Ultrastructural studies of H-1 parvovirus replication. V. Immunocytochemical demonstration of separate chromatin-associated and inclusion-associated antigens.

Electron microscopy and immunocytochrome c staining were used to define the phenotypes of several temperature-sensitive (ts) H-1 mutants. They were classified into three separate groups based on the properties of their capsids at the restrictive temperature (rT): (class 1) ts2 did not assemble capsids but produced spherical and irregular amorphous inclusions; (class 2) ts1 and ts7 exclusively synthesized empty particles which all aggregated and crystallized; and (class 3) ts8 and ts10 formed noncrystalline aggregates of empty virions, but many individual full, as well as empty, capsids were associated with euchromatin. Synthesis of progeny DNA and hemagglutinin at rT were normal for class 3 mutants, but defective for those in classes 1 and 2. The immunospecific staining patterns of these mutants indicated that the H-1 capsid proteins probably form two separate intranuclear antigens: (i) a thermostable chromatin-associated antigen present in proteins that have not formed capsids and are concentrated on heterochromatin and nucleolar-associated chromatin and (ii) a thermolabile inclusion-associated antigen found in the proteins of assembled empty capsids that compose H-1 inclusions.     

Replication process of the parvovirus H-1. X. Isolation of a mutant defective in replicative-form DNA replication.

A temperature-sensitive mutant of H-1, ts14, that is partially defective in replicative-form (RF) DNA synthesis has been isolated. ts14 H-1 is characterized by a decrease in plaque-forming ability and production of infectious virus at the restrictive temperature of 39.5 degrees C. RF DNA synthesis of ts14 is reduced to 3 to 7% of that of wild-type H-1 at either the restrictive or the permissive temperature. A complementation analysis of RF synthesis of ts14 and a viable defective H-1 virus, DI-1, or wild-type H-3 indicates that the defective RF DNA synthesis of ts14 is cis-acting. ts14, unlike wild-type H-1, causes a multiplicity-dependent inhibition of DI-1 or H-3, but not LuIII, RF DNA synthesis. Mixed infections of cells with two parvoviruses also exhibited a cross-interference for viral protein synthesis that was multiplicity dependent, ts14 inhibited infectious virus production of H-1 or H-3, but not LuIII. LuIII-or H-3-pseudotype particles were produced by coinfection with H-1. H-3 and H-1 showed similar interactions with ts14, and H-3 DNA was more homologous to H-1 than was LuIII by comparative physical mapping studies. The results suggest that ts14 is a mutant with a defect in a regulatory sequence of its DNA that influence RF DNA replication.     

Mutant ts1 of bacteriophage PM2 is defective in the major capsid protein and fails to package its DNA.

Infection of Alteromonas espejiana at restrictive temperature with mutant ts1 of bacteriophage PM2 resulted in the intracellular accumulation of virus-sized empty-appearing membrane vesicles. The DNA associated with purified vesicles was fully susceptible to digestion with DNase. Sedimentation analysis and electron microscopy suggested a full-length linear form of the normally circular viral genome. A pulse-chase-shift experiment suggested that [3H]thymidine-labeled DNA made under restrictive conditions is assembled into virions after shift to permissive temperature. A defective structural protein in the ts1 virion appears to be the cause of a rapid rate of thermal inactivation of infectivity. Analysis of the proteins of ts1 by isoelectric focusing indicated a more alkaline isoelectric mobility of the major capsid protein, sp27. Six spontaneous revertants of ts1 showed reversion to the wild-type isoelectric form of sp27. These results identify sp27 as the defective gene product of ts1. Taken together, these results suggest that the membrane of PM2 is formed without the aid of an inner core or an outer scaffolding.     

Control of membrane morphogenesis in bacteriophage PM2

The regulation of membrane formation in bacteriophage PM2 serves as a simple model for changes in membrane structure in eukaryotic cells. Prior to Pseudomonas host lysis, wild-type virions mature to an icosahedral morphology at the inner face of the cytoplasmic membrane. The proliminary charcterization of two temperature-sensitive mutants of PM2 is described. In cells infected at the restrictive temperature with ts 1, an abundance of “empty” virus-size membrane vesicles are seen. Synthesis of DNA is also reduced in ts 1 infected cells. The preponderance of vesicles is not sen in cells infected with wil-type virus or with ts 1 at the permissive temperature. The “empty” appearance of the viral membranes suggests that viral DNA is not encapsulated. The major viral capsid protein (MW 26,000) is located just out side the viral membrane and normallyl sediments with host and virus membranes; insted, large amounts of capsid protein can be precipitated from the supernatant with TCA. Compared to cells infected with wild type virus, cells infected with is 5 at th restrictive temperature produce inside the cell an aboundance of virus-soze membrane vesicles. Taken Together, These results with viral mutants suggest that formation of a viral membrane of the proper size does not require a DNA core around which to form, or an outer scaffolding of coat protein against which to form a spherical bilayer.     

Host Cell Participation in Small Virus Replication I. Replication of M-13 in a Strain of Escherichia coli with a Temperature-sensitive Lesion in Deoxyribonucleic Acid Synthesis

The replication of M-13 in a strain of Escherichia coli with a thermosensitive lesion in deoxyribonucleic acid synthesis was studied. M-13 failed to replicate at the restrictive temperature, even when the parental replicative form was allowed to form at the permissive temperature. When cells which were actively producing phage at the permissive temperature were shifted to the restrictive temperature, phage production continued. The incorporation of radioactive label into phage particles at 42 C indicated that continued single-strand synthesis was unaffected by the lesion in the host cell.     

Evidence for a separate signal sequence for the carboxy-terminal envelope glycoprotein E1 of Semliki forest virus.

When Semliki Forest virus temperature-sensitive mutant ts-3 was grown at the restrictive temperature an aberrant nascent cleavage of the 130,000-dalton structural polyprotein took place relatively frequently. This cleavage yielded an abnormal 86,000-dalton fusion protein (p86) consisting of the amino-terminal capsid protein linked to the amino acid sequences of envelope protein p62 (a precursor of E3 and E2). The other cleavage product was the carboxy-terminal envelope protein E1. p86 was not glycosylated and was sensitive to the action of protease in the microsomal fraction, whereas E1 was glycosylated and protected from proteases, indicating that it had been segregated into the cysternal side of the microsomal vesicles. All attempts to show the E1 protein at the cell surface have failed so far, suggesting that it remains associated with intracellular membranes. When ts-3-infected cells labeled at the restrictive temperature were shifted to the permissive temperature the only labeled protein released with the virus particles was E1, indicating that E1, synthesized at the restrictive temperature, was competent to participate in the virus assembly. These results suggest strongly that there are two separate signal sequences for the envelope proteins of Semliki Forest virus. One follows the capsid protein as shown previously, and the other is for the carboxy-terminal E1. Even if the insertion of the amino-terminal envelope protein (p62) fails due to a cleavage defect, the other signal sequence can operate independently to guide the E1 through the endoplasmic reticulum membrane.     

Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus.

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.     

Defective Particles in BHK Cells Infected with Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Defective particles were the major product after undiluted passage of certain temperature-sensitive (ts) mutants of the Indiana C strain of vesicular stomatitis virus in BHK-21 cells at the permissive temperature (31 C). Essentially homogeneous preparations of defective particles were obtained with the wild-type and individual ts mutants. The defective particles associated with some of the ts mutants, however, were morphologically and physically distinguishable from wild type and from each other. All varieties of defective particle interfered with the multiplication of mutant and wild-type virus at the permissive temperature at early times of infection but failed to complement virions of different complementation groups at the restrictive temperature (39 C) at any time during infection.     

Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types.     

Characterization of a temperature-sensitive mutant of human adenovirus type 7.

The properties of a naturally occurring temperature-sensitive (ts) mutant of human adenovirus type 7 (Ad7) were studied. Mutant Ad7 (19), or E46-, was the nonhybrid adenovirus component derived from the defective simian virus 40 (SV40)-Ad7 hybrid (PARA). Growth of the mutant was restricted at 40.5 degrees C, and the ratios of virus yields in KB cells at 40.5 and 33 degrees C were 10(-2) to 10(-3). Viral DNA synthesis and the synthesis of adenovirus-specific antigens (tumor, capsid, hexon, and penton antigens) appeared normal at the restrictive temperature. The assembly of virus particles was aberrant, as determined by thin-section of infected cells. The infectivity of mutant virions was heat labile at 50 degrees C, suggesting a ts defect in a structural component of the viron. Analysis by polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in mutant-infected cells suggested that at least the major virion polypeptides were synthesized at the restrictive temperature. A lack of inhibition of host protein synthesis late in mutant infections, as compared with wild-type (WT) infections at both the permissive and nonpermissive temperatures, made quantitation of infected-cell polypeptides difficult. Analysis of the assembly of capsomeres from cytoplasmic extracts of infected cells on sucrose gradients and by non-dissociating polyacrylamide gel electrophoresis suggested that hexon capsomeres were made at 40.5 degrees C. The hexon capsomeres made by the mutant at either 33 or 40.5 degrees C displayed a decreased migration in the non-dissociating gels compared with the WT hexon capsomeres. The molecular weights of the mutant and WT hexon polypeptides were identical. These results suggest that the ts lesion of this group B human Ad7 mutant may be reflected in altered hexons. The mutant Ad7 interfered with the replication of adenovirus types 2 and 21 at the elevated temperature.     

Biochemical transformation by temperature-sensitive mutants of herpes simplex virus type 1.

Biochemical transformation assays of herpes simplex virus type 1 temperature-sensitive (ts) mutants distinguished three groups of mutants with regard to their thymidine kinase (TK) transforming ability: those incapable of transferring the TK gene at either the permissive or restrictive temperatures (group I); those resembling the wild-type virus, and therefore able to transform at both the permissive and nonpermissive temperatures (group II); and those that failed to transform or exhibited very low transformation frequencies at the permissive temperature but were able to transform at the nonpermissive temperature (group III). Two mutants in group II exhibited greatly enhanced transformation efficiency at the permissive temperature. The ts lesions in the majority of the mutants tested map between 0.30 and 0.60 units on the viral genome. Mutants with TK-positive (TK+), but DNA-negative, phenotypes at the nonpermissive temperature produced no TK+ transformants at the permissive temperature and only unstable transformants at the nonpermissive temperature. This suggests that a function which is required for viral DNA synthesis is also required to obtain stable expression or to transfer the TK+ gene or both when transfer is mediated by the entire viral genome.     

Structure of the lipid-containing bacteriophage PRD1: disruption of wild-type and nonsense mutant phage particles with guanidine hydrochloride.

The lipid-containing bacteriophage PRD1 was disrupted, and the subviral particles were studied. Guanidine treatment released two phage proteins (P3 and P5). These proteins form the polyhedral capsid. The remaining phage proteins were associated with the phage membrane vesicle. The vesicle was capable of forming a tubular structure. The isolated phage membrane vesicles aggregated readily. We found that aggregation and tube formation were associated with specific phage proteins (P11 and P18, respectively) by using protease treatment and an analysis of nonsense mutant phage particles. In addition, the possibility that free vesicles might be precursors to empty virions was studied.     

Role of simian virus 40 gene A function in maintenance of transformation.

Mouse, hamster, and human cells were transformed at the permissive temperature by mutants from simian virus 40 (SV40) complementation group A in order to ascertain the role of the gene A function in transformation. The following parameters of transformation were monitored with the transformed cells under permissive and nonpermissive conditions: morphology; saturation density; colony formation on plastic, on cell monolayers, and in soft agar; uptake of hexose; and the expression of SV40 tumor (T) and surface (S) antigens. Cells transformed by the temperature-sensitive (ts) mutants exhibited the phenotype of transformed cells at the nonrestrictive temperature for all of the parameters studied. However, when grown at the restrictive temperature, they were phenotypically similar to normal, untransformed cells. Growth curves showed that the (ts) A mutant-transformed cells exhibited the growth characteristics of wild-type virus-transformed cells at the permissive temperature and resembled normal cells when placed under restrictive conditions. There were 3-to 51-fold reductions in the levels of saturation density, colony formation, and uptake of hexose when the mutant-transformed cells were the elevated temperature as compared to when they were grown at the permissive temperature. Mutant-transformed cells from the nonpermissive temperature were able to produce transformed foci when shifted down to permissive conditions, indicating that the phenotypically reverted cells were still viable and that the reversion was a reversible event. SV40 T antigen was present in the cells at both temperatures, but S antigen was not detected in cells maintained at the nonpremissive temperature. All of the wild-type virus-transformed cells exhbited a transformed cells exhibited a transformed phenotype when grown under either restrictive or nonrestrictive conditions. Thers results indicate that the SV40 group A mutant-transformed cells are temperature sensitive for the maintenance of growth properties characteristics of transformation. Virus rescued from the mutant-transformed cells by the transfection method was ts, suggesting that the SV40 gene A function, rather than a cellular one, is responsible for the ts behavior of the cells.     

Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation.

The maturation of pseudorabies virus DNA from the replicative concatemeric form to molecules of genome length was examined using nine DNA+ temperature-sensitive mutants of pseudorabies virus, each belonging to a different complementation group. At the nonpermissive temperature, cells infected with each of the mutants synthesized concatemeric DNA. Cleavage of the concatemeric DNA to genome-length viral DNA was defective in all the DNA+ ts mutants tested, indicating that several viral gene products are involved in the DNA maturation process. In none of the ts mutant-infected cells were capsids with electron-dense cores (containing DNA) formed. Empty capsids with electron-translucent cores were, however, formed in cells infected with six of the nine temperature-sensitive mutants; in cells infected with three of the mutants, no capsid assembly occurred. Because these three mutants are deficient both in maturation of DNA and in the assembly of viral capsids, we conclude that maturation of viral DNA is dependent upon the assembly of capsids. In cells infected with two of the mutants (tsN and tsIE13), normal maturation of viral DNA occurred after shiftdown of the cells to the permissive temperature in the presence of cycloheximide, indicating that the temperature-sensitive proteins involved in DNA maturation became functional after shiftdown. Furthermore, because cycloheximide reduces maturation of DNA in wild-type-infected cells but not in cells infected with these two mutants, we conclude that a protein(s) necessary for the maturation of concatemeric DNA, which is present in limiting amounts during the normal course of infection, accumulated in the mutant-infected cells at the nonpermissive temperature. Concomitant with cleavage of concatemeric DNA, full capsids with electron-dense cores appeared after shiftdown of tsN-infected cells to the permissive temperature, indicating that there may be a correlation between maturation of DNA and formation of full capsids. The number of empty and full capsids (containing electron-dense cores) present in tsN-infected cells incubated at the nonpermissive temperature, as well as after shiftdown to the permissive temperature in the presence of cycloheximide, was determined by electron microscopy and by sedimentation analysis in sucrose gradients. After shiftdown to the permissive temperature in the presence of cycloheximide, the number of empty capsids present in tsN-infected cells decreased with a concomitant accumulation of full capsids, indicating that empty capsids are precursors to full capsids.     

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.     

Partial reversion of conditional transformation correlates with a decrease in the sensitivity of rat cells to killing by the parvovirus minute virus of mice but not in their capacity for virus production: effect of a temperature-sensitive v-src oncogene.

The cytolytic effect of the autonomous parvovirus minute virus of mice, prototype strain (MVMp), was studied in cultures of ts 339/NRK rat cells that display a temperature-sensitive transformed phenotype as a result of their transformation with a Rous sarcoma virus strain matured in the v-src oncogene. A shift from restrictive (39.5 degrees C) to permissive (34.5 degrees C) temperature was associated with a marked sensitization of these cells to killing by MVMp. In contrast, ts 339/NRK cell derivatives supertransformed with a wild-type src oncogene were sensitive to MVMp at both temperatures, suggesting that the expression of a functional oncogene product may determine, at least in part, the extent of the parvoviral cytopathic effect. Although ts 339/NRK cells were quite resistant to parvoviral attack at 39.5 degrees C, they were similarly proficient in MVMp uptake, viral DNA and protein synthesis, and infectious particle production at both permissive and restrictive temperatures. Consistently, electron microscopic examination of infected ts 339/NRK cultures incubated at 39.5 degrees C revealed the presence, in the majority of the cells, of numerous full and empty virions that were predominantly located in autophagic-type vacuoles. Thus, in this system, the reversion of transformed and MVMp-sensitive phenotypes appears to correlate with the setting up of a noncytocidal mode of parvovirus production. These results raise the possibility that the physiological state of host cells may affect their susceptibility to parvoviruses by modulating not only their capacity for virus replication but also cellular processes controlling the cytopathic effect of viral products.     

Adenovirus L1 52- and 55-kilodalton proteins are required for assembly of virions.

A variant of adenovirus type 5 that contained a mutation within the L1 52- and 55-kilodalton (52/55K) protein-coding region was isolated. The mutant, termed ts369, produced L1 52/55K proteins with a two-amino-acid substitution and was temperature sensitive. Temperature-shift experiments indicated that the ts369 defect was late in the viral growth cycle. DNA replication and synthesis of late proteins occurred normally in ts369-infected cells at the nonpermissive temperature, but mature virions were not produced. Rather, capsidlike particles associated with the left-terminal region of the viral chromosome accumulated. These incomplete particles could not be chased into mature virions when the infected cells were shifted to the permissive temperature. However, previously synthesized proteins could be assembled into virions in the presence of a protein synthesis inhibitor upon shiftdown from the nonpermissive temperature, suggesting that the inactivation of the L1 52/55K proteins was reversible. These results indicate that the adenovirus L1 52/55K proteins play a role in the assembly of infectious virus particles.     

Analysis of the protein-protein interactions in the parvovirus H-1 capsid.

The structure of the icosahedral capsid of the H-1 parvovirus was probed by chemical cross-linking methods. Treatment of empty capsids with high-molecular-weight polyethylene glycols resulted in irreversible aggregation of the minor capsid protein VP1. Multimers of VP1 containing at least five and perhaps six molecules were obtained, but only with empty capsids and not with the full, DNA-containing virus. Cross-linking of the empty capsids with dimethylsuberimidate confirmed the assignments of the products formed after treatment with polyethylene glycol. With dimethylsuberimidate the most abundant product was a heterologous dimer containing VP1 and the major capsid protein VP2'. A small amount of homologous VP2' dimer was also obtained, but the majority of VP2' remained unreacted even at high concentrations of dimethylsuberimidate. The capsid proteins of the full virus, on the other hand, were completely unreactive to dimethylsuberimidate. The data suggest that the minor protein VP1 may be clustered in the capsid and perhaps composes one or two of the morphological units of the icosahedral shell.