Formation of influenza virus particles lacking hemagglutinin on the viral envelope.

We investigated the intracellular block in the transport of hemagglutinin (HA) and the role of HA in virus particle formation by using temperature-sensitive (ts) mutants (ts134 and ts61S) of influenza virus A/WSN/33. We found that at the nonpermissive temperature (39.5 degrees C), the exit of ts HA from the rough endoplasmic reticulum to the Golgi complex was blocked and that no additional block was apparent in either the exit from the Golgi complex or post-Golgi complex transport. When MDBK cells were infected with these mutant viruses, they produced noninfectious virus particles at 39.5 degrees C. The efficiency of particle formation at 39.5 degrees C was essentially the same for both wild-type (wt) and ts virus-infected cells. When compared with the wt virus produced at either 33 or 39.5 degrees C or the ts virus formed at 33 degrees C, these noninfectious virus particles were lighter in density and lacked spikes on the envelope. However, they contained the full complement of genomic RNA as well as all of the structural polypeptides of influenza virus with the exception of HA. In these spikeless particles, HA could not be detected at the limit of 0.2% of the HA present in wt virions. In contrast, neuraminidase appeared to be present in a twofold excess over the amount present in ts virus formed at 33 degrees C. These observations suggest that the presence of HA is not an obligatory requirement for the assembly and budding of influenza virus particles from infected cells. The implications of these results and the possible role of other viral proteins in influenza virus morphogenesis are discussed.     

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein.

We describe a procedure that enriches for temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV), Indiana serotype, which are conditionally defective in the biosynthesis of the viral glycoprotein. The selection procedure depends on the rescue of pseudotypes of known ts VSV mutants in complementation group V (corresponding to the viral G protein) by growth at 39.5 degrees C in cells preinfected with the avian retrovirus Rous-associated virus 1 (RAV-1). Seventeen nonleaky ts mutants were isolated from mutagenized stocks of VSV. Eight induced no synthesis of VSV proteins at the nonpermissive temperature and hence were not studied further. Four mutants belonged to complementation group V and resembled other ts (V) mutations in their thermolability, production at 39.5 degrees C of noninfectious particles specifically deficient in VSV G protein, synthesis at 39.5 degrees C of normal levels of viral RNA and protein, and ability to be rescued at 39.5 degrees C by preinfection of cells by avian retroviruses. Five new ts mutants were, unexpectedly, in complementation group IV, the putative structural gene for the viral nucleocapsid (N) protein. At 39.5 degrees C these mutants also induced formation of noninfectious particles relatively deficient in G protein, and production of infectious virus at 39.5 degrees C was also enhanced by preinfection with RAV-1, although not to the same extent as in the case of the group V mutants. We believe that the primary effect of the ts mutation is a reduced synthesis of the nucleocapsid and thus an inhibition of synthesis of all viral proteins; apparently, the accumulation of G protein at the surface is not sufficient to envelope all the viral nucleocapsids, or the mutation in the nucleocapsid prevents proper assembly of G into virions. The selection procedure, based on pseudotype formation with glycoproteins encoded by an unrelated virus, has potential use for the isolation of new glycoprotein mutants of diverse groups of enveloped viruses.     

Temperature-sensitive defect of vesicular stomatitis virus in complementation group II.

The prototype member of the complementation group II temperature-sensitive (ts) mutants of vesicular stomatitis virus, ts II 052, has been investigated. In ts II 052-infected HeLa cells at the restrictive temperature (39.5 degrees C), reduced viral RNA synthesis was observed by comparison with infections conducted at the permissive temperature (30 degrees C). It was found that for an infection conducted at 39.5 degrees C, no 38S RNA or intracytoplasmic nucleocapsids were present. For nucleocapsids isolated from ts II 052 purified virions or from ts II 052-infected cells at 30 degrees C, the RNA was sensitive to pancreatic RNase after an exposure at 39.5 degrees C in contrast to the resistance observed for wild-type virus. The nucleocapsid stability of wild-type virus when heated to 63 degrees C or submitted to varying pH was not found in nucleocapsids extracted from ts II 052 purified virions. The data suggest that for ts II 052 there is an altered relationship between the viral 38S RNA and the nucleocapsid protein(s) by comparison with wild-type virus. Such results argue for the complementation group II gene product being N protein, so that the ts defect in ts II 052 represents an altered N protein.     

Polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of Madin-Darby canine kidney cells infected with temperature-sensitive viruses

The intracellular route followed by viral envelope glycoproteins in polarized Madin-Darby canine kidney cells was studied by using temperature-sensitive mutants of vesicular stomatitis virus (VSV) and influenza, in which, at the nonpermissive temperature (39.5 degrees C), the newly synthesized glycoproteins (G proteins) and hemagglutinin (HA), respectively, are not transported out of the endoplasmic reticulum. After infection with VSV and incubation at 39.5 degrees C for 4-5 h, synchronous transfer of G protein to the plasma membrane was initiated by shifting to the permissive temperature (32.5 degrees C). Immunoelectron microscopy showed that under these conditions the protein moved to the Golgi apparatus and from there directly to a region of the lateral plasma membrane near this organelle. G protein then seemed to diffuse progressively to basal regions of the cell surface and, only after it had accumulated in the basolateral domain, it began to appear on the apical surface near the intercellular junctions. The results of these experiments indicate that the VSV G protein must be sorted before its arrival at the cell surface, and suggest that passage to the apical domain occurs only late in infection when tight junctions are no longer an effective barrier. In complementary experiments, using the temperature-sensitive mutant of influenza, cultures were first shifted from the nonpermissive temperature (39.5 degrees C) to 18.5 degrees C, to allow entrance of the glycoprotein into the Golgi apparatus (see Matlin, K.S., and K. Simons, 1983, Cell, 34:233-243). Under these conditions HA accumulated in Golgi stacks and vesicles but did not reach the plasma membrane. When the temperature was subsequently shifted to 32.5 degrees C, HA rapidly appeared in discrete regions of the apical surface near, and often directly above, the Golgi elements, and later diffused throughout this surface. To ensure that the anti-HA antibodies had access to lateral domains, monolayers were treated with a hypertonic medium to dilate the intercellular spaces. Some labeling was then observed in the lateral plasma membranes soon after the shift, but this never increased beyond 1.0 gold particle/micron, whereas characteristic densities of labeling in apical surfaces soon became much higher (approximately 10 particles/micron). Our results suggest that the bulk of HA follows a direct pathway leading from the Golgi to regions of the apical surface close to trans-Golgi cisternae.     

Characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain A59 with maturation defects in the spike protein.

Two temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59, ts43 and ts379, have been described previously to be ts in infectivity but unaffected in RNA synthesis (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). We present a detailed analysis of the protein synthesis of the mutant viruses at the permissive (31 degrees C) and nonpermissive (39.5 degrees C) temperatures. It was found that synthesis of the nucleocapsid protein N and the membrane protein M of both viruses was insensitive to temperature. However, the surface protein S of both viruses was retained in the endoplasmic reticulum at the nonpermissive temperature. This was shown first by analysis of endoglycosidase H-treated and immunoprecipitated labeled S proteins. The mature Golgi form of S was not present at the nonpermissive temperature for the ts viruses, in contrast to wild-type (wt) virus. Second, gradient purification of immunoprecipitated S after pulse-chase labeling showed that only wt virus S was oligomerized. We conclude that the lack of oligomerization causes the retention of the ts S proteins in the endoplasmic reticulum. As a result, ts virus particles that were devoid of S were produced at the nonpermissive temperature. This result could be confirmed by biochemical analysis of purified virus particles and by electron microscopy.     

Morphological and biochemical characterization of viral particles produced by the tsO45 mutant of vesicular stomatitis virus at restrictive temperature.

The growth at restrictive temperature of tsO45, a group V (glycoprotein) conditional lethal mutant of vesicular stomatitis virus (VSV), was demonstrated to result in the production of large numbers of noninfectious viral particles. The infectivity of these tsO45 particles could be enhanced by procedures known to promote membrane fusion. Morphologically and biochemically these particles differed from wild-type VSV by their lack of viral glycoprotein. The other structural proteins of VSV were present and indistinguishable by size and relative proportion from those of virus grown at the permissive temperature. Examination of glycoprotein maturation at the restrictive temperature (39.5 degrees C) in tsO45-infected cells demonstrated the synthesis of normal viral glycoprotein but failed to demonstrate the presence of this glycoprotein in either the cell membrane or the envelope of free virions. The further absence of soluble viral glycoprotein from the supernatants of such cells strongly suggests that viral glycoprotein may not be necessary for the successful budding of VSV.     

Ubiquitin-activating enzyme, E1, is associated with maturation of autophagic vacuoles

The ubiquitin-activating enzyme, E1, is required for initiating a multi-step pathway for the covalent linkage of ubiquitin to target proteins. A CHO cell line containing a mutant thermolabile E1, ts20, has been shown to be defective in stress-induced degradation of proteins at restrictive temperature (Gropper et al., 1991. J. Biol. Chem. 266:3602-3610). Parental E36 cells responded to restrictive temperature by stimulating lysosome-mediated protein degradation twofold. Such a response was not observed in ts20 cells. The absence of accelerated degradation in these cells at 39.5 degrees C was accompanied by an accumulation of autolysosomes. The fractional volume of these degradative autophagic vacuoles was at least sixfold greater than that observed for either E36 cells at 30.5 degrees or 39.5 degrees C, or ts20 cells at 30.5 degrees C. These vacuoles were acidic and contained both acid phosphatase and cathepsin L, but, unlike the autolysosomes observed in E36 cells, ubiquitin-conjugated proteins were conspicuously absent. Combined, our results suggest that in ts20 cells, which are unable to generate ubiquitin-protein conjugates due to heat inactivation of E1, the formation and maturation of autophagosomes into autolysosomes is normal, but the conversion of autolysosomes into residual bodies is disrupted.     

Mutational changes in the vesicular stomatitis virus glycoprotein affect the requirement of carbohydrate in morphogenesis.

The role of carbohydrate in the morphogenesis of vesicular stomatitis virus was studied, using the antibiotic tunicamycin to inhibit glycosylation. It has been reported previously (Gibson et al., J. Biol. Chem. 254:3600-3607, 1979) that the San Juan strain of vesicular stomatitis virus requires carbohydrate for efficient migration of the glycoprotein (G) to the cell surface and for virion formation, whereas the prototype or Orsay strain of vesicular stomatitis virus is less stringent in its carbohydrate requirement at 30 degrees C. However, there are many differences between the two strains. We found that mutational changes within the G protein of the same strain of virus (prototype or Orsay) alters the requirement for carbohydrate at 30 degrees C. Group V or G protein mutants tsO45 and tsO44, like their prototype parent, did not require carbohydrate for efficient morphogenesis. In contrast, the G protein of another group V mutant, tsO110, was totally dependent upon carbohydrate addition for migration to the cell surface. Furthermore, no tsO110 particles were released in the absence of glycosylation. The wild-type prototype strain did require carbohydrate at 39.5 degrees C for insertion of the G protein into the plasma membrane and virion formation. However, a pseudorevertant of tsO44 (tsO44R), unlike the prototype parent, no longer exhibited this temperature-sensitive requirement for carbohydrate. At 39.5 degrees C in the presence of tunicamycin, tsO44R-infected cells released normal yields of particles and the unglycosylated G reached the cell surface very efficiently. In contrast to tsO110, which absolutely requires carbohydrate, mutational change in the tsO44R G protein has eliminated the requirement for carbohydrate. Thus, simple mutational changes, as opposed to many changes in the molecule, are sufficient to alter the carbohydrate requirement.     

Role of glycosylation in transport of vesicular stomatitis virus envelope glycoprotein. A new class of mutant defective in glycosylation and transport of G protein

A temperature-sensitive mutant (ts gamma 1) of the Cocal serotype of vesicular stomatitis virus synthesizes at the permissive temperature (32 degrees C) a glycoprotein G whose size is smaller (Mr 68,000) than the wild-type (Mr 71,000) and that renders the virion thermolabile. At the nonpermissive temperature (39 degrees C), reduced amounts of noninfectious virus-like particles deficient in G protein were produced. The size of the intracellular G protein was further decreased (Mr 64,000) at the nonpermissive temperature. Biochemical studies including sugar labeling, tryptic peptide analysis, and NH2-terminal sequence analysis of the various glycoproteins suggest that at 32 degrees C a G protein containing a single glycosidic moiety is synthesized. The G protein containing only 1 oligosaccharide residue is transported to the cell surface and is incorporated in infectious virus particles. In contrast, the G protein synthesized at 39 degrees C is nonglycosylated and fails to reach the cell surface. These results suggest that glycosylation of G protein is essential for its transport to the cell surface, and the presence of a single carbohydrate chain is sufficient for this purpose.     

Adenovirus DNA-binding protein in cells infected with wild-type 5 adenovirus and two DNA-minus, temperature-sensitive mutants, H5ts125 and H5ts149.

Studies have been done to characterize further H5ts125, an adenovirus type 5 conditionally lethal, temperature-sensitive (ts) mutant defective in initiation of DNA synthesis and to investigate whether the single-strand-specific DNA-binding (72,000 molecular weight) protein is coded by the mutated viral gene. When H5ts125-infected cells were labeled with [35S]methionine at 32 degrees C and then incubated without isotope at 39.5 degrees C, the mutant's nonpermissive temperature, the 72,000 molecular weight polypeptide was progressively degraded. Immunofluorescence examination of cells infected with wild-type virus, H5ts125, and H5ts149 (a second, unique DNA-minus mutant) showed that immunologically reactive DNA-binding protein was barely detectable in H5ts125-infected cells at 39.5 degrees C, whereas this protein was present in wild-type- and H5TS149-infected cells, that the protein made at 32 degrees C in H5ts125-infected cells lost its ability to bind specific DNA-binding protein antibody when the infected cells were shifted to 39.5 degrees C, and that if H5ts125-infected cells were shifted from the restrictive temperature to 32 degrees C, even in the presence of cycloheximide to stop protein synthesis, immunologically reactive DNA-binding protein reappeared.     

Restitution of infectivity to spikeless vesicular stomatitis virus by solubilized viral components.

Noninfectious spikeless particles have been obtained from vesicular stomatitis virus (VSV, Indiana serotype) by bromelain or Pronase treatment. They lack the viral glycoprotein (G) but contain all the other viral components (RNA, lipid, and other structural proteins). Triton-solubilized VSV-Indiana glycoprotein preparations, containing the viral G protein as well as lipids (including phospholipids), have been extracted from whole virus preparations, freed from the majority of the detergent, and used to restore infectivity to spikeless VSV. The infectivity of such particles has been found to be enhanced by poly-L-ornithine but inhibited by Trition or homologous antiserum pretreatment. Heat-denatured glycoprotein preparations were not effective in restoring the infectivity to spikeless VSV. Heterologous glycoprotein preparations from the serologically distinct VSV-New Jersey serotype were equally capable of making infectious entities with VSV-Indiana spikeless particles, and the infectivity of these structures was inhibited by VSV-New Jersey antiserum but not by VSV-Indiana antiserum. Purified, detergent-free glycoprotein selectively solubilized from VSV-Indiana by the dialyzable detergent, octylglucoside, also restored infectivity of spikeless virions of VSV-Indiana and VSV-New Jersey.     

Uukuniemi virus glycoproteins accumulate in and cause morphological changes of the Golgi complex in the absence of virus maturation.

We have studied the transport of the Uukuniemi virus membrane glycoproteins in baby hamster kidney and chick embryo cells by using a temperature-sensitive mutant (ts12). Uukuniemi virus assembles in the Golgi complex, where both glycoproteins G1 and G2 and nucleocapsid protein N accumulate (E. Kuismanen, B. B?ng, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984). At the restrictive temperature (39 degrees C), the glycoproteins of ts12 were transported to the Golgi complex as in wild-type, virus-infected cells, whereas the nucleocapsid protein failed to accumulate there. Pulse-chase labeling followed by immunoprecipitation and treatment with endo-beta-N-acetylglucosaminidase H showed that G1 synthesized at 39 degrees C in ts12-infected cells had an altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a lack of terminal glycosylation. The typical Uukuniemi virus-induced vacuolization and expansion of the Golgi complex could be seen also in ts12-infected cells at 39 degrees C, although no virus particles were formed. This suggests that the morphological changes were induced by the Uukuniemi virus glycoproteins. In wild-type virus- or ts12-infected cells, G1 and G2 could not be chased out from the Golgi complex even after 6 h of treatment with cycloheximide. The glycoproteins were thus retained in the Golgi even under conditions when no virus maturation took place and when nucleocapsids did not accumulate in the Golgi region. Accordingly, the glycoproteins of Uukuniemi virus were found to have properties resembling those of Golgi-specific proteins. This virus model system may be useful in studying the synthesis and transport of membrane proteins that are transported to and retained in the Golgi.     

Transformation-associated changes in nuclear-coded mitochondrial proteins in 3T3 cells and SV40-transformed 3T3 cells

Comparative two-dimensional gel electrophoretic studies were performed on mitochondrial proteins in nontransformed mouse 3T3 cells and in SV40-transformed 3T3 cells, SV-T2. Two polypeptides, of 58 and 40 kDa, were present in increased amounts in SV40-transformed cells. These polypeptides were demonstrated to be nuclear-coded mitochondrial proteins by their absence in mitochondrial preparations, when labeling was performed in the presence of a mitochondrial-specific inhibitor, Rhodamine 6G. Temperature-sensitive mutants for transformation were derived from 3T3 cells by transfection with cloned SV40 DNA containing the ts A58 mutation. Increased amounts of the 58 kDa protein were apparent in these cells at the permissive temperature (33 degrees C) compared to the restrictive temperature (39.5 degrees C).     

Inhibition of Bcl-2-dependent cell survival by a caspase inhibitor: a possible new pathway for Bcl-2 to regulate cell death.

The REtsAF cell line expresses a temperature-sensitive mutant of the SV40 large tumor antigen. At restrictive temperature (39.5 degrees C), the cells undergo p53-mediated apoptosis, which can be inhibited by Bcl-2. Here, we show that Z-VAD-fmk, a caspase inhibitor, can suppress the Bcl-2-dependent cell survival at 39.5 degrees C. This result suggests that a caspase-like activity can act as an inhibitor of apoptosis in this model, downstream of Bcl-2. Our results also suggest that this activity may be up-regulated by Bcl-2 and may be responsible for cleavage of the tumor suppressor Rb protein.     

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.     

Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus

We have studied the role of a previously described tubulovesicular compartment near the cis-Golgi apparatus in endoplasmic reticulum (ER)-to-Golgi protein transport by light and immunoelectron microscopy in Vero cells. The compartment is defined by a 53-kDa transmembrane protein designated p53. When transport of the vesicular stomatitis virus strain ts045 G protein was arrested at 39.5 degrees C, the G protein accumulated in the ER but had access to the p53 compartment. At 15 degrees C, the G protein was exported from the ER into the p53 compartment which formed a compact structure composed of vesicular and tubular profiles in close proximity to the Golgi. Upon raising the temperature to 32 degrees C, the G protein migrated through the Golgi apparatus while the p53 compartment resumed its normal structure again. These results establish the p53 compartment as the 15 degrees C intermediate of the ER-to-Golgi protein transport pathway.     

The phenotype in vitro and in infected cells of herpes simplex virus 1 alpha trans-inducing factor (VP16) carrying temperature-sensitive mutations introduced by substitution of cysteines.

alpha trans-inducing factor (alpha TIF, VP16, Vmw65) is an essential structural protein of herpes simplex virus, being required for virion assembly. The protein also forms complexes with host proteins and a response element and transactivates the alpha genes which carry this element. The protein contains an acidic carboxyl terminus required for transactivation and a much larger amino-terminal domain required for promoter recognition. We report the first set of temperature-sensitive (ts) mutations deliberately introduced into the protein by substitution of the cysteine codons with those specifying glycine at positions 78, 102, and 176, either singly or in combinations. We report the following results. (i) All mutated proteins synthesized in vitro formed complexes with the DNA response element at room temperature. However, the mutant with the triple substitution and two mutants with substitutions in two of the three cysteines exhibited a ts phenotype at 33 and 37 degrees C, and one exhibited a ts phenotype only at 37 degrees C. (ii) Replacement of wild-type alpha TIF with genes carrying substitutions in any two cysteines conferred a ts phenotype for replication at 39.5 degrees C. Shift-down experiments indicated that the 10(4)- to 10(5)-fold reduction in virus yield at the nonpermissive temperature was due to the disfunction of alpha TIF late in infection, presumably in virion maturation. (iii) The alpha TIF expressed in cells infected with mutant viruses exhibited the same ts phenotype in protein-DNA complex formation as those expressed in vitro from mutated plasmids. Although the virus carrying the alpha TIF substitutions at Cys-102 and Cys-176 failed to induce a reporter gene linked to the alpha 4 promoter at 39.5 degrees C, it replicated as well as the parent virus in cells maintained for the first 10 h of infection at 39.5 degrees C. We conclude the following. (i) Formation of DNA-protein complexes containing alpha TIF is a poor prognosticator of alpha TIF function. (ii) The data presented here and in the literature strongly support the hypothesis that the secondary structure of the alpha TIF is very sensitive to deletions or insertions which probably affect the interaction of alpha TIF with both viral proteins in the virion and cellular proteins during infection. As a consequence, deletion-insertion mutagenesis may not shed useful information on the role of transactivating function of alpha TIF in infection. (iii) Since cysteines may play a role in stabilizing the secondary structure of proteins, substitutions of cysteines may be a powerful technique for site-specific construction of ts mutants in essential viral proteins.     

Bypass of the ts block of tsJT60, a G0-specific ts mutant from rat fibroblasts, by fetal bovine serum and epidermal growth factor

tsJT60, a temperature-sensitive (ts) G0-mutant cell line from a Fischer rat, grows normally in the exponential growth phase at 34 degrees C and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS), from the G0 phase they reenter the S phase at 34 degrees C but not at 39.5 degrees C. The ts-block was bypassed when G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus epidermal growth factor (EGF). The presence of EGF for the first 6 h after serum stimulation caused tsJT60 cells to enter the S phase in the presence of FBS at 39.5 degrees C. When EGF was added 6 h after serum stimulation, entrance into the S phase was delayed by about 6 h. The sequential presence of two growth factors, EGF without FBS for 6 h then FBS without EGF, or the reversed sequence, failed to initiate DNA synthesis at 39.5 degrees C. The binding of EGF was not temperature sensitive. The amounts of RNA and protein present doubled after stimulation with both FBS and EGF at 39.5 degrees C. These and other findings suggest that EGF bypasses only some specific event in the entire prereplicative process that operates operating in serum-stimulated cells at 39.5 degrees C.     

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.     

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.