Kenneth L. Powell

RSV604, a Novel Inhibitor of Respiratory Syncytial Virus Replication

ABSTRACT Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections worldwide, yet no effective vaccine or antiviral treatment is available. Here we report the discovery and initial development of RSV604, a novel benzodiazepine with submicromolar anti-RSV activity. It proved to be equipotent against all clinical isolates tested of both the A and B subtypes of the virus. The compound has a low rate of in vitro resistance development. Sequencing revealed that the resistant virus had mutations within the nucleocapsid protein. This is a novel mechanism of action for anti-RSV compounds. In a three-dimensional human airway epithelial cell model, RSV604 was able to pass from the basolateral side of the epithelium effectively to inhibit virus replication after mucosal inoculation. RSV604, which is currently in phase II clinical trials, represents the first in a new class of RSV inhibitors and may have significant potential for the effective treatment of RSV disease.

Identification of the herpes simplex virus DNA polymerase gene.

THERE is at present much interest in the herpes viruses because they have been associated with human cancer and cause latent infections with recurrent disease. They are also of interest because they form a relatively simple but apt model for the processes of transcription and DNA replication in eukaryotic cells. We have studied the virus-induced proteins involved in DNA replication, and present here results that give definitive evidence that the virus-induced DNA polymerase is encoded in the virus genome, that locate the gene for virus DNA polymerase, and also show that the enzyme is essential for virus replication.

Polypeptides synthesized in herpes simplex virus type 2-infected HEp-2 cells

Abstract The proteins induced by herpes simplex virus type 2 in infected HEp-2 cells were studied by high-resolution polyacrylamide slab-gel electrophoresis. A total of 51 infected cell-specific polypeptides (ICSP) were detected, accounting for three-fourths of the coding capacity of the virus. The induced polypeptides were allocated to three groups based on their time of synthesis in the virus growth cycle. Cycloheximide treatment of infected cells during the early stage of virus growth was found to cause irreversible inhibition of protein synthesis. Herpes simplex virus type 2 polypeptides which underwent considerable posttranslational modification were detected.

Herpesvirus-induced antigens in squamous-cell carcinoma in situ of the vulva.

Antigens induced by herpes simplex virus Type 2 (HSV2) were found to be associated with squamous-cell carcinoma in situ of the vulva in nine of 10 patients. The HSV2-induced antigens are DNA-binding proteins that are normally present in the nuclei of infected cells, but in the cells of the carcinomas in situ they were found in the cytoplasm. Whole-virion structural antigens were not present, although there was serologic evidence of previous HSV2 infection in patients tested for the presence of antibodies. The observations reported here and the recent parallel rise in the prevalence of both HSV2 infections and vulvar carcinoma in situ, particularly in women under 40 years of age, suggest an association of HSV2 infection with this type of neoplasia, the nature of which remains to be determined.

Herpes simplex virus non-structural proteins. III. Function of the major DNA-binding protein.

The herpes simplex virus type 2 major DNA-binding protein has been functionally characterized using temperature-sensitive mutants in the complementation group 2-2. The mutants were shown to be defective in the DNA-binding protein gene by mapping the mutants to the area of the genome known to code for the protein, and by demonstrating alterations in the major DNA-binding protein induced in mutant-infected cells. The mutants were shown to be defective in the replication of virus DNA. The nature of this defect was examined by studying virus DNA synthesis in vitro and by the examination of virus enzymes. An effect of mutation in the DNA-binding protein was to destabilize both the DNA polymerase and the alkaline exonuclease.

Studies on the herpes simplex virus alkaline nuclease: detection of type-common and type-specific epitopes on the enzyme.

Five monoclonal antibodies to the alkaline nuclease of herpes simplex virus (HSV) types 1 and 2 have been used in immunoperoxidase tests to demonstrate the nuclear localization of the enzyme within HSV-1- and HSV-2-infected cells and to purify the enzyme from cells infected with either virus by immunoadsorbant chromatography. Affinity chromatography with a 32P-labelled extract of HSV-2-infected cells has enabled us to demonstrate that the nuclease eluting from the immunoadsorbant is a phosphoprotein, hence confirming the nuclease to be identical to the phosphorylated polypeptide previously referred to as ICSP 22 (HSV-2) or ICP 19 (HSV-1). In addition, the results clearly demonstrate that monoclonal antibodies Q1, CC1 and CH2 are directed against HSV type-common epitopes while V1 and T2T1 antibodies are against HSV-2-specific epitopes on the enzyme. Using the type-specific monoclonal antibodies in an immunoperoxidase test, the enzyme specified in cells infected with intertypic recombinants has been typed; correlation of these data with restriction endonuclease maps of the recombinants has enabled us to map the position of the active site of the nuclease gene to map units 0.168 to 0.184 on the genomes of both HSV-1 and HSV-2. Taken with the data mapping the mRNA encoding this enzyme, the nuclease active site can be mapped to 0.168 to 0.175 on the genome. Finally, the use of monoclonal antibodies in immunofluorescence tests on infected cells has demonstrated that the nuclease is synthesized within 2 h post-infection.

Herpes Simplex Virus Non-structural Proteins. IV. Purification of the Virus-induced Deoxyribonuclease and Characterization of the Enzyme Using Monoclonal Antibodies

The alkaline nucleases induced by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) have been purified from high salt extracts of virus-infected cells. The purification used three types of column chromatography and resulted in apparently homogeneous DNase preparations with good recovery. The enzyme from HSV-2-infected cells has been characterized. It had both exonuclease and endonuclease activity, each with an unusually high pH optimum. The enzyme had an absolute requirement for magnesium which could not be replaced by other divalent cations. Analysis of the sedimentation characteristics and electrophoretic properties of the purified enzyme indicated that it was composed of a single subunit of mol. wt. 85 000. The purified HSV-2 enzyme was used as an immunogen to prime BALB/c mice which were used to prepare monoclonal antibodies. Three monoclonal antibodies were shown by several criteria to react with the enzyme. Thus, we were able to confirm that the 85K polypeptide did indeed have nuclease activity. This polypeptide was designated ICSP 22 in earlier studies and is a major polypeptide of virus-infected cells.

DNA-binding proteins of cells infected by herpes simplex virus type 1 and type 2.

Proteins showing affinity for DNA in HSV-1-and HSV-2-infected cells were compared by DNA-cellulose chromatography and PAGE. The proteins observed depended on the type of virus used to infect the cell; however, several examples of analogous polypeptides were present in cells infected by both virus types. Proteins showing highest affinity for DNA-cellulose were similar in molecular size in cells infected by both virus types.

The synthesis of herpes simplex virus proteins in the absence of virus DNA synthesis

Summary When herpes simplex virus infected cells were blocked from synthesizing DNA, alterations in the production of certain classes of herpes simplex virus specific proteins were detected. Some proteins were made in excess, some in normal amounts, some in reduced amounts and some were not made. An example of each type was chosen, and its kinetics of synthesis studied. Possible explanations of the phenomena are discussed.

Characterization of factors involved in human papillomavirus type 16-mediated immortalization of oral keratinocytes

We have examined intrinsic and external factors that influence human papillomavirus type 16 (HPV-16)-mediated immortalization of oral keratinocytes. The efficiency with which HPV can immortalize human oral keratinocytes was quantified and a considerable difference in the transfection and immortalization competence of the cells was detected. The ability of HPV-16 to immortalize oral cells appeared to be linked to the age of the culture upon transfection. The addition of dexamethasone to the transfected cultures increased the efficiency of immortalization, possibly indicating a role for a critical level of HPV gene expression in initial outgrowth of immortalized colonies. We also document in detail the changes in the oral keratinocyte induced by HPV-16 immortalization. These include alterations associated with crisis and feeder independence as well as basic changes in keratin expression and differentiation.