Persistent baculovirus infection results from deletion of the apoptotic suppressor gene p35

Infection with the wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) results in complete death of Spodoptera frugiperda (Sf) cells. However, infection of Sf cells with AcMNPV carrying a mutation or deletion of the apoptotic suppressor gene p35 allowed the cloning of surviving Sf cells that harbored persistent viral genomes. Persistent infection established with the virus with p35 mutated or deleted was blocked by stable transfection of p35 in the host genome or by insertion of the inhibitor of apoptosis (iap) gene into the viral genome. These artificially established persistently virus-infected cells became resistant to subsequent viral challenge, and some of the cell lines carried large quantities of viral DNA capable of early gene expression. Continuous release of viral progenies was evident in some of the persistently virus-infected cells, and transfection of p35 further stimulated viral activation of the persistent cells, including the reactivation of viruses in those cell lines without original continuous virus release. These results have demonstrated the successful establishment of persistent baculovirus infections under laboratory conditions and that their establishment may provide a novel continuous, nonlytic baculovirus expression system in the future.     

Formation of a Sindbis virus nonstructural protein and its relation of 42S mRNA function

Chicken embryo fibroblasts infected with an RNA- temperature-sensitive mutant (ts24) of Sindbis virus accumulated a large-molecular-weight protein (p200) when cells were shifted from the permissive to nonpermissive temperature. Appearance of p200 was accompanied by a decrease in the synthesis of viral structural proteins, but [35S]methionine tryptic peptides from p200 were different from those derived from a 140,000-molecular-weight polypeptide that contains the amino acid sequences of viral structural proteins. Among three other RNA- ts mutants that were tested for p200 formation, only one (ts21) produced this protein. The accumulation of p200 in ts24- and ts21-infected cells could be correlated with a shift in the formation of 42S and 26S viral RNA that led to an increase in the relative amounts of 42S RNA. These data indicate that p200 is translated from the nonstructural genes of the virion 42S RNA and further suggest that this RNA does not function effectively in vivo as an mRNA for the Sindbis virus structural proteins.     

A single point mutation of hamster aminoacyl-tRNA synthetase causes apoptosis by deprivation of cognate amino acid residue

BACKGROUND: We have isolated a series of temperature-sensitive mutants for cell-proliferation from the BHK21 cell line derived from the golden hamster (Nishimoto & Basilico 1978; Nishimoto et al. 1982). Using these mutants as a recipient of DNA-mediated gene transfer, we have been cloning human genes which complement these ts mutants. RESULTS: Cultures of tsBN269 cells, a temperature-sensitive mutant of the BHK21 cell line, underwent apoptosis at 39.5 degrees C, a nonpermissive temperature. The gene complementing the tsBN269 cells was cloned and found to encode lysyl-tRNA synthetase. Indeed, tsBN269 cells were found to have a single cytosine to a thymine point mutation at the first nucleotide of codon 542 in hamster lysyl-tRNA synthetases. Due to this mutation, the activity of lysyl-tRNA synthetase was reduced--even at 33.5 degrees C, a permissive temperature. Consistent with these findings, while supplementation with lysine permitted tsBN269 cells to grow at a nonpermissive temperature, the deprivation of lysine caused apoptosis in tsBN269 cells, even at 33.5 degrees C. Cycloheximide inhibited the apoptosis caused by lysine starvation at 33.5 degrees C, but not at 39.5 degrees C. We also found that another hamster temperature-sensitive mutant, tsBN250, which is defective in histidyl-tRNA synthetase, entered apoptosis with the deprivation of histidine. CONCLUSION: Our data suggested that the defect in aminoacyl-tRNA synthetase turned on the cascade of apoptosis that was already present in the cells.     

The polyproline region of p53 is required to activate apoptosis but not growth arrest

p53 is a pivotal regulator of apoptosis but its mechanism of action is obscure. We report that the polyproline (PP) region located between p53's transactivation and DNA binding domains is necessary to induce apoptosis but not cell growth arrest. The PP region was dispensable for DNA binding, inhibition of SAOS-2 tumor cell growth, suppression of E1A + RAS cell transformation, and cell cycle inhibition. A temperature-sensitive dominant inhibitory p53 mutant lacking PP (p53ts deltaPP) retained its ability to cooperate with adenovirus E1A in transformation of primary BRK cells. However, while activation of wt p53 induced apoptosis in E1A + p53ts-transformed cells, activation of p53 deltaPP induced cell cycle arrest but not apoptosis in E1A + p53ts deltaPP-transformed cells. Similarly, PP deletion abolished apoptosis in LoVo colon carcinoma cells, which are killed by wt p53 overexpression. Transactivation was largely unaffected by PP deletion. Significantly, BAX induction was intact, indicating that additional events are required for p53 to induce apoptosis. As a recently described site for familial mutation in at least one breast cancer family, the PP region represents a domain that may be altered in human tumors. We concluded that p53's ability to induce apoptosis is dispensable for inhibiting cell growth and transformation and that the PP region plays a crucial role in apoptotic signaling.     

Adenovirus transcription. IV. Synthesis of viral-specific RNA in human cells infected with temperature-sensitive mutants of adenovirus 5

Cytoplasmic RNA sequences produced in HeLa cells infected with the adeno-virus 5 temperature-sensitive mutants ts1, ts2, ts9, ts17, ts18, ts19, ts20, ts22, ts49, ts36, and ts125 were characterized by hybridization to DNA probes generated by strand separation of restriction endonuclease fragments of adenovirus 5 DNA. Two "early' mutants defective in DNA synthesis, ts125 and ts36, fail to make wild-type levels of all previously reported classes of late RNA at the nonpermissive temperature. At 40.5 degrees C, both ts125 and ts36 synthesize a wild-type complement of early cytoplasmic RNA 16 h after infection. Under these conditions, no "late' cytoplasmic RNA sequences were observed. Similarly, nuclear RNA present in these cells resembled early cytoplasmic RNA rather than late nuclear RNA. All the late adenovirus 5 temperature-sensitive mutants synthesized normal wild-type levels of late cytoplasmic RNA at the nonpermissive temperature, except ts2, which appears to overproduce certain cytoplasmic species.     

Characterization of a cell cycle mutant derived from hamster fibroblast: reversion analysis

K12 is a temperature-sensitive (ts) mutant cell line derived from Chinese hamster fibroblasts. When incubated at the nonpermissive temperature, K12 cells exhibit the following properties: (a) the cells cannot initiate DNA synthesis;o (b) the synthesis of cytosol thymidine kinase is suppressed; and (c) the synthesis of three cellular proteins of molecular weights 94, 78, and 58 kdaltons is greatly enhanced. Here we characterize a spontaneous revertant clone, R12, derived from the K12 cells. We selected the revertant clone for its ability to grow at the nonpermissive temperature. Our results indicate that all the traits which constitute the K12 mutant phenotype are simultaneously reverted to the wild type in the revertant cell line, suggesting that the ts mutation of the K12 cells is of regulatory nature and exerts multiple effects on the expressed phenotypes.     

Control of protein synthesis by a temperature-sensitive mutant of reovirus 3. I. Temperature-sensitive function of ts261-b mutant

The ability of a temperature-sensitive (ts) mutant of reovirus, ts261-b, to synthesize virus-specific RNAs and proteins during infection at the nonpermissive temperature (37 degrees C) was investigated. The relative amounts of the mutant virus-specific single-stranded (ss) RNA's and double-stranded (ds) RNA's synthesized in cells at 37 degrees C were 20 to 25% as much as those synthesized in the wild-type virus-infected cells. The 10 segments of the mutant ds RNAs and the three size classes of the ss RNAs were synthesized in the usual proportions. The methylation of the mutant viral mRNA's (ss RNAs) was not blocked at 37 degrees C in infected cells. A striking temperature-sensitive restricted function of the ts261-b mutant was expressed in the synthesis of the viral proteins. This study, which uses an in vitro protein-synthesizing system reconstituted with an endogenous polysomal fraction and a postribosomal supernatant from reovirus-infected cells, has demonstrated that the endogenous polysomes obtained from ts261-b mutant-infected cells at 37 degrees C are not active in the synthesis of the viral polypeptides of known molecular weights, and the amounts of the mutant viral polypeptides synthesized in vitro by these polysomes are 5 to 9% of those synthesized by the corresponding fraction from wild-type-infected cells. The impaired protein-synthesizing capacity of the mutant virus-specific polysomes can be restored during maintenance of the infected cells at 30 degrees C after shift-down from 37 degrees C. The in vitro synthesis of viral polypeptides of known size by the active endogenous polysomes derived from cells infected at the permissive temperature is accelerated by the addition of the postribosomal supernatant obtained from cells infected at the permissive temperature. The postribosomal supernatant from mutant-infected cells at 37 degrees C did not have a stimulatory effect, but rather, it inhibited in vitro viral protein synthesis.     

In vivo and in vitro analysis of baculovirus ie-2 mutants

Upon transient expression in cell culture, the ie-2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: trans activation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived from Spodoptera frugiperda but not in a cell line derived from Trichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. The ie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form of ie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus, ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.     

Baculovirus p33 binds human p53 and enhances p53-mediated apoptosis

In vertebrates, p53 participates in numerous biological processes including cell cycle regulation, apoptosis, differentiation, and oncogenic transformation. When insect SF-21 cells were infected with a recombinant of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) overexpressing human p53, p53 formed a stable complex with the product of the AcMNPV orf92, a novel protein p33. The interaction between p53 and p33 was further confirmed by immunoprecipitation studies. When individually expressed in SF-21 cells, human p53 localized mainly in the nucleus whereas baculovirus p33 displayed diffuse cytoplasmic staining and punctuate nuclear staining. However, coexpression of p33 with p53 resulted in exclusive nuclear localization of p33. In both SF-21 and TN-368 cells, p53 expression induced typical features of apoptosis including nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Coexpression of p53 with a baculovirus inhibitor of apoptosis, p35, OpIAP, or CpIAP, blocked apoptosis, whereas coexpression with p33 enhanced p53-mediated apoptosis approximately twofold. Expression of p53 in SF-21 cells stably expressing OpIAP inhibited cell growth in the presence or absence of p33. Thus, human p53 can influence both insect cell growth and death and baculovirus p33 can modulate the death-inducing effects of p53.     

Factors regulating baculovirus late and very late gene expression in transient-expression assays

Eighteen genes of Autographa californica nuclear polyhedrosis virus are necessary and sufficient to transactivate expression from the late vp39 promoter in transient-expression assays in SF-21 cells. These 18 genes, known as late expression factor genes (lefs), are also required to transactivate the very late promoter of the polyhedrin gene, polh, but expression from this promoter is relatively weak compared with expression from the vp39 promoter. To further define the factors required for late and very late promoter expression, we first determined that the eighteen lefs were also required for expression from two other major baculovirus promoters: the late basic 6.9-kDa protein gene, p6.9, and the very late 10-kDa protein gene, p10. We next examined the effect of the very late expression factor 1 gene (vlf-1), a gene previously identified by analysis of a temperature-sensitive mutant, in the transient expression assay and found that vlf-1 specifically transactivated the two very late promoters but not the two late promoters. We then surveyed the Autographa californica nuclear polyhedrosis virus genome for additional genes which might specifically regulate very late gene expression; no additional vlf genes were detected, suggesting that VLF-1 is the primary regulator of very late gene expression. Finally, we found that the relative contribution of the antiapoptosis gene p35, which behaves as a lef in these transient-expression assays, depended on the nature of the other viral genes provided in the cotransfection mixtures, suggesting that other viral genes also contribute to the ability of the virus to block apoptosis.     

p53 status does not determine outcome of E1B 55-kilodalton mutant adenovirus lytic infection

The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G1. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39 degreesC, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39 degreesC. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.