The transposon TnphoA was used to generate fusions between phoA, the gene for alkaline phosphatase (PhoA), and genes encoding proteins that are secreted by Vibrio cholerae. One of the PhoA' mutants isolated showed a dramatic reduction in its ability to colonize the intestines of suckling mice. This mutant no longer produced a 20.5-kDa protein (TcpA) that we show is the major subunit of a V. cholerae pilus. Amino-terminal sequence analysis of the TcpA pilus subunit showed that it shares amino acid homology with the pilins produced by several other pathogenic bacteria. The TcpA pilus was coordinately expressed with cholera toxin under various culture conditions, and this effect appeared to be dependent on the transcriptional activator encoded by the toxR gene. We conclude that the toxR gene plays a central role in the transcriptional regulation of multiple virulence genes of V. cholerae.
We were interested in screening a series of isolates of the protozoan Leishmania for the presence of viruses. The experimental procedure we used was based on an enzymatic assay originally developed for viral RNA-dependent RNA polymerases. Simultaneously, total promastigote nucleic acid preparations were analyzed for the presence of viral genome and/or transcripts. Two isolates, both classified as L. braziiensis guyanensis, were found to be positive for RNA polymerase activity and to carry a large (6 kilobases) RNA species. The polymerase reaction products hybridized to the 6-kilobase RNA, believed to be the viral genome. In conjunction with electron microscopical observations these results indicate the presence of an RNA virus in these Leishmania isolates.The study of bacteriophages and viruses has provided important leads in our understanding of the control of gene expression, RNA processing, and translation. The successful use of viruses as molecular tools and models has been facilitated by their small genome size and small number of genes, as well as by the abundance of viral transcripts and proteins in infected cells. These characteristics motivated us to search for the presence of viruses in the protozoan parasite Leishmania.There have been several reports on the molecular characterization of viruses in parasitic protozoa. Wang and Wang (1) reported the presence of a double-stranded RNA virus in Giardia lamblia. A double-stranded RNA virus has been associated with another parasitic protozoan, Trichomonas vaginalis (2). This virus has a linear genome of approximately 5 kilobases (kb). Recently, Stuart and co-workers (3) reported observations consistent with a viral infection in a Leishmania isolate and named this putative virus LR1.As classical virological techniques based on plaque assays for the detection of viruses are not applicable to protozoans, we have screened Leishmania isolates for RNA polymerase activity that could indicate viral replication. The use of enzymatic assays for viral enzymes has proven in the past to be the most sensitive method for the isolation of new viral systems, such as vaccinia virus with viral RNA polymerase (4) and retroviruses with RNA-dependent DNA polymerase (5, 6). As replication of both negative-and positive-stranded RNA viruses depends on viral RNA polymerase, this enzymatic activity can point to the presence of such viruses, regardless of their genomic polarity.We describe here a series of experiments aimed at detecting viral genome and RNA polymerase activity in a set of Leishmania isolates. Polymerase activity was found in two isolates and correlated with the presence of a 6-kb nucleic acid species. These observations suggest the existence of a virus in these isolates. MATERIALS AND METHODSParasites. Leishmania isolates were grown in vitro as promastigotes in Schneider's medium supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 4 mM glutamine, and 0.01% gentamycin sulfate (GIBCO). Isolate code M4147 is abbreviated from the official World Hea...
Previous studies have shown that cells infected with the herpes simplex virus 1(HFEM) mutant tsB7 and maintained at the nonpermissive temperature fail to accumulate viral polypeptides. Analyses of intertypic recombinants generated by marker rescue of tsB7 with herpes simplex virus 2 DNA fragments localized the mutation between 0.46 and 0.52 map units on the viral genome (Knipe et al., J. Virol. 38:539-547, 1981). In this paper we report that the mutation in tsB7 affects several aspects of the reproductive cycle of the virus at the nonpermissive temperature. Thus, (i) viral capsids accumulate at the nuclear pores and do not release viral DNA for at least 6 h postinfection at 39°C. The DNA was released within 30 min after a shift to the permissive temperature. (ii) Experiments involving shifts from the permissive to the nonpermissive temperature indicated that viral protein synthesis was not sustained in cells maintained at the permissive temperature for less than 4 h. (iii) Viral DNA synthesis was delayed at the permissive temperature for as long as 8 h. Once initiated, it continued at 39°C. (iv) Marker rescue of tsB7 by transfection with herpes simplex virus 1(F) DNA fragments localized the mutation to between 0.501 and 0.503 map units on the viral genome. These results are consistent with the tsB7 lesion being in a gene coding for a virion component which affects release of viral DNA from capsids and onset of viral DNA synthesis. Herpes simplex virus 1 (human herpesvirus 1; HSV-1) mutant tsB7 of strain HSV-1(HFEM) was reported to complement other temperaturesensitive mutants in doubly infected cells (8, 14). However, viral gene products were not detected in cells infected and maintained at the nonpermissive temperature. The mutation was mapped by marker rescue between 0.46 and 0.52 map units on the prototype arrangement of the DNA (8). In this paper, we report evidence that tsB7 is defective in several functions, such as release of viral DNA from capsids, viral DNA synthesis, and late gene expression, even though fine mapping of the lesion indicates that it is contained within a 300-base-pair region of the DNA. MATERIALS AND METHODS Viruses and cells. HSV-1(HFEM) tsB7 syn+ was isolated by bromodeoxyuridine mutagenesis of HSV-1(HFEM)-infected cells (4), and the properties of this mutant were described elsewhere (8). Virus stocks were prepared and titrated on Vero or HEp-2 cells as previously described (12). Rabbit skin cells were originally obtained from J. McLaren. Human embryonic lung (HEL) cells were originally obtained from Flow Laboratories, Inc., Rockville, Md. Enzymes and radioisotopes. The restriction endonucleases BamHI, Sall, and Xhol were obtained from New England Biolabs (Waverly, Mass.) and Bethesda Research Laboratories (Rockville, Md.). T4 DNA ligase was purchased from P-L Biochemicals, Inc., Milwaukee, Wis. The radioisotopes [methyl-3H]thymidine (59 Ci/mmol) and ['4C]leucine, [14C]sioleucine, and [(4C]valine (each >250 mCi/mmol) were purchased from New England Nuclear Corp., Boston, Mass. Electron...
The core of the herpes simplex virion consists of an electron-dense toroidal structure 50 nm high, with an inside diameter of 18 nm and an outside diameter of 70 nm, penetrated by a less dense cylindrical mass. The toroid contains deoxyribonucleic acid (DNA), as evident from the observation that uranyl ion staining is removed by ethylenediaminetetraacetic acid under conditions which result in the extraction of uranyl ions bound to nuclear DNA. Studies on negatively stained preparations of purified capsids suggest that the toroid consists of DNA arranged as if it were spooled around the cylindrical mass.
In herpes simplex virus-infected cells, coreless capsids accumulate at the nuclear pores soon after infection, but subsequently disappear, suggesting that, as in adenovirus-infected cells (S. Dales and Y. Chardonnet, Virology 56:465-483, 1973), the release of viral DNA from nucleocapsids takes place at the nuclear pores. A nonlethal mutant, HSV-1(50B), produced by mutagenesis of HSV DNA fragments and selected for delayed production of plaques at 31 degrees C, accumulated coreless capsids at the nuclear pores late in infection in contrast to wild-type viruses. Recombinants selected for ability to produce plaques at 31 degrees C by marker rescue with digests of herpes simplex virus 2 DNA and selected clone fragments of HSV-1 DNA no longer accumulated empty capsids at nuclear pores late in infection. These results suggest that herpes simplex viruses encode a function which prevents accumulation of coreless capsids at nuclear pores, presumably by preventing uptake, unenvelopment, and DNA release from progeny virus, and indicate that the cold sensitivity of plaque formation and accumulation of coreless capsids might be related or comap in the S component of the genome.
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