A persistent infection of Vero cells by egg-adapted mumps virus

Serial subculture of Vero cells infected with the chick embryo adapted Enders strain of mumps virus gives rise to a low level productive though persistent infection. Persistently infected cultures exhibit minimal cytopathology; however, widely dispersed foci of multinucleate cells are almost always present. Neither infected cell nor virus growth is temperature sensitive. Biological and biochemical evidence indicate that defective interfering particles are replicated along with infectious (nondefective) virus during the course of the persistent infection, although the plaque-purified inoculum virus stock contained only genome size RNA. With continued cell passage a heterogeneous, changing population of subgenomic sized viral RNA accumulates, suggesting that defective interfering (DI) RNA species are evolving from virion RNA, with no single DI RNA predominating. Since such factors as interferon, antibody, and temperature-sensitive mutants are absent from this system, DI particles are the likely factor modulating this persistent infection.     

Persistent infection by a temperature-sensitive mutant isolated from a Sendai virus (HVJ) carrier culture: Its initiation and maintenance without aid of defective interfering particles

Previous studies showed that there is a selective survival of is mutants in BHK-Sendai virus (HVJ) carrier culture. In addition, it is now demonstrated that the carrier culture synthesizes defective interfering (DI) size RNAs. Thus, both is mutants and DI particles have been suggested to play roles in the maintenance of the persistent infection. The present study was undertaken to clarify the role of is mutants. is virus was isolated from the carrier culture, purified so as not to contain detectable DI particles, and examined for its capacity to establish persistent infections in normal cells as well as for its interaction with wild-type Sendai virus. The is virus has extremely low cytopathogenicity, allows the infected LLCMK₂ cells to survive, and readily establishes a long-term persistent infection. The established carrier culture is resistant to challenge by wild-type Sendai virus while sensitive to a heterologous virus infection. Nearly 100% of the carrier cells exhibit virus-specific antigens. The intracellular nucleocapsids isolated from the carrier cells contain only 50 S virion-size RNA but no detectable DI RNAs at each subculture of analysis. Original is characteristics have been expressed in the carrier cells as demonstrated by temperature-dependent production of HN and M polypeptides and progeny viruses. Persistent infections were also established in a variety of the other cell lines without added DI particles. These results indicate that the to virus is able to establish persistent infection without requirement of DI particles for either its initiation or maintenance. Further, the is virus itself strongly interfered with the replication of wild-type virus and mixed infection of LLCMK₂ cells with is and wild-type viruses also readily established a long-term persistent infection.     

Characteristics of a persistent respiratory syncytial virus infection in HeLa cells

Persistent infection with respiratory syncytial (RS) virus has been established in HeLa cells. The persistently infected cell line (HeLa_RS) continues to produce virus (RSpi) at low levels after 230 passages during 3 years. The cells are morphologically similar to the parental HeLa cell line, and susceptible to vesicular stomatitis virus, but resist superinfection with standard RS virus (RS_wt). The block in RS_wt replication is not at attachment. Although infectious center and immunofluorescence assays suggested that only 5 to 30% of the cells in the culture were infected, 30 of 32 clones isolated from HeLa_RS contained some cells with virus antigen and 23 of those clones produced virus. All the clones, including the 2 that appeared not to be infected, were more resistant to RS_wt than HeLa. The clones that produced virus were significantly more resistant than the others. RS_pi is a small-plaque mutant, but not a temperature-sensitive mutant, of RS_wt. Although prior infection of HeLa with RS_pi interferes with RS_wt replication, the interference appears not to be caused by defective interfering particles, but by RS_pi virions. RS_pi initiates a persistent infection in HeLa only after a cytolytic phase similar to that which preceded the establishment of HeLa_RS.     

A temperature-sensitive mutant of tobacco mosaic virus deficient in synthesis of single-stranded RNA.

RNA synthesis by a temperature-sensitive, replication-deficient mutant of tobacco mosaic virus (TMV), IV-35, was examined in temperature-shift experiments. Viral RNA synthesis occurred normally at 25°, but upon a shift to 35°, synthesis of single-stranded genome RNA ceased immediately while production of replicative form and replicative intermediate RNAs continued normally. The plus strand:minus strand ratios of IV-35 double-stranded RNAs labeled at 35° were the same as those of wild type. The ability to produce single-stranded RNA was not irreversibly lost at 35° because upon a shift back to 25°, synthesis of single-stranded RNA immediately resumed. The in vitro activity of membrane-bound replicase was not temperature sensitive. However, the stabilities in vitro and in vivo at 35° and the optimum temperature profile of the enzyme from IV-35-infected leaves were altered from those of the wild-type enzyme preparation.     

Further genetic analysis of a temperature-sensitive mutant of cowpea mosaic virus

The two reciprocal hybrids of wild-type cowpea mosaic virus (Sb) and the temperature-sensitive mutant N168 were constructed. Symptoms of both hybrids differed from those of the parent strains in three differential hosts indicating that N168 carries mutations in both components and that symptoms result from interactions between components. The occurrence of one or more temperature-sensitive mutations in the middle-component RNA was confirmed. In vitro recombination of the hybrids yielded isolates indistinguishable from the parent strains.     

A temperature-sensitive lesion affecting levels of transformation-specific viral RNA in a mutant of avian sarcoma virus PRCII

LA46 is a mutant of avian sarcoma virus PRCII that is temperature sensitive (ts) in the maintenance of oncogenic transformation and in the production of infectious virus. These ts properties are independent of the helper virus. At the nonpermissive temperature LA46-infected cells also fail to produce significant quantities of the transformation-specific protein, and the amount of intracellular transformation-specific viral RNA is reduced at least 100-fold. The probable primary defect of LA46, a temperature sensitivity in transformation-specific viral RNA, can account for the phenotype of the mutant. The molecular mechanism of this defect is currently investigated.     

An avian sarcoma virus mutant which produces an aberrant transformation affecting cell morphology

ST 529 is a temperature-sensitive mutant of Rous sarcoma virus (RSV) strain SR-A, which causes an unusual pattern of phenotypic changes in cells that it transforms. At 35°, ST 529-transformed cells exhibit an elongated, fusiform morphology (morph^f), and also possess an aberrant and “unlinked” phenotypic pattern of transformation-related properties. ST529-transformed cells, at 35°, resemble classically transformed cells in respect to density-independent growth, sugar uptake, protease levels, and ability to form soft agar colonies. However, they differ from classically transformed cells in respect to cell morphology, fibronectin levels, adhesiveness, and organization of actin stress fibers. At a nonpermissive temperature (42°), ST529-infected cells appear phenotypically normal. pp60^src isolated from ST529-infected cells at 42° possesses little or no associated protein kinase activity. Kinase activity becomes detectable rapidly, however, within 30 min after a shift to 35°, and reaches maximal levels within 3 hr after the shift. It is probable that an unusual, mutant src gene product is responsible for the novel pattern of transformation-related changes observed in ST529-infected cells. Previous studies utilizing mutants or variants of RSV have suggested that there are probably at least two biologically significant targets for pp60^src. The present experiments provide additional evidence for a multifunctional src gene product.     

Characterization of a temperature-sensitive mutant of frog virus 3 defective in DNA replication

We have characterized the defect of a temperature-sensitive (ts) DNA^? mutant (ts 6642) of frog virus 3 (FV 3). At the nonpermissive temperature (30°) ts 6642 synthesized <3% of the viral DNA that was synthesized at the permissive temperature (23°). When ts 6642-infected cells were incubated at 30° for 4.0 hr and then shifted to permissive temperature, viral DNA synthesis started immediately even when protein synthesis was inhibited at the time of shiftdown. This result implies that at 30°, ts 6642 synthesized all the proteins required for viral DNA replication but that one of these was nonfunctional at the nonpermissive temperature. Further characterization revealed that ts 6642 was probably defective in the initiation of DNA replication. This conclusion was based on the following data: When ts 6642-infected cells incubated at 23° for 4.0 hr were shifted to 30°, there was a gradual decrease in viral DNA synthesis. By 1 to 1.5 hr after the shiftup, viral DNA synthesis was completely inhibited. Analysis of the density of the DNA synthesized after a shiftup in the presence of BUdR and FUdR suggested that residual viral DNA synthesis represented chain elongation, and not initiation of new rounds of DNA replication. The defective protein was therefore involved in the initiation process. Both wild-type FV 3 (FV 3⁺) and ts 6642 induced the synthesis of thymidine kinase and DNA polymerase at 30°. Therefore, neither of these enzymes was involved in the DNA replication defect of ts 6642.At the nonpermissive temperature, ts 6642 synthesized all the viral proteins that were detectable at the permissive temperature. However, synthesis of late proteins was delayed, and never reached wild-type levels. Furthermore, the rate of synthesis of late proteins at 30° became dependent upon the multiplicity of infection. These results reinforce our previous conclusion (R. Goorha and A. Granoff, 1974, Virology60, 237–250) that in FV3⁺-infected cells late proteins (and by implication late mRNAs) were synthesized in the absence of viral DNA replication.     

A mutant of influenza virus with a temperature-sensitive defect in the posttranslational processing of the hemagglutinin.

A mutant of fowl plague virus (ts 227) with a temperature-sensitive defect in the hemagglutinin [Scholtissek, C., and Bowles, A. L. (1975). Virology67, 576–587] has been analyzed. At the nonpermissive temperature, the hemagglutinin is synthesized and incorporated into the rough endoplasmic reticulum, but is not transported to the smooth endoplasmic reticulum and to the cell surface. As a result of this defect in migration, proteolytic cleavage of the hemagglutinin does not occur and glycosylation is incomplete, in that mannose and glucosamine are incorporated into the glycoprotein, whereas galactose and fucose are attached only at the permissive temperature. Another step involved in glycosylation, which occurs also only at the permissive temperature, is partial removal of mannose in a trimming process. The data suggest that the hemagglutinin has a structural feature which, after insertion of the molecule into the rough endoplasmic reticulum, promotes its transport to the cell surface. The hemagglutinin incorporated into the rough endoplasmic reticulum at the nonpermissive temperature does not show hemagglutinating activity. After solubilization with detergent, it reacts with antiserum specified against wild-type hemagglutinin. When migration of the hemagglutinin is blocked, synthesis of the neuraminidase proceeds normally, indicating that there is little interdependence in the processing of both envelope glycoproteins.     

Role of protease in mouse hepatitis virus-induced cell fusion. Studies with a cold-sensitive mutant isolated from a persistent infection

A plaque mutant was isolated from Kirsten mouse sarcoma virus-transformed BALB/c cells persistently infected with a mouse hepatitis virus strain MHV-S. While the wild type produced large plaques consisting of fused cells (fusion type) both at 39 and at 32°, the mutant produced small fusion-type plaques at 39°, and at 32°, only after longer incubation, plaques consisting of round dead cells (non-fusion type) were obtained. The mutant grew equally well at both temperatures. Thus, the mutant was cold sensitive in inducing cell fusion, but not in replication or in ultimate cell killing. The cold sensitivity was overcome by the addition of trypsin (0.04 to 0.05%) to the culture medium, but not by treating the virions with trypsin. SDS-polyacrylamide gel electrophoresis of the virion proteins failed to detect differences between the wild type and the mutant. In intracellular viral proteins immunoprecipitated with anti-MHV-S rabbit serum, a protein of 68,000 daltons (68K protein) which was present in the wild type-infected cells was absent in the mutant-infected cells, but trypsin treatment or incubation at 39° of the mutant-infected cells failed to induce the 68K protein. After six to seven undiluted passages, the mutant segregated varieties of mutants which were partially or totally reverted to the wild type in phenotype, and also those whose cell fusion induction was absent even at 39°. All these mutants failed to induce the 68K protein in the infected cells. Thus, there was no linkage between the presence of 68K protein and the fusion induction. Trypsin treatment of the infected cells enabled MHV-S to form fusion plaques on otherwise resistant cells, and MHV-2, a producer of non-fusion-type plaques, to form fusion-type plaques. Protease appears to play an important role in MHV-induced cell fusion in general.     

Partial characterization of a Moloney murine sarcoma virus 85,000-dalton polypeptide whose expression correlates with the transformed phenotype in cells infected with a temperature-sensitive mutant virus

The viral proteins specified by a Moloney murine sarcoma virus (Mo-MuSV) with a temperature-sensitive mutation in its transforming gene were examined. Normal rat kidney cells infected with this replication-defective virus have transformed cell characteristics at 33° but revert to a normal phenotype at 40°. At the temperature permissive for transformation, the cells contained an 85,000-dalton protein (P85) which had antigenic determinants of p15, pp12, and p30, and also tryptic peptides characteristic of p15 and p30 as well as additional unidentified tryptic peptides. P85 was only detectable at the permissive temperature. A 58,000-dalton protein (P58) was also detected. It had both antigenic determinants and tryptic peptides of p15, pp12, and p30. P58 was seen at both temperatures. Phosphorylation experiments indicated that P58 is a phosphoprotein whereas ³²P-labeled P85 was not observed. Temperature shift experiments showed that newly synthesized P85 was first detected between 2 and 3 hr following transfer of cultures to 33°. Morphological and biochemical changes indicative of transformation occurred 8 or more hr after temperature shift. These results are consistent with the interpretation that P85 contains peptide sequences derived from both the gag gene and the MuSV-acquired or src gene sequences.     

Normal DBA/2 mouse cells synthesize a glycoprotein which interferes with MCF virus infection

The mechanism of resistance to Friend leukemia virus-induced [mink cell focus-inducing, (MCF)-mediated] leukemogenesis in DBA/2 mice was investigated in cell culture systems. DBA/2 fibroblasts were found to be resistant to infection with MCF viruses but not to ecotropic or amphotropic murine leukemia viruses (MuLV's). Since this resistance has been correlated with the presence of an MCF virus-related gp70 constitutively present on the surface of DBA/2 cells, it seemed possible that the mechanism of resistance in this system involved the saturation of MCF-specific cell surface receptors with the gp70 in analogy to viral interference. Two inhibitors of glycoprotein synthesis, 2-deoxy-d-glucose and tunicamycin, which have been shown to reduce retrovirus-induced interference in productively infected cells, significantly decreased the resistance of DBA/2 cells to productive infection with MCF viruses. This decrease in resistance to MCF virus infection could be correlated with a decrease in the expression of MCF-related gp70 at the cell surface. Cells from several other mouse strains showed neither resistance to MCF virus infection nor enhancement of MCF infectivity following drug treatment. These data indicate that DBA/2 cells are resistant to MCF infection in vitro and, by implication, to MCF-mediated leukemogenesis in vivo by a process analogous to viral interference. Ecotropic pseudotypes of MCF virus were able to productively infect untreated DBA/2 cells, indicating that the cell surface interference-like process described here is the only restriction mechanism responsible for resistance to MCF infection in DBA/2 cells. The results are consistent with the idea that Friend leukemia virus actually causes disease via MCF intermediates.     

A temperature-sensitive (TS) mutant of frog virus 3 (FV3) is defective in second-stage DNA replication

It has been suggested that FV3 DNA replication occurs in two stages [R. Goorha (1982) J. Virol. 43, 519-528]. First-stage DNA synthesis is restricted to the nucleus, where the replicating DNA ranges from genome to twice genome size; second-stage DNA replication occurs exclusively in the cytoplasm, and the replicating DNA is concatameric. A temperature-sensitive mutant (ts 12488) of FV3, at a nonpermissive temperature (30 degrees), synthesized DNA in the nucleus only, and the size of the replicative complex (as determined by neutral sucrose gradient analysis) was between genome and twice genome length. These characteristics establish that at nonpermissive temperature, ts 12488 is arrested in the first stage of DNA replication. Temperature shift-down (30 degrees----25 degrees) of ts 12488-infected cells at 4 hr postinfection showed that, within 30 min of the shift, the replicative complex became very large (more than 10 times genome size). Furthermore, newly synthesized DNA was now found in the cytoplasmic fraction also. These results suggest that ts 12488, upon shift-down, enters into the second stage of DNA replication where progeny DNA is synthesized as a large concatamer. In shift-down experiments, de novo protein synthesis was not required to initiate second-stage DNA replication, strongly suggesting that the thermosensitive protein is directly involved in second-stage DNA replication. This genetic evidence establishes the previous biochemical findings of a two-stage replication scheme for FV3 DNA.     

A group of temperature-sensitive mutants of Moloney leukemia virus which is defective in cleavage of env precursor polypeptide in infected cells also induces hind-limb paralysis in newborn CFW/D mice

A group of temperature sensitive mutants of Moloney murine leukemia virus (MoMuLV) designated as ts1, ts7, and ts11 rapidly and invariably induce hind-limb paralysis ranging from 28 to 52 days postinjection of neonatal CFW/D mice. These temperature-sensitive mutants are defective in the processing of the precursor of the env protein, gPr80env in infected cells, resulting in the accumulation of gPr80env in the infected cell and production of virions with reduced amounts of gp70, p15E, and p12E when compared to that of the wild-type virion. In contrast, two nonparalytogenic ts mutants, ts3 and ts10, like the wild-type virus, show normal processing of gPr80env in infected cells and production of virions with a similar amount of env proteins to that of the wild-type virion.     

A temperature-sensitive mutant of the baculovirus Autographa californica nuclear polyhedrosis virus defective in an early function required for further gene expression

Fifteen temperature-sensitive (ts) mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) have been analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins synthesized in infected cells. One of the mutants, tsB821, was found to be defective in a very early function. Seven virus-induced proteins were synthesized by 2 hr postinfection. In marked contrast to wild-type virus and the other 14 ts mutants, the synthesis of further virus-induced proteins did not occur in tsB821-infected cells at the restrictive temperature (33 degrees ). Host protein synthesis continued as normal after transient expression of the seven early proteins. Viral-specific DNA synthesis was blocked or significantly delayed in tsB821-infected cells at 33 degrees . The relative synthesis of certain viral-induced proteins, particularly P31, P32, P42, P66, and P69, varied considerably in the remaining 14 mutants at 33 degrees. Three mutants exhibited alterations in specific polypeptides; P75 was approximately 1 kDa smaller in tsB1075, P40 was approximately 1 kDa smaller in tsB951, and P25 was greatly reduced in quantity or altered in tsB305.     

A new endonuclease restriction site which is at the locus of a temperature-sensitive mutation in vaccinia virus is associated with true and pseudoreversion

A temperature-sensitive mutant of vaccinia IHD-W, designated is 9251, possesses a novel EcoRI restriction endonuclease site in the fragment D of the parental genome. Spontaneous ts⁺ revertants of ts 9251 fall into two distinct categories: the majority of revertants reacquired the parental EcoRI restriction profile, while one isolate maintained the mutant cleavage site. Using two-dimensional gel analysis of viral polypeptides induced by vaccinia virus in infected cytoplasms, it was observed that the fingerprint of mutant ts 9251 differed from the parental IHD-W in that a single viral protein of a molecular weight of 37,000 daltons migrated to a new isoelectric point. The revertants which had regained the wild-type restriction profile now encoded for a 37K polypeptide identical to that of the wild-type virus while the single revertant which maintained the novel EcoRI site possessed a 37K protein of a charge intermediate between ts 9251 and wild type. We conclude that ts 9251 can revert to the ts⁺ phenotype either by true reversion at the original mutant locus or by a second independent mutation within the same gene.     

Activation of cellular stress protein genes by herpes simplex virus temperature-sensitive mutants which overproduce immediate early polypeptides

This paper describes the activation of cellular genes after infection of chick embryo fibroblasts (CEF) with temperature-sensitive (ts) mutants of herpes simplex virus type 1. One mutant, tsK, has a mutation in the gene sequences which encode the immediate-early (IE) polypeptide Vmw175. After infection of CEF with tsK at the nonpermissive temperature (38.5°), IE polypeptides are overproduced but early and late viral gene products cannot be detected. In addition, the synthesis of three cellular polypeptides is stimulated in a manner which closely resembles the “stress” or “heat-shock” response. Peptide mapping confirmed that the tsK-induced polypeptides are stress proteins. No induction was observed when viral gene expression was prevented, leading to the conclusion that one or more IE polypeptides is responsible for the stress response. Two other mutants, tsD and tsT, which have mutations in different regions of the Vmw175 coding sequences, also induce stress proteins at 38.5°.     

A temperature-sensitive mutant of western equine encephalitis virus with an altered envelope protein E1 and a defect in the transport of envelope glycoproteins

A temperature-sensitive (ts) mutant, ts-39, of western equine encephalitis virus had been shown to have a defect in hemagglutinating activity. The hemagglutinating protein E1 synthesized in ts-39-infected cells migrated faster than that of wild type of SDS-polyacrylamide gel electrophoresis. The altered E1 protein (designated as El¹) was synthesized in cells infected with the mutant even at the permissive temperature and incorporated into mature virus. In addition to E1¹, the B protein synthesized in ts-39-infected cells appeared to migrate faster than that made during wild type infection. This suggests that the altered mobility of E1¹ protein is not due to an altered cleavage from its precursor. The difference in electrophoretic mobility was not ascribed to incomplete or altered glycosylation since the unglycosylated form of E1¹ synthesized in the presence of tunicamycin also had a greater mobility compared with that of E1. In peptide mapping analyses, we could not detect any deleted peptide spots in the maps of E1¹ protein as compared with those of E1 protein.Ten ts+ revertants of the mutant were isolated from plaques formed at the restrictive temperature. The revertants occurred at a frequency of about 10^?6 and restored the thermostability of hemagglutinating activity as well. All of the revertants synthesized E1 and B proteins indistinguishable from those of wild type virus, suggesting that the alteration of E1 protein is genetically linked to the is lesion of the mutant. E1¹ protein could be immunoprecipitated with antiserum for purified E1 protein. Immunofluorescence studies showed that in the cells infected with ts-39 at the restrictive temperature E1¹ protein was not transported to the cell surface; neither was PE2 (or E2) protein. When the cultures were shifted to the permissive temperature, those glycoproteins came to reach the cell surface. Thus ts-39 virus was shown to have a reversible defect in the transport of the envelope glycoproteins.