feed storage areas (OR, 33.4; 95% CI, 1.7-666.0). Case-patients were more likely than near controls to plant( OR, 6.2; 95% CI, 1.1-34.0) and more likely than far controls to clean animal sheds( OR, 11.9;95% CI, 1.4-103.0). Peridomestic cleaning, agricultural activities, and an increased number of small rodents at the household were associated with HPS

Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed