Complete, circular papovavirus genomes in the cells of hamsters exposed to a horizontally transmitted lymphomagenic agent.

Contagious lymphomas were produced in a colony of Syrian golden hamsters by an unknown agent that also caused fatal ulcerative bowel disease (UBD) lesions prior to lymphoma development. A low percentage of these animals developed epitheliomas of the skin independently of the UBD or lymphomas. Previous work has shown that the epitheliomas contain numerous hamster papovavirus (HaPV) particles, whereas lymphomas do not. Cells from both kinds of tumors do contain HaPV DNA sequences, however. In this study, Southern blot hybridization showed that complete, circular HaPV genomes were present in these cells. Complete, circular HaPV genomes also were found in the cells of animals with UBD. Electron microscopy revealed the presence of HaPV particles in UBD lesions. These results, together with previous data, indicate that in the hamster, lymphomas contain complete, circular papovavirus genomes in the absence of virus particles, whereas epitheliomas and UBD lesions contain these genomes in the presence of virus particles.

Papovavirus in epitheliomas appearing on lymphoma-bearing hamsters: lack of association with horizontally transmitted lymphomas of Syrian hamsters.

Several epizootics of lymphoma occurred in a colony of LVG hamsters contaminated with an unusual, horizontally transmitted, subviral, lymphomagenic agent. Hamsters with horizontally transmitted lymphoma, or others housed with these hamsters, occasionally developed epitheliomas bearing an unclassified papovavirus. The possibility that the virus present in the wart-like structures in our hamster colony could activate lymphoma was tested, and a search was conducted for mature virions in passaged epitheliomas and lymphomas. The agent responsible for the skin epitheliomas in our hamster facility was an icosahedral, 36-nm virion compatible with the morphology of a polyomavirus or simian virus 40. Horizontally transmitted lymphoma cells and epitheliomas contained hamster papovavirus (HaPV) DNA sequences detected by dot hybridization; however, such sequences were not found in extracts of lymphomas with oncogenic potential. In contrast to reports by other investigators, infection of hamsters with the papovavirus present in primary epitheliomas produced epitheliomas in good yield but was not reproducibly associated with lymphoma induction. These data confirm the observation that the HaPV is the causative agent of epitheliomas, but they suggest clearly that HaPV is not the agent responsible for lymphomagenesis.

Effect of phosphonoacetate on Marek's disease virus replication.

Phosphonoacetate (PA), but not any of its analogues tested, effectively inhibited avian herpesvirus replication and viral DNA synthesis in cell cultures. At 100 mug/ml culture medium, PA completely inhibited the replication of Marek's disease virus (MDV), herpesvirus of turkeys, and owl herpesvirus, but had no measurable effect on normal cell growth. PA also inhibited DNA polymerases induced by these avian viruses. Enzyme inhibition was 50% at a PA concentration of 0.2 mug/ml. At a concentration of 3-6 mug/ml, the compound also effected a 50% inhibition of alpha (maxi) enzyme of the host DNA polymerase. It had no effect on the host beta (mini) enzyme. When administered to chickens, PA did not inhibit the replication of MDV, nor did it prevent the development of lymphoma.

A phosphonoacetate-resistant mutant of herpesvirus of turkeys.

A phosphonoacetate (PA)-resistant mutant of the herpesvirus of turkeys (HVT) was isolated and characterized. The mutant of HVT resistant to PA (HVTpa) replicated in duck embryo fibroblast (DEF) culture in media containing 300 microgram PA/ml, whereas the replication of the wild type of HVT (HVTwt) was completely inhibited in DEF culture in media containing 100 microgram PA/ml. The HVTpa was distinct from the HVTwt in plaque morphology, but was indistinguishable antigenically and showed in vitro temperature sensitivity at 41 degrees C (3741 degrees C efficiency of replication was about 5). It replicated poorly in chickens and failed to provide complete protection against challenge with Marek's disease virus (MDV). The HVTpa-induced DNA polymerase had an apparent inhibition constant for PA, an apparent inhibition constant for pyrophosphate, and an apparent Michaells constant for dCTP about 10, 2, and 2.5 times, respectively, greater than the constants for the HVTwt-induced enzyme and was also more thermolabile.

Characterization of the single-stranded DNA-binding domain of the herpes simplex virus protein ICP8.

The DNA-binding protein, ICP8, of herpes simplex virus type 1 (HSV-1) is multifunctional in vivo and binds preferentially to single-stranded DNA (ssDNA) in vitro. To define the ssDNA-binding domain of ICP8, peptides were produced and analyzed. Portions of the ICP8 gene were cloned into the transcription vector pSP64, and RNA was synthesized in vitro. Translation of this RNA in rabbit reticulocyte lysates produced peptides of 29, 35 and 30 kDa, representing amino-acid residues 332-564, 571-899 and 900-1196, respectively, of intact ICP8 (128 kDa, 1196 amino acids). These peptides were analyzed by ssDNA-cellulose column chromatography. About 55% of the 29 kDa peptide bound to ssDNA-cellulose columns, and the majority which bound eluted with 1.0 M NaCl. About 5% of the 35 kDa peptide and 12% of the 30 kDa peptide bound and eluted with 0.3 M NaCl. Thus, three regions of ICP8 were associated with ssDNA-binding activity. The ssDNA-binding domain of ICP8 was not completely defined, however, because a 95 kDa peptide which included these regions did not bind to or elute from ssDNA-cellulose in the same way as intact ICP8. Amino-acid residues 332-564 and 571-899 not only were associated with ssDNA-binding activity but also contain the altered amino acids of four ICP8 molecules which are deficient in DNA binding.

A potent, cost-effective RNase inhibitor.

The performance characteristics of a potent new RNase inhibitor have been evaluated, as have applications for its use in molecular biology. PRIME Inhibitor is a protein, not related to the commonly available human placental RNase inhibitors (HPRI). It has a high specific activity, enhanced temperature stability, broad reaction pH range and significantly greater cost-effectiveness than commercial HPRI. PRIME Inhibitor is suitable for use in in vitro transcription, in vitro translation, first- and second-strand cDNA synthesis, preparation of RNA and mRNA, and reverse transcription-polymerase chain reaction.

A carboxyl-terminal peptide of the DNA-binding protein ICP8 of herpes simplex virus contains a single-stranded DNA-binding site.

The DNA-binding protein ICP8 of herpes simplex virus is a multifunctional protein which is required for viral replication. To identify the single-stranded DNA-binding domain of the protein, recombinant plasmids containing the 5' or 3' coding portion of the ICP8 gene or the intact gene were constructed and transcribed using SP6 RNA polymerase. The resulting RNA was translated in vitro to produce a 62,000-Da amino-terminal peptide, a 69,000-Da carboxyl-terminal peptide, or the intact protein. When these were analyzed by single-stranded DNA-cellulose column chromatography, large amounts of the intact ICP8 bound to the columns while small amounts of the carboxyl-terminal peptide and undetectable amounts of the amino-terminal peptide bound. The majority of the carboxyl-terminal peptide which bound eluted from the columns with the same salt concentration as the intact ICP8. The in vitro synthesized intact protein had the same affinity for single-stranded DNA-cellulose as ICP8 purified from infected cells. These results suggest that the carboxyl-terminal portion of ICP8 contains a single-stranded DNA-binding site.

Herpes simplex virus type 1 infection of isogenic Epstein-Barr virus genome-negative and -positive Burkitt's lymphoma-derived cell lines.

The Epstein-Barr virus (EBV) genome-negative Burkitt's lymphoma-derived cell lines BJAB and Ramos and their in vitro EBV-converted sublines BJAB-B1, BJAB-A5, BJAB-B95-8, and AW-Ramos were infected with high multiplicities of herpes simplex virus type 1 (HSV-1; 10 to 70 PFU/cell). Cultures were monitored for cell growth and HSV-1 DNA synthesis. EBV-converted BJAB cultures were more permissive for HSV-1 infection than BJAB cultures. Significant cell killing and HSV-1 DNA synthesis were observed during the first 48 h of infection in the EBV-converted BJAB cultures but not in the BJAB cultures. The EBV-converted BJAB-B1 cell line contains an appreciable fraction of EBV-negative cells. Therefore, it was cloned. EBV-positive and -negative cells were identified by using EBV-determined nuclear antigen anti-complement immunofluorescence. Two types of subclones were identified: (i) those which contained both EBV-determined nuclear antigen-positive and -negative cells and (ii) those which contained only EBV-determined nuclear antigen-negative cells. When levels of HSV-1 DNA synthesis were measured in these subclones, it was found that the former were more permissive for HSV-1 infection than the latter. Thus, the presence of the EBV genome in BJAB cells correlates with increased permissiveness of these cells for HSV-1 during the first 48 h of infection. Nonetheless, persistent HSV-1 infections were established in both BJAB and EBV-converted BJAB-B1 cultures. No differences in extent of permissiveness for HSV-1 infection were found for Ramos and EBV-converted AW-Ramos cells.

Identification and characterization of deoxyribonucleoprotein complexes containing the major DNA-binding protein of herpes simplex virus type 1.

Nuclear extracts were prepared from cells infected with herpes simplex virus type 1 (HSV-1) and fractionated by sucrose gradient centrifugation to identify deoxyribonucleoprotein complexes involved in viral replication. Large amounts of an HSV-1 induced protein with a molecular weight of about 133,000 sedimented as a broad peak in the 25 S region of the gradient and cosedimented with 13 S DNA fragments. The sedimentation of both the protein and DNA decreased upon treatment of nuclear extracts with DNase. This result indicated that the protein and DNA were associated in deoxyribonucleoprotein complexes. The protein was identified as the HSV-encoded major DNA-binding protein ICP8 based on its molecular weight, its association with DNA in nuclear extracts, and its immunoprecipitation with monospecific antiserum and monoclonal antibody to ICP8. Deoxyribonucleoprotein complexes containing ICP8 could be immunoprecipitated from nuclear extracts. When DNA was extracted from these immunoprecipitates, fractionated by agarose gel electrophoresis, transferred to nitrocellulose paper, and hybridized to 32P-labeled HSV-1 or cell DNA, both HSV-1 and cell DNA sequences were identified. Cesium chloride gradient analysis of the immunoprecipitated DNA indicated that duplex DNA was present in the complexes. Thus, the major DNA-binding protein of HSV-1 is associated with both duplex HSV-1 and cell DNA in vivo.

The structure of herpes simplex virus type 1 DNA as probed by micrococcal nuclease digestion.

Micrococcal nuclease digestion was used to probe the structures in which herpes simplex virus type I (HSV-I) DNA is found during virus replication. Parental DNA, progeny DNA and DNA in nucleocapsids were analysed. Parental DNA was examined after infection of Vero cells with 32P- or 3H-thymidine-labelled HSV-I. Progeny DNA was examined after HSV-I-infected Vero cells were pulse-labelled with 3H-thymidine during HSV-I DNA synthesis. In both cases, nuclei were isolated and digested with micrococcal nuclease. Digestion products were analysed by agarose or polacrylamide gel electrophoresis (PAGE). Most parental DNA remained as intact molecules. However, a small amount was degraded into fragments which were heterogeneous in size or the size of nucleosomal cell DNA. These two classes of fragments were also produced upon digestion of progeny DNA. The heterogeneous fragments and nucleosomal fragments comprised major and minor fractions, respectively, of digested progeny DNA. When digested DNA from HSV-I-infected cells was transferred from composite polyacrylamide-agarose gels to diazobenzyloxymethyl paper, nucleosomal fragments hybridized to 32P-labelled HSV-I DNA as well as to 32P-labelled Vero cell DNA.. Therefore, nucleosomal fragments contained HSV-I DNA sequences. HSV-I DNA in nucleocapsids was analysed by micrococcal nuclease digestion after nucleocapsids were disrupted with PH 9.3 buffer, pyridine, Sarkosyl or NcCl/urea. Only fragments of heterogeneous size were produced. Thus, HSV-I DNA is found predominantly in structures other than nucleosomes during virus replication.

Clinical applications of DNA probes in the diagnosis of genetic diseases.

Currently, advances in molecular technology involving recombinant DNA have led to dramatic breakthroughs in genetic diseases, cancer research, and identification of foreign DNA. Of particular interest is the impact these tools have made and will make on the clinical laboratory. We describe the techniques and their effects on clinical testing in the chemistry laboratory by using selected examples of available applications. Specific examples include carrier detections and prenatal diagnosis in cystic fibrosis and hemophilia, and sickle cell anemia.

Deoxyribonucleoprotein complexes and DNA synthesis of herpes simplex virus type 1.

Two temperature-sensitive mutants of herpes simplex virus type 1 in complementation group 1-1 were analyzed to determine if the major DNA-binding protein they produced was thermolabile. Cells infected with these mutants were analyzed for deoxyribonucleoprotein complexes containing the DNA-binding protein. These complexes were found in cells infected at the permissive temperature but not at the nonpermissive temperature. In temperature shift-up experiments with mutant virus infected cells, the levels of the deoxyribonucleoprotein complexes decreased with time of incubation at the nonpermissive temperature. Viral DNA synthesis terminated in cells infected with these mutants after temperature shift-up. The kinetics of termination of viral DNA synthesis were similar to the kinetics of dissociation of the deoxyribonucleoprotein complexes. These results indicate that two mutants in complementation group 1-1 produce a thermolabile DNA-binding protein and that this protein is required for viral DNA synthesis. Furthermore, they suggest that the major DNA-binding protein of herpes simplex virus type 1 functions in viral DNA synthesis as a component of deoxyribonucleoprotein complexes.

Lymphoma-associated ulcerative bowel disease in the hamster (Mesocricetus auratus) induced by an unusual agent.

In a hamster model of non-Hodgkin's lymphoma which closely parallels the disease in man, and which is induced by an unusual agent(s), a diarrheal bowel disease was a major cause of mortality. This study was initiated to characterize this bowel disease and its relation to lymphoma induction and to natural diseases seen in the hamster. The studies showed that the bowel disease was an ulcerative process and was distinct from natural diseases. The incidence of the bowel disease correlated directly with that of the lymphoma in repeated epizootics, in titration studies, and in agent inactivation tests. The ulcerative bowel lesions were seen at the same stage of the disease as acute and chronic inflammatory infiltrates with necrosis in the thymus and mesenteric lymph nodes. Since necrosis in the gut-associated lymphoid tissue can lead to perforation and sepsis, these bowel lesions were lethal, whereas similar necrosis in other lymphoid tissues (thymus and lymph nodes) could be clinically undetectable. Similar lesions have been reported in man. The ulcerative bowel disease was a reliable early clinical marker for exposure of hamsters to this lymphomagenic agent(s).

Altered biological and biochemical properties of a phosphonoacetate-resistant mutant of herpesvirus of turkeys.

A phosphonoacetate (PA)-resistant mutant of the herpesvirus of turkeys (HVT) was isolated and characterized. The mutant (HVTpa) replicates in growth medium containing 300 microgram/ml of PA and shows in vitro temperature sensitivity at 41 degrees C (its 37 degrees C/41 degrees C efficiency of replication is about 5). HVTpa replicates poorly in chickens and fails to provide complete protection against MDV challenge. The HVTpa-induced DNA polymerase has an apparent inhibition constant for PA 10 times as great, an apparent inhibition constant for pyrophosphate, twice as great, and an apparent Michaelis constant for dCTP 2.5 times as great as the respective figures for the HVTwt-induced enzyme. The HVTpa-induced enzyme is also more thermolabile.

Mechanism of phosphonoacetate inhibition of herpesvirus-induced DNA polymerase.

Phosphonoacetate was an effective inhibitor of both the Marek's disease herpesvirus- and the herpesvirus of turkey-induced DNA polymerase. Using the herpesvirus of turkey-induced DNA polymerase, phosphonoacetate inhibition studies for the DNA polymerization reaction and for the deoxyribonucleoside triphosphate-pyrophosphate exchange reaction were carried out. The results demonstrated that phosphonoacetate inhibited the polymerase by interacting with it at the pyrophosphate binding site to create an alternate reaction pathway. A detailed mechanism and rate equation for the inhibition were developed. For comparison to phosphonoacetate, pyrophosphate inhibition patterns and apparent inhibition constants were determined. Twelve analogues of phosphonoacetate were tested as inhibitors of the herpesvirus of turkey-induced DNA polymerase. At the concentrations tested, only one, 2-phosphonopropionate, was an inhibitor. The apparent inhibition constant for it was about 50 times greater than the corresponding apparent inhibition constant for phosphonoacetate. DNA polymerase alpha of duck embryo fibroblasts, the host cell for the herpesviruses, was inhibited by phosphonoacetate. The apparent inhibition constants for the alpha polymerase were about 10-20 times greater than the corresponding inhibition constants for the herpesvirus-induced DNA polymerase. Duck DNA polymerase beta, Escherichia coli DNA polymerase I, and avian myeloblastosis virus reverse transcriptase were not inhibited by phosphonoacetate.

Substrate specificity of the NS3 serine proteinase of hepatitis C virus as determined by mutagenesis at the NS3/NS4A junction.

Hepatitis C virus (HCV) encodes a polyprotein that is processed to produce the structural and nonstructural proteins of the virus. Nonstructural protein 3 (NS3) is a serine proteinase that cleaves the polyprotein to release the NS4A, NS4B, NS5A, and NS5B proteins. To characterize the substrate specificity of NS3, we synthesized by in vitro translation the polyprotein NS2*-NS3-NS4*P that includes 70% of the NS2 protein, the complete NS3 protein, and 25% of the NS4 protein region attached to substance P, an epitope tag. We demonstrated that NS3 cleaves at the NS3/NS4A junction to release the NS4*P protein. Subsequently, we used this reaction to evaluate the importance of conserved amino acids that flank the NS3/NS4A junction. We replaced amino acids in the P6, P1, and P1' positions of the scissile bond of this junction using site-directed mutagenesis. When the P6 aspartic acid was changed to asparagine, lysine, or serine, NS3-mediated cleavage occurred. When threonine in the P1 position was replaced with other polar amino acids or with amino acids having aliphatic side chains, cleavage occurred, although it was not detected when arginine or tyrosine was present. Replacement of serine in the P1' position with other polar amino acids, with amino acids having aliphatic side chains, or with arginine resulted in NS3-mediated cleavage. Thus, since fewer amino acids in the P1 position supported cleavage than in the P6 or P1' positions, the P1 position of the scissile bond may play a more important role in defining the substrate specificity of the HCV NS3 proteinase.