Conserved polynucleotide sequences among the genomes of papillomaviruses.

The DNAs of different members of the Papillomavirus genus of papovaviruses were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm - 28 degrees C), no homology was detectable among the genomes of human papillomavirus type 1 (HPV-1), bovine papillomavirus type 2 (BPV-2), or cottontail rabbit (Shope) papillomavirus (CRPV). However, under less stringent conditions (i.e., Tm - 43 degrees C), stable hybrids were formed between radiolabeled DNAs of CRPV, BPV-1, or BPV-2 and the HindIII-HpaI A, B, and C fragments of HPV-1. Under these same conditions, radiolabeled CRPV and HPV-1 DNAs formed stable hybrids with HincII B and C fragments of BPV-2 DNA. These results indicate that there are regions of homology with as much as 70% base match among all these papillomavirus genomes. Furthermore, unlabeled HPV-1 DNA competitively inhibited the specific hybridization of radiolabeled CRPV DNA to bpv-2 DNA fragments, indicating that the homologous DNA segments are common among these remotely related papillomavirus genomes. These conserved sequences are specific for the Papillomavirus genus of papovaviruses as evidenced by the lack of hybridization between HPV-1 DNA and either simian virus 40 or human papovavirus BK DNA under identical conditions. These results indicate a close evolutionary relationship among the papillomaviruses and further establish the papillomaviruses and polyoma viruses as distinct genera.     

Evolutionary relationships of the primate papovaviruses: base sequence homology among the genomes of simian virus 40, stump-tailed macaque virus, and SA12 virus.

Physical maps of the genomes of the two newly discovered primate papovaviruses, SA12 and stump-tailed macaque virus (STMV), were generated by restriction endonuclease analysis. The base sequence homologies among the genomes of SA12, stump-tailed macaque virus, and simian virus 40 (SV40) were studied by heteroduplex analysis. Heteroduplexes between SA12 and SV40 DNAs and stump-tailed macaque virus and SV40 DNAs were constructed and mounted for electron microscopy in various amounts of formamide to achieve a range of effective temperatures. At each effective temperature, the regions of duplex DNA in the heteroduplexes were measured and localized on the SV40 physical and functional maps. By analyzing the data from this study and rom our previous study (N. Newell, C. J. Lai, G. Khoury, and T. J. Kelly Jr., J. Virol. 25:193-201, 1978) on the base sequence homology between the genomes of BK virus and SV40, some general conclusions have been drawn concerning the evolutionary relationships among the genomes of the primate papovaviruses. The extent of homology among the viral genomes does not reflect the phylogenetic relationships of their hosts. At comparable effective temperatures Tm - 33 degrees C), the heteroduplexes between the DNAs of BK virus and SV40 contained the largest amount of duplex (about 90%). The heteroduplexes made between SA12 and SV40 DNAs were slightly less homologous, containing about 80% duplex. The heteroduplexes made between SV40 and stump-tailed macaque virus DNAs were only 20% duplex under the same conditions. When the various heteroduplexes were mounted for microscopy at effective temperatures greater than Tm - 33 degrees C, the fraction of the duplex DNA decreased in each case, indicating the existence of considerable base mismatching in the homologous regions. When specific coding or noncoding regions of the viral genomes were compared, the data indicated that the extent of sequence divergence differed markedly from one region to another. In all the heteroduplexes studied, there were two regions, located near the junctions between early and late regions on the SV40 map, which were essentially nonhomologous. All of the heteroduplexes studied showed significantly greater homology in the late region than in early region. Within the late region, the sequences coding for the major capsid polypeptide, VP1, were the most highly conserved.     

Temperature-sensitive adult liver cell line dependent on glucocorticoid for differentiation.

A clonal rat adult hepatocyte cell line (RALA255-10G) was shown to be temperature sensitive (ts) for growth and differentiation. Glucocorticoid was necessary to maintain the maximal levels of differentiated functions in these cells. The RALA255-10G cell line was established by transforming primary adult hepatocytes with simian virus 40 tsA255 virus that is temperature sensitive for maintenance of transformation. At the permissive temperature (33 degrees C), RALA255-10G cells showed characteristics of malignant transformation, synthesized low levels of albumin and transferrin, and contained low levels of functional receptors for glucagon. At the nonpermissive temperature (40 degrees C), these cells regain the normal differentiated phenotype, and the levels of these three hepatic functions were increased. Induction of albumin and transferrin production by RALA255-10G cells at 40 degrees C was shown to be the result of the increase in the biosynthesis of these proteins. Furthermore, the albumin and transferrin produced by these cells were immunologically and electrophoretically indistinguishable from authentic rat albumin and transferrin. Glucocorticoid, which reduced the growth rate and saturation density of RALA255-10G cells at 33 degrees C, was absolutely required by these cells to synthesize albumin at both temperatures. This hormone also enhanced transferrin production and glucagon response. Our data indicate that glucocorticoid hormone is one of the factors that maintain adult hepatocytes in a differentiated state.     

A mutated membrane protein of vesicular stomatitis virus has an abnormal distribution within the infected cell and causes defective budding.

Two temperature-sensitive (ts) mutants of the M protein of vesicular stomatitis virus (tsG31 and tsG33) are defective in viral assembly, but the exact nature of this defect is not known. When infected cells are switched from nonpermissive (40 degrees C) to permissive (32 degrees C) temperatures in the presence of cycloheximide, tsG33 virus release increased by 100-fold, whereas tsG31 release increased only by 10-fold. Thus, the tsG33 defect is more reversible than that of tsG31. Therefore, we investigated how the altered synthesis and cellular distribution of tsG33 M protein correlates with the viral assembly defect. At 32 degrees C tsG33 M protein is stained diffusely in the cell cytoplasm and later at the budding sites. In contrast, at 40 degrees C the mutant M protein formed unusual aggregates mostly located in the perinuclear regions of virus-infected cells and partially colocalized with G protein in this region. In temperature shift-down experiments, M can be disaggregated and used to some extent for nucleocapsid coiling and budding, which correlates with the virus titer increase. M aggregates also formed after shift-up from 32 to 40 degrees C, indicating a complete dependence of M aggregation on the temperature. Biochemical analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that at 40 degrees C M protein is detected exclusively in pellet fractions (nuclear and cytoskeleton components), whereas at 32 degrees C M protein is mainly in the cytoplasmic soluble fractions. Furthermore, when the temperature is raised from 32 to 40 degrees C, the distribution of M protein tends to shift from the soluble to the pellet and cytoskeletal fractions. Electron micrographs of immunoperoxidase-labeled M protein showed that at 40 degrees C M aggregates are often associated with the outer nuclear membranes as well as with vesicular structures. No nucleocapsid coiling was observed in these cells, whereas coiling and budding were seen at 32 degrees C in cells where M protein was partly associated with the plasma membrane. We suggest that the tsG33 M protein mutation may produce a reversible conformational alteration which causes M protein to aggregate at 40 degrees C, therefore inhibiting the proper association of M protein with nucleocapsids and budding membranes.     

Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis.

We have cloned from Bacillus subtilis a novel protease gene (nprB) encoding a neutral protease by using a shotgun cloning approach. The gene product was determined to have a molecular mass of 60 kDa. It has a typical signal peptide-like sequence at the N-terminal region. The expression of nprB can be stimulated by using a B. subtilis strain, WB30, carrying a sacU(h)h mutation. Expression of this protease gene results in production of a 37-kDa protease in the culture medium. The first five amino acid residues from the N terminus of the mature protease were determined to be Ala-Ala-Gly-Thr-Gly. This indicates that the protease is synthesized in a preproenzyme form. The purified protease has a pH optimum of around 6.6, and its activity can be inhibited by EDTA, 1,10-phenanthroline (a zinc-specific chelator), and dithiothreitol. It retained 65% of its activity after treatment at 65 degrees C for 20 min. Sequence comparison indicates that the mature form of this protease has 66% homology with the two thermostable neutral proteases from B. thermoproteolyticus and B. stearothermophilus. It also shares 65, 61, and 56% homology with the thermolabile neutral proteases from B. cereus, B. amyloliquefaciens, and B. subtilis, respectively. The zinc-binding site and the catalytic residues are all conserved among these proteases. Sequence homology extends into the "propeptide" region. The nprB gene was mapped between metC and glyB and was not required for growth or sporulation.     

Isolation and characterization of a heat-inducible simian virus 40 mutant.

We have isolated a new type of temperature-sensitive mutant of simian virus 40 (SV40) that is capable of productive infection in permissive cells but not of maintenance of viral DNA integration in transformed cells at the conditional temperature. Virus development is induced when cells transformed by this mutant are shifted to temperatures above 39 degrees C, but is not induced below this temperature. The plaque-purified, temperature-sensitive mutant virus confers heat inducibility to new host cells, indicating that the conditional function is a property of the viral genome. Unlike previously described temperature-sensitive SV40 mutants, in (ts)-1501 is capable of productive infection in permissive cells at the conditional temperature. The morphology, growth, and oncogenicity of in (ts)-1501-transformed cells at 37 degrees C are similar to those of cell lines transformed by wild-type SV40. HK10-c2(in(ts)-1501), a cloned cell line, transformed at 37 degrees C by the mutant virus, exhibits a transient increase in DNA synthesis before cell death at the conditional temperature. Many properties of in(ts)-1501 are analogous to those of the heat-inducible mutants of bacteriophages in which a heat-inactivated protein is responsible for the stable integration of the prophage in the bacterial chromosome.     

Phosphorylation of T-antigen and control T-antigen expression in cells transformed by wild-type and tsA mutants of simian virus 40.

Chinese hamster lung (CHL) cells transformed by wild-type simian virus 40 (cell line CHLWT15) or transformed by the simian virus 40 mutants tsA30 (cell lines CHLA30L1 and CHLA30L2) or tsA239 (cell line CHLA239L1) were used to determine the rates of turnover and synthesis of the T-antigen protein and the rate of turnover of the phosphate group(s) attached to the T-antigen at both the permissive and restrictive temperatures. The phosphate group turned over several times within the lifetime of the protein to which it was attached, with the exception of the phosphate group in the tsA transformants at 40 degrees C, which turned over at the same rate as the T-antigen protein. The steady-state levels of the T-antigens (molecular weights, 92,000 [92K] and 17K) and the amount of simian virus 40-specific RNA was also determined in each of the lines. The CHLA30L1 line contained two to three times more early simian virus 40 RNA than the CHLA30L2 line; although neither line formed colonies in agar at 40 degrees C, CHLA30L1 overgrew a normal monolayer at 40 degrees C. The rate of 92K-T-antigen synthesis was 1.5 times faster in CHLA30L1 than in CHLA30L2 at 33 degrees C and 4 times faster at 40 degrees C. The different phenotype of these two presumably isogenic cell lines seem to be related to the levels of the T-antigens. The ratios of the 92K T-antigen to the 17K T-antigens were similar in the two lines. Transformed CHL cell lines, unlike transformed mouse 3T3 cell lines, were found to contain very small amounts of the 56K T-antigen.     

Prolongation of herpes simplex virus latency in cultured human cells by temperature elevation.

Treatment of herpes simplex virus type 2 (HSV-2)-infected human fibroblast cells with cytosine arabinoside (ara-C) at 25 microgram/ml resulted in complete inhibition of virus replication. Removal of ara-C after 7 days of treatment ultimately resulted in renewed virus replication, but after a delay of at least 5 days. If however, the temperature was elevated from 37 degrees C to 39.5 to 40 degrees C at the time of ara-C reversal, infectious HSV-2 did not reappear. As long as the cultures were maintained at 39.5 to 40 degrees C (up to at least 128 days), HSV-2 was latent and infectious virus was undetectable. If the temperature was reduced to 37 degrees C at any time during the latent period, infectious virus was always reactivated, but only after a period of incubation at 37 degrees C of a least 11 days. Infectious-center assays performed with latent cultures indicated that only a very small fraction of cells could reactivate virus. The infectious-center titer did not show significant changes during much of the period of latency. This seemed to argue against the possibility that the latent cultures were synthesizing very small amounts of infectious virus. Additional studies were aimed at determining the minimum incubation period at 37 degrees C required to reactivate infectious HSV-2. Latent cultures reduced from 39.5 to 40 degrees C to 37 degrees C for less than 96 h did not yield infectious HSV-2, but those incubated at 37 degrees C for 96 h or more did.     

Consequences of herpes simplex virus type 2 and human cell interaction at supraoptimal temperatures.

The consequences of herpes simplex virus type 2 (HSV-2) and human embryonic fibroblast cell interaction at different temperatures (37, 40, and 42 degrees C) were investigated. Incubation at 37 or 40 degrees C was permissive for HSV-2 inhibition of host DNA synthesis, induction of virus-specific DNA replication, and infectious virus production. The amount of [methyl-3H]thymidine incorporated into viral DNA and the final yield of new infectious virus were significantly reduced at 40 degrees C compared to 37 degrees C. At 42 degrees C, detectable virus-specific DNA synthesis was totally blocked. Maximum stimulation of host cell DNA synthesis at 42 degrees C was measured after a multiplicity of infection of 0.5 to 1.0 PFU/cell. By autoradiography, data indicated that HSV-2 stimulates host cell chromosomal DNA synthesis. Stimulation of thymidine kinase activity with thermostability properties in common with a virus enzyme was detected during the first 24 h of infection at 42 degrees C, after 24 h the enhanced thymidine kinase activity had properties in common with host cell isozymes. The data obtained during this investigation indicated that stimulation of host cell DNA synthesis does not require viral DNA synthesis.