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PubMed1993

The acidic amino-terminal region of herpes simplex virus type 1 alpha protein ICP27 is required for an essential lytic function.

The herpes simplex virus type 1 (HSV-1) alpha protein ICP27 regulates the transition between the delayed-early and late phases of the viral infection. Previous genetic analyses have suggested that the important functional domains of ICP27 map to its carboxyl-terminal half. One striking feature of the primary sequence of ICP27, however, is an extremely acidic region near its amino terminus. To determine whether this region is required for ICP27 function, we deleted the sequences in the ICP27 gene which encode it (codons 12 through 63). In transient expression assays, the deletion mutant was unable to efficiently repress the expression of a cotransfected reporter gene or to efficiently complement the growth of d27-1, an HSV-1 ICP27 null mutant. These results suggested that the acidic region of ICP27 is involved in a regulatory function required for lytic growth. To test this possibility further, we introduced the mutant allele into the HSV-1 genome by marker transfer. Two independently derived isolates of the mutant virus, designated d1-2a and d1-2b, were recovered and analyzed. Both isolates were defective for growth in Vero cells, exhibiting a 100-fold reduction in virus yield compared with the wild-type infection. Vero cells infected with the d1-2 isolates showed a three- to eightfold reduction in viral DNA replication, a moderate reduction in the expression of viral gamma genes, and a delay in the repression of beta genes. The phenotype of the d1-2 isolates differs substantially from the phenotypes of previously isolated ICP27 mutants, which show much more severe defects in viral gene expression. Our results demonstrate that the amino-terminal half of ICP27 participates in its regulatory activities in both infected and transfected cells.

Animals↗
PubMed1982

Characterization of human cells persistently infected with cytomegalovirus and exposed to a chemical carcinogen.

A non-productive, persistent human cytomegalovirus (CMV) infection was developed in human embryo lung (LU) cells by initially using supraoptimal temperatures to restrict the replication of a recent CMB isolate (76-24). Although CMV replication was observed in some cells within a few weeks of shifting the cultures to 37 degrees C and subculturing, CMV cytopathic effects and infectious CMV were not detected with further subculturing during the next 5 weeks. CMV-specific nuclear antigens were, however, observed in most cells (designated LU-76-24) for 38 additional culture passages made during the following 12 months. Although the LU-76-24 cells were morphologically distinct from LU cell cultures carried in parallel as controls, the LU-76-24 were contact-inhibited in spite of a somewhat disoriented growth pattern. Treatment of LU-76-24 cells with 0.05 microgram/ml of the chemical carcinogen 4-nitroquinoline 1 oxide (NQO) induced a lytic CMV infection from which a clone of cells (designated LUC-NQO) was isolated. No effect was seen when LU cells were treated with the same dose of NQO. LUC-NQO cells were morphologically altered from LU-76-24 cells and had lost contact inhibition. Even though CMV-specific nuclear antigens were observed in LUC-NQO cells, infectious CMV was not detected in lysates of these cells, nor was CMV rescued by co-cultivation of these cells with susceptible cells or by induction with 5-iodo-2'-deoxyuridine. LUC-NQO, but not LU or LU-76-24 cells, plated with good efficiency in semi-solid medium. The results indicate that long-term, non-productive, persistent CMV infections can be established in vitro and suggest that such cells may be more susceptible than non-infected cells to the action of chemical carcinogens.

4-Nitroquinoline-1-oxide↗
PubMed2002

Gene products encoded in the ninR region of phage lambda participate in Red-mediated recombination.

BACKGROUND: The ninR region of phage lambda contains two recombination genes, orf (ninB) and rap (ninG), that were previously shown to have roles when the RecF and RecBCD recombination pathways of E. coli, respectively, operate on phage lambda. RESULTS: When lambda DNA replication is blocked, recombination is focused at the termini of the virion chromosome. Deletion of the ninR region of lambda decreases the sharpness of the focusing without diminishing the overall rate of recombination. The phenotype is accounted for in large part by the deletion of rap and of orf. Mutation of the recJ gene of the host partially suppresses the Rap- phenotype. CONCLUSION: ninR functions Orf and Rap participate in Red recombination, the primary pathway operating when wild-type lambda grows lytically in rec+ cells. The ability of recJ mutation to suppress the Rap- phenotype indicates that RecJ exonuclease can participate in Red-mediated recombination, at least in the absence of Rap function. A model is presented for Red-mediated RecA-dependent recombination that includes these newly identified participants.

Bacterial Proteins↗
PubMed1990

Recombination of genomic terminus of bovine herpesvirus type 1 with cellular DNA.

Bovine herpesvirus 1 (BHV-1) has a linear DNA genome of about 135 kb which appears as two isomers, resulting from its short unique segment being present in the two possible orientations with respect to the large unique segment. BHV-1 also circularizes its DNA to form replicative molecules. Definition of the target sequences at the genomic termini involved in the recombination events during genomic replication and isomerization, as well as virus maturation, led to the discovery that 10% of the genome molecules have additional DNA sequences attached to the right-hand terminus, as shown by electron microscopy. Three such tails have been cloned molecularly; they differ in length and nucleotide sequence, and hybridization experiments demonstrate the cellular origin of two of the three tails. The evidence presented here is consistent with a proportion of the BHV-1 genomes recombining their DNA with cellular DNA during lytic infection.

Animals↗
PubMed1985

Both trans-acting factors and chromatin structure are involved in the regulation of transcription from the early and late promoters in simian virus 40 chromosomes.

We isolated simian virus 40 (SV40) chromosomes from lytically infected CV-1 cells at various times during the late phase and transcribed them in vitro with either whole-cell or nuclear extracts of HeLa cells. The late promoter was 3- to 10-fold more active than the early promoter. With bare SV40 DNA templates, the early promoter was up to 10-fold stronger than the late promoter. The relative strengths of the early and late promoters on SV40 chromosomes were essentially independent of template concentration or length of the replicative phase of the infection. When monoclonal antibodies or antisera against T antigen (T Ag) were added to SV40 chromosomes or when T Ag, both free and chromatin bound, was removed by immunoprecipitation with anti-T, the activity of the late promoter remained essentially unchanged. Washing with 0.4 M NaCl removed T Ag from more than 90% of the mature chromosomes associated with T Ag. Transcription from the late promoter still predominated in the salt-washed T Ag-depleted chromosomes, even though there was a marked increase in early promoter activity. The depression of the early promoter could be reversed by adding the T Ag-containing extract back to the depleted chromosomes. Extraction of SV40 chromosomes with 1.5 M NaCl resulted in a decrease in the activity of the late promoter and a further increase in the activity of the early promoter so that the relative amounts of early and late RNA synthesized were similar to those for bare SV40 DNA templates. Late RNA synthesis from bare SV40 DNA templates was stimulated by high-speed supernatants prepared from nuclear extracts of SV40-infected cells but not from those of uninfected cells. Pretreatment of the supernatants with anti-T did not alter the result. Our findings indicate that the activity of the early and late SV40 promoters is regulated by at least two different mechanisms at the chromosomal level. One is mediated by a subclass of T Ag bound to SV40 chromosomes which represses early SV40 transcription but has no effect on late transcription. A second level of regulation, involving a tightly bound trans-acting chromosomal factor and a stable nucleoprotein structure, favors the late promoter over the early promoter by up to 10-fold.

Animals↗
PubMed1987

Specific interaction of simian virus 40 large T antigen with cellular chromatin and nuclear matrix during the course of infection.

We analyzed the subnuclear distribution of the simian virus 40 (SV40) large tumor (large T) antigen during the course of viral infection. Three distinct nuclear subclasses were detected in SV40 lytically infected TC7 cells (large T antigen in the nucleoplasm, at the cellular chromatin, and at the nuclear matrix). During the course of infection the relative subnuclear distribution of large T antigen changed significantly at about the switch from the early to late phase of infection: at early times postinfection, large T antigen was present mainly in the nucleoplasm and at the cellular chromatin, and nuclear-matrix-associated large T antigen was barely detectable. Concomitant with the onset of viral DNA replication, the amount of nuclear-matrix-associated large T antigen increased drastically. During the further course of infection large T antigen accumulated at the cellular chromatin and nuclear matrix, paralleling the increase in viral DNA synthesis. The biological significance of this correlation was corroborated by analysis of cells infected with the SV40 mutant tsA58 at permissive (32 degrees C) and restrictive (39 degrees C) temperatures. tsA58 large T antigen failed to initiate viral DNA replication in infected cells kept at the restrictive temperature and also failed to associate with the cellular chromatin and nuclear matrix. By blocking viral DNA synthesis with aphidicolin, an inhibitor of DNA polymerase alpha, we were able to show that the accumulation of large T antigen at these structures does not result from the binding of large T antigen to viral chromatin but reflects an association with cellular components of the chromatin and nuclear matrix of infected cells.

Animals↗
PubMed1980

Method to detect asymptomatic carriers of simian hemorrhagic fever virus.

Evidence was obtained that mononuclear phagocytic cells are the target cells for simian hemorrhagic fever virus replication. Using peritoneal macrophages from rhesus monkeys in an in vitro, 18 of 20 asymptomatic chronically infected patas monkeys were detected from coded samples. The two chronically infected patas monkeys not detected by the test, nevertheless, contained virus. This was determined by inoculating macrophage cultures with plasma from macaques dying as a result of inoculation with plasma from these chronically infected animals. in addition to virus found in chronically infected animals, all isolates of simian hemorrhagic fever virus tested previously described epizootics lytically infected rhesus monkey macrophages. These data suggested that the highly fatal nature of simian hemorrhagic fever in macaques was related to the extreme sensitivity of their mononuclear phagocytic cells to infection and lysis.

Animals↗
PubMed1976

UV sensitivity of a nonrepressor regulatory protein of bacteriophage P22.

The product of phage P22 gene c1 has two functions: it promotes synthesis of P22 repressor and it retards expression of some lytic genes. We present evidence that this product is inactivated in UV-irradiated hosts. The conditions for inactivation of c1 product include a functional DNA recombination system involving the host recA gene.

Bacterial Proteins↗
PubMed1985

Small and middle T antigens contribute to lytic and abortive polyomavirus infection.

Using three different polyomavirus hr-t mutants and two polyomavirus mlT mutants, we studied induction of S-phase by mutants and wild-type virus in quiescent mouse kidney cells, mouse 3T6 cells, and FR 3T3 cells. At different times after infection, we measured the proportion of T-antigen-positive cells, the incorporation of [3H]thymidine, the proportion of DNA-synthesizing cells, and the increase in total DNA, RNA, and protein content of the cultures. In permissive mouse cells, we also determined the amount of viral DNA and the proportion of viral capsid-producing cells. In polyomavirus hr-t mutant-infected cultures, onset of host DNA replication was delayed by several hours, and a smaller proportion of T-antigen-positive cells entered S-phase than in wild-type-infected cultures. Of the two polyomavirus mlT mutants studied, dl-23 behaved similarly to wild-type virus in many, but not all, parameters tested. The poorly replicating but well-transforming mutant dl-8 was able to induce S-phase, and (in permissive cells) progeny virus production, in only about one-third of the T-antigen-positive cells. From our experiments, we conclude that mutations affecting small and middle T-antigen cause a reduction in the proportion of cells responding to virus infection and a prolongation of the early phase, i.e., the period before cells enter S-phase. In hr-t mutant-infected mouse 3T6 cells, production of viral DNA was less than 10% of that in wild-type-infected cultures; low hr-t progeny production in 3T6 cells was therefore largely due to poor viral DNA replication.

Animals↗
PubMed1987

Three mutants of herpes simplex virus type 2: one lacking the genes US10, US11 and US12 and two in which Rs has been extended by 6 kb to 0.91 map units with loss of Us sequences between 0.94 and the Us/TRs junction.

In the process of generating restriction endonuclease site deletion mutants, we have isolated and characterized three mutants of herpes simplex virus type 2 (HSV-2), strain HG52, with large genomic deletions in Us and TRs. The deleted sequences (7.5 kb) extend from 0.94 map coordinates (m.c.) to 0.99 m.c. and are presumed to contain the HSV-2 gene equivalents of US10, 11 and 12, one copy of immediate early (IE) gene 3 and one copy of an origin of replication (ORIs). One of the mutants (HG52X163X12) has a simple deletion whereas in the two others (HG52X163X14 and HG52X163X21) the deleted sequences have been replaced by inverted duplications of Us/IRs sequences between 0.83 and 0.91 m.c. such that the molecules have short region inverted repeats extended by 6 kb on either side. All three are viable, stable and grow in tissue culture indicating that the polypeptides coded by the HSV-2 genes equivalent to US10, 11 and 12 are non-essential for lytic growth in BHK21/C13 cells. In addition the lack of one copy of the HSV-2 equivalent of IE gene 3 and ORIs in HG52X163X12 shows that only one copy of each suffices for viability. However the temperature restriction of the mutants at 38.5 degrees C suggests that one or more of the polypeptides coded by the deleted sequences may be required in conjunction with another polypeptide(s) for viral growth or stability at the higher temperature.

Animals↗
PubMed1988

The relationship of receptor occupancy to the kinetics of cell death mediated by tumor necrosis factor.

An analysis of the relationship between TNF receptor occupancy and the kinetics of cell death has been conducted in the sensitive L929.10 murine fibrosarcoma cell line. The receptor affinity and number, measured by competitive and saturation binding assays with human 125I-TNF, is consistent with a single class of binding sites having a Kd = 1.5 nM and 7,000 sites/cell. Human lymphotoxin competed equally well with TNF for binding to the receptor. Determination of the kinetics of L929.10 cell death as a function of TNF concentration revealed three sequential phases to the cytolytic process: An induction phase, that lasted 4 to 9 hrs after the addition of TNF, in which no cell death occurred; the burst phase in which cells died at a rate of 12 to 14% per hr; and the third phase, termed the slow lytic phase, was characterized by a rate of lysis of 1 to 2% per hr. Two parameters, the length of the induction phase and the percentage of cells killed in the burst phase, were dependent on TNF receptor occupancy. Both of these receptor dependent processes were maximized at 10% receptor occupancy. Half maximal lysis of the L929.10 clone was achieved at 10 pM or 0.67% receptor occupancy. In contrast, the rates of cell death during the burst phase and the slow lytic phase were independent of the concentration of TNF. Mitomycin C, an antitumor drug that alkylates and crosslinks DNA, enhanced the sensitivity of the L929.10 cells to TNF mediated lysis by modulating both of the TNF receptor dependent processes and by doubling the (apparent) rate of cell death (22 to 25%/hr) during the burst phase. These results suggest a direct relationship exists between the strength of the TNF-receptor signal and nuclear events, perhaps those associated with processes involved in DNA replication or cell division, during the cytotoxic reaction induced by these inflammatory cytokines.

Animals↗
PubMed1988

Recognition of simian virus 40 T antigen synthesized during viral lytic cycle in monkey kidney cells expressing mouse H-2Kb- and H-2Db-transfected genes by SV40-specific cytotoxic T lymphocytes leads to the abrogation of virus lytic cycle.

Simian virus 40 (SV40)-encoded tumor or T antigen localizes in the membranes in addition to the nucleus of SV40-infected permissive monkey cells and SV40-transformed nonpermissive cells. The surface T antigen in SV40-transformed mouse cells provides a target for the cytotoxic T lymphocytes (CTL) which recognize SV40 T antigen in association with murine K/D, class I H-2 antigens. In order to demonstrate that SV40 T antigen synthesized in SV40-infected permissive monkey kidney cells (TC-7) may also function as a target for CTL, cloned murine H-2Db and H-2Kb genes were expressed in TC-7 cells by DNA transfection and TC-7 cell lines expressing high levels of either H-2Kb or H-Db antigens were established after cell sorting. SV40-infected TC-7/H-2Kb and TC-7/H-2Db cells became susceptible to lysis by SV40-specific H-2b restricted CTL. The susceptibility of these transfected SV40-infected monkey cells to anti-SV40 bulk culture CTL and SV40-specific H-2Db- and H-2Db-restricted CTL clones depended upon the synthesis of SV40 T antigen and the expression of the appropriate H-2Kb or H-2Db restriction elements. Treatment of SV40-infected TC-7/H-2Db and TC-7/H-2Kb with CTL clones abrogated the virus lytic cycle indicating that CTL may play an important role in limiting papovavirus infection in the natural host.

Animals↗
PubMed2004

Molecular and transcriptional analysis of the temperate lactococcal bacteriophage Tuc2009.

The genome of bacteriophage Tuc2009 consists of 38347 base pairs on which 57 open reading frames (ORFs) were identified, divided in two oppositely transcribed regions. The leftward-transcribed region harbors three ORFs, two of which are involved in the establishment of lysogeny. The rightward-transcribed region contains 54 ORFs, which are assumed to be required for the lytic life cycle. An exception to the above organization is ORF 10, of unknown function, located within the rightward-transcribed region that has an orientation opposite to the ORFs surrounding it. Transcriptional analysis of the Tuc2009 genome following infection of a sensitive host revealed that most ORFs are transcribed in a sequential manner. ORFs that are presumed to form (part of) the genetic switch along with the superinfection exclusion-encoding gene are transcribed immediately after infection, followed by transcription of the presumed replication region. Subsequent to this, several small transcripts could be identified followed by a single 24-kb transcript. This latter transcript was shown to specify most of the identified structural proteins as well as two proteins required for host lysis. Interestingly, the 24-kb mRNA was shown to undergo splicing through the activity of a type I intron whose removal from the mRNA resulted in the formation of an ORF specifying a major structural protein. Primer extension analysis was employed to identify the 5' ends of mRNA transcripts and the genome and transcriptional data are discussed in relation to other lactococcal bacteriophages.

Bacteriophages↗
PubMed1978

Polyoma virus-specific RNA synthesis in an inducible line of polyoma virus-transformed rat cells.

Viral RNA present in the inducible LPT clone 1A of polyoma virus-transformed rat cells was characterized before and after mitomycin C induction by hybridization with 32P-labeled separated E and L strands of polyoma viral DNA restriction endonuclease fragments. In clone 1A cells maintained under normal growth conditions, the cytoplasm contained a transcript of the E-strand DNA from the "early" region similar to that previously identified in lytically infected cells, as well as minor quantities of RNA complementary to less than one-half of the L- and the E-strand DNA from the "late" region. Nuclei of normally growing cells contained the same species found in the cytoplasm, as well as an additional abundant RNA complementary to one-half of the L-strand DNA of the late region. No significant changes occurred in the cytoplasmic viral RNA after mitomycin C treatment before the onset of viral DNA replication, but the concentration of the nuclear L-strand DNA transcript diminished. After the onset of viral DNA replication after mitomycin C treatment, transcripts of virtually the entire L-strand DNA were found in the nuclei, and a 10-fold increase was observed in the abundance of RNA transcribed from the E strand of the early region. In the cytoplasm, the abundance of the early RNA increased about 25-fold and late RNA complementary to the L-strand DNA of the late region was found in a similar quantity. The synthesis of both the early and the late RNA species was inhibited if viral DNA replication was blocked with 5-fluorodeoxyuridine. We conclude that the induction of viral DNA replication in LPT cells is not determined at the level of mRNA synthesis.

Animals↗
PubMed1985

Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids.

We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.

Animals↗
PubMed1983

Interaction of minute virus of mice with differentiated cells: strain-dependent target cell specificity is mediated by intracellular factors.

The prototype strain of minute virus of mice and the immunosuppressive strain are unable to grow lytically in each other's murine host cell type. To characterize these strain-dependent virus-host cell interactions further, we have compared the early events of both productive and restrictive infections. Each virus binds to specific receptors on the surface of both productive and restrictive cell types. Competition experiments show that both viruses recognize the same receptor on each cell type. Penetration and uncoating are presumed to be similar in both productive and restrictive infections, since incoming viral genomes are converted to parental replicative form DNA independent of the final outcome of the virus-host cell interaction. In contrast to the majority of other systems studied to date, these differences in minute virus of mice target cell specificity are not mediated at the cell surface, but by the interaction of a strain-specific viral determinant with intracellular host factors that are expressed in particular cell types as a function of differentiation. These cellular factors catalyze a step in viral replication which occurs after the initiation of viral DNA synthesis, but before the detectable expression of the viral capsid polypeptide genes.

Animals↗
PubMed2026

Repurposing anti-phage defenses to differentially arrest the viral lifecycle reveals the regulatory logic of a parasitic satellite.

Mobile genetic elements frequently encode defense mechanisms to protect their bacterial hosts from viral attack. In Vibrio cholerae, these defensive elements include phage-inducible chromosomal island-like elements (PLEs), which are phage satellites that act as highly specialized parasites of the lytic phage ICP1. While PLE transcriptional activation upon ICP1 infection is known to be temporally regulated, the underlying regulatory logic and dependencies on the progression of the phage's developmental program required for activation remain unclear. In this study, we took a novel approach to define these dependencies by introducing independent anti-phage defense systems, BREX and DarTG, as molecular roadblocks to impede the ICP1 lifecycle. We discovered that, for both ICP1 and PLE, late-stage gene expression is fundamentally uncoupled from genome replication, representing a striking departure from the standard paradigm for double-stranded DNA phages. While BREX restricts ICP1 to an immediate-early transcriptional state that stalls PLE activation, DarTG allows the phage to execute its full transcriptional cascade despite the total block in DNA replication. This permissive environment provides the necessary cue(s) for complete PLE induction, revealing that robust PLE activation is profoundly dependent on the transcriptional progression of its helper phage.IMPORTANCEBacteria and their viruses (phages) are locked in perpetual evolutionary conflict. Some bacteria harbor phage satellites, specialized parasites that are activated to hijack the phage's components to spread all the while inhibiting viral production. While some satellites respond to a single viral trigger, the regulation of many satellites, including clinically relevant phage-inducible chromosomal island-like elements (PLEs) in Vibrio cholerae, remains poorly understood. Here, we used bacterial defense systems as molecular roadblocks to probe how PLE activation depends on its helper phage. We found that severe disruptions to viral transcription stall PLE activation. Unexpectedly, both the virus and the satellite can execute their full transcriptional programs even when DNA replication is completely blocked, challenging a fundamental paradigm in virology. These insights reveal a sophisticated level of phage-satellite coordination, illustrating how satellite activation is tightly linked to the transcriptional state of its helper phage, a dependency that ultimately drives the dissemination of mobile genetic elements.

Vibrio cholerae↗