Solon L. Rhode

Canine Parvovirus: a Biochemical and Ultrastructural Characterization

A canine virus derived from a diseased dog has been plaque-purified and characterized in detail. Analysis of infected cells demonstrated that virus antigen accumulated in the nucleus at 12 to 24 h post-infection and the cytopathology at the ultrastructural level was diagnostic of a parvovirus infection. The purified virus particles were 23 to 26 nm in diam. and banded at a density 1.44 g/ml in CsCl. Detailed biochemical analysis revealed a single-stranded DNA genome and three structural proteins of mol. wt. 82,300, 67,300 and 63,500. All of the data presented are consistent with the classification of this virus as a parvovirus.

Parvovirus H-1 P38 promoter requires the trans-activation region (tar), an SP1 site, and a TATA box for full activity

In the parvovirus H-1 P38 promoter, there are sequences identified as a TATA box, an SP1 site, and a trans-activation responsive element (tar). It was previously shown that the parvovirus H-1 nonstructural protein NS1 positively regulates the expression of the P38 promoter for the viral capsid protein gene via the tar. To characterize the tar element further, a series of single-point mutations of the tar was constructed and the mutants were compared to wild-type for the trans-activation of the P38 promoter using a cat reporter gene. Most of the tar mutations had a negative effect on the P38 promoter and some of them reduced activity as much as 70%. However, when several mutants with multiple-point mutations in the tar were tested, no significant additive effect was observed. We examined the function of the SP1 site in the trans-activation of the P38 promoter by replacing the wild-type SP1 sequence with synthetic DNA fragments, OSP1 or 2SP1, containing no SP1 or two SP1 sites respectively, in a P38 construct with a cat reporter gene. The results indicate that P38 expression varies in proportion to the number of SP1 sites, suggesting a role for the SP1 site during trans-activation by NS1. The role of the TATA box on the P38 promoter was also examined by mutagenizing TATA to CACG. The activity of this promoter was reduced to 43%. When a construct mutated at both the SP1 and TATA box sites was tested for its activity, about 22% of the wild-type activity remained, implying that this remaining activity was contributed largely by the tar element. A model is proposed for how the tar element activates the wild-type and SP1-TATA minus promoters in the presence of NS1.

Encapsidation of a recombinant LuIII parvovirus genome by H1 virus and the fibrotropic or lymphotropic strains of minute virus of mice

We previously constructed a recombinant LuIII parvovirus genome lacking viral coding sequences and used it to generate luciferase-transducing virions, by cotransfection of cells with a helper plasmid expressing LuIII viral proteins. Here, we describe similar cotransfections using alternative, replication-defective helpers encoding the non-structural and capsid proteins of parvovirus H1, or of either the fibrotropic or lymphotropic parvovirus strain of minute virus of mice [MVM(p) or MVM(i)]. Each cotransfection generated transducing virus which directed luciferase expression after infection of HeLa cells. The transducing activity of virus produced using either LuIII or H1 helper plasmids could be specifically neutralized by antiserum raised against the corresponding infectious virus. When the recombinant LuIII parvovirus was pseudotyped with MVM(p) or MVM(i), the resulting virions efficiently expressed luciferase after infection in human or murine cells known to be permissive for both MVM strains. The MVM(p) pseudotyped virus also expressed this reporter efficiently when infected into the murine A9 fibroblast line. In contrast, the recombinant virus generated with an MVM(i) helper gave luciferase expression that was barely detectable after infection of A9 cells which are highly restrictive for MVM(i) productive infection. These results support the notion that the allotropic determinant of these MVM strains functions through their capsid proteins. Pseudotyping of recombinant parvovirus genomes should be useful in controlling their host range as vectors, and in studying mechanisms influencing the permissiveness of parvovirus infections.

Characterization of the HeLa cell single-stranded DNA-dependent ATPase/DNA helicase II.

A single-stranded DNA-dependent ATPase activity, consisting of two subunits of 83 kDa (p90) and 68 kDa (p70), was previously purified from HeLa cells (Vishwanatha, J.K. and Baril, E.F. (1990) Biochem 29, 8753–8759). Homology of the two subunits of single-stranded DNA-dependent ATPase with the human Ku protein (Caoet al. (1994) Biochem 33, 8548–8557) and identity of the Ku protein as the human DNA helicase II (Tutejaet al. (1994) EMBO J. 13, 4991–5001) have been reported recently. Using antisera raised against the subunits of the HDH II, we confirm that the Hela single-stranded DNA-dependent ATPase is the HDH II. Similar to the activity reported for Ku protein, ssDNA-dependent ATPase binds to double-stranded DNA and the DNA-protein complex detected by gel mobility shift assay consists of both the ATPase subunits. The p90 subunit is predominantly nuclear and is easily dissociated from chromatin. The p70 is distributed in cytosol and nucleus, and a fraction of the nuclear p70 protein is found to be associated with the nuclear matrix. Both the p90 and p70 subunits of the ATPase are present in G1 and S phase of the cell cycle and are rapidly degraded in the G2/M phase of the cell cycle.

Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13

SummaryComparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.

Trans-activation of H-1 parvovirus P38 promoter is correlated with increased binding of cellular protein(s) to the trans-activation responsive element (tar)

The parvovirus H-1 P38 promoter contains a trans-activation responsive element (tar). It was previously shown that the parvovirus H-1 nonstructural protein NS1 positively regulates the expression of the P38 promoter for the viral capsid protein gene via the tar (Rhode and Richard, 1987, J. Virol. 61, 2807-2515). To characterize the mechanism of trans-activation by the tar, we used gel shift assays to demonstrate that there exist proteins in virus-infected cellular extracts which have higher binding activity than that found in mock-infected extracts. These observations in vitro are consistent with the expression by P38 constructs with the wild-type promoter linked to a reporter gene, chloramphenicol acetyl transferase (cat), in vivo. We also provide evidence that the protein(s)-tar complex has a molecular mass of approximately 75 kDa in an SDS-polyacrylamide gel, which is less than NS1, and this complex cannot be precipitated by NS1 antibody, which suggests that NS1 mediates the trans-activation by inducing an alteration in the binding activity of some cellular protein(s) in an indirect manner. These data support our previous hypothesis for the activation of the P38 promoter, in which the trans-activator(s) interacts with the tar effectively in the presence of NS1, leading to the formation of the transcription initiation complex by protein-protein associations (Gu, Chen, and Rhode, 1992, Virology 187, 10-17).

Altered Expression of Oncogenes in Mouse Epidermis Following Exposure to Benzo(A)Pyrene Diol Epoxides

There now exists a great deal of evidence which indicates that all normal eukaryotic cells contain endogenous, highly-conserved DNA sequences known as proto-oncogenes (1). The normal functions of these cellular oncogenes are not yet known, although it has been hypothesized that they may be important in cellular differentiation, fetal development and control of cell proliferation (2–4). Since a significant proportion of human cancers are presumed to be the result of exposure to environmental chemicals, extensive research efforts have focused on determining the effects of chemical carcinogens and tumor promoting agents on the expression of these c-onc sequences. Studies by Barbacid and coworkers using the rat mammary carcinoma model have shown that the Ha-ras oncogene is activated in rat mammary carcinomas induced by N-methylnitrosourea (5). Sequencing of the activated rat c-Ha-ras oncogene in individual mammary adenocarcinomas indicated that the rat proto-oncogene had undergone a point mutation in the 12th codon, resulting in a glycine-for-valine substitution in the ras P21 protein product. In similar studies, Balmain and Pragnell employed the two stage model of initiation and promotion in mouse skin to demonstrate that a percentage of papillomas and carcinomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) and promotion with 12-0-tetradecanoyl phorbol-13acetate (TPA) contained elevated levels of Ha-ras transcripts compared with normal mouse epidermis. Furthermore, DNA from papillomas and squamous cell carcinomas caused morphological transformation of NIH/3T3 cells in vitro (6,7). Southern blot hybridization studies demonstrated that the transforming properties of the DNA were due to transfection of an activated cellular Ha-ras oncogene.