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PubMed1984

Replication of murine coronaviruses in somatic cell hybrids between murine fibroblasts and rat schwannoma cells.

The replication of the murine coronaviruses MHV3 and JHM has been studied in somatic cell hybrids formed between murine fibroblast L2 cells which support lytic infections with both these agents, and rat RN2 Schwannoma cells which support the replication of JHM in a temperature-sensitive, persistent manner but are restrictive to the replication of MHV3. The results described in this report indicate that the totally permissive state is dominant over the persistent or restricted state since the hybrid cells permit the replication of both these viral agents in a lytic manner.

Animals↗

PubMed1997

Assembly of complete, functionally active herpes simplex virus DNA replication compartments and recruitment of associated viral and cellular proteins in transient cotransfection assays.

Early during the herpes simplex virus (HSV) lytic cycle or in the presence of DNA synthesis inhibitors, core viral replication machinery proteins accumulate in intranuclear speckled punctate prereplicative foci, some of which colocalize with numerous sites of host cellular DNA synthesis initiation known as replisomes. At later times, in the absence of inhibitors, several globular or large irregularly shaped replication compartments are formed; these compartments also contain progeny viral DNA and incorporate the IE175(ICP4) transcription factor together with several cellular proteins involved in DNA replication and repair. In this study, we demonstrate that several forms of both prereplication foci and active viral replication compartments that display an appearance similar to that of the compartments in HSV-infected cells can be successfully assembled in transient assays in DNA-transfected cells receiving genes encoding all seven essential HSV replication fork proteins together with oriS target plasmid DNA. Furthermore, bromodeoxyuridine (BrdU)-pulse-labeled DNA synthesis initiation sites colocalized with the HSV single-stranded DNA-binding protein (SSB) in these replication compartments, implying that active viral DNA replication may be occurring. The assembly of complete HSV replication compartments and incorporation of BrdU were both abolished by treatment with phosphonoacetic acid (PAA) and by omission of any one of the seven viral replication proteins, UL5, UL8, UL9, UL42, UL52, SSB, and Pol, that are essential for viral DNA replication. Consistent with the fact that both HSV IE175 and IE63(ICP27) localize within replication compartments in HSV-infected cells, the assembled HSV replication compartments were also able to recruit both of these essential regulatory proteins. Blocking viral DNA synthesis with PAA, but not omission of oriS, prevented the association of IE175 with prereplication structures. The assembled HSV replication compartments also redistributed cotransfected cellular p53 into the viral replication compartments. However, the other two HSV immediate-early nuclear proteins IE110(ICP0) and IE68(ICP22) did not enter the replication compartments in either infected or transfected cells.

Animals↗

PubMed1986

Identification and characterization of mutations in Escherichia coli that selectively influence the growth of hybrid lambda bacteriophages carrying the immunity region of bacteriophage P22.

Mutations in two Escherichia coli genes, sipA and sipB, result in a specific inhibition of the growth of certain hybrid lambdoid bacteriophages, lambda immP22, that have the early regulatory regions and adjacent genes from bacteriophage P22. The sipB391 mutation maps near minute 56 and exerts the strongest inhibitory effect on the growth of the hybrid phages. The sipA1 mutation maps near minute 72 and plays an auxiliary role: enhancing the action of sipB391. Such a role is not limited to sipA1, since there is a similar enhancement by the nusA1 and nusE71 mutations. The Sip-imposed restriction on the growth of lambda immP22 phages is not observed if the phage carries a mutation in the c1 gene. Perhaps this reflects the fact that the c1 product regulates phage DNA replication and is a major determinant in the decision governing whether the phage takes the lytic or lysogenic pathway. Consistent with this idea is the observation that lambda immP22 DNA replication is severely inhibited in bacteria carrying the sipB391 mutation. It is suggested that sip mutations exaggerate the normal role of c1 in limiting lytic growth. This causes a failure in the expression of sufficient amounts of some or all of the lytic gene products required for phage growth.

Bacteriophage lambda↗

PubMed2001

Restricted growth of avirulent avian reovirus strain 2177 in macrophage derived HD11 cells.

The replication of two pathotypes of avian reovirus, 1733 and 2177 in transformed chicken lymphoid and myeloid cell lines was examined, showing that only the macrophage cell line, HD11, supports replication. The virulent strain 1733 causes a lytic infection producing 100-1000 fold more virus than the avirulent strain 2177. Cells infected with strain 2177 display delayed viral RNA and protein synthesis as well as a suppressed expression of the major capsid protein muB. These features may contribute to the lower virulence of the strain 2177 in their natural host in vivo.

Animals↗

PubMed2000

Disruption of PML-associated nuclear bodies by IE1 correlates with efficient early stages of viral gene expression and DNA replication in human cytomegalovirus infection.

In human cytomegalovirus (HCMV) infection, both of the major immediate-early proteins IE1(IE68, UL123) and IE2(IE86, UL122) target to PML protein-associated nuclear bodies known as PODs or ND10 at very early times after infection. IE1 causes a redistribution of both PML and IE1 from the PODs into a nuclear diffuse form, whereas IE2 initially localizes adjacent to PODs but later associates with viral DNA replication compartments. The peripheries of PODs are also believed to be sites for initiation of both viral IE transcription and DNA replication. However, because IE1 is nonessential at high multiplicity of infection (m.o.i.) in HF cells, the exact role of these processes in viral infection has been enigmatic. Therefore, we investigated the effects of overexpression of PML in the presence or absence of IE1 on the intranuclear distribution of IE2 and formation of viral DNA replication compartments, as well as on the levels of delayed-early and late viral transcription and protein accumulation. Infection with wild-type HCMV(Towne) and the IE1-deleted derivative HCMV(CR208), which fails to disrupt PODs, was compared in a pair of related astrocytoma/glioblastoma cell lines, the U373-Neo control and a variant U373-PML that constitutively overexpresses PML(560) in much larger than normal PODs. IFA studies on the localization patterns for IE1, IE2, and PML showed that, although the numbers of IE2-positive cells were not significantly reduced in either the wild-type virus-infected U373-PML cell line or in DeltaIE1-infected control cells, POD disruption by IE1 in wild-type virus infection was delayed by up to 6 h in U373-PML cells compared to control cells. Furthermore, there was considerable enhancement of IE2 colocalization with PODs in Delta IE1-infected U373-PML cells. Formation of viral DNA replication compartments in the U373-PML cell line was also greatly delayed, measured at fivefold lower after wild-type virus infection and 12-fold lower after infection with Delta IE1 than in the control cell line at 48 h at an m.o.i. of 1.0. The levels of representative early and late viral proteins detected by Western blotting were suppressed by fivefold and 22-fold at 24 and 72 h, respectively, in the U373-PML cell line, even with high m. o.i. wild-type HCMV infection. Decreased viral protein levels also occurred when control cells were infected with the Delta IE1 virus and these two effects were additive in the U373-PML cell line. Similarly, when U373-PML cells were infected with recombinant HCMV expressing an extragenic luciferase reporter gene under the control of viral early (Pol) or late (pp28) promoters, their transcriptional activation was reduced up to fivefold at both high and low m.o.i. compared to that of the control cells. Overall, these results suggest that POD disruption by IE1 and subsequent redistribution of both PML and IE1 at very early times after infection may play an important role in the efficient utilization of cellular transcription and replication machinery by HCMV and contribute to rapid progression of the HCMV lytic cycle.

Cytomegalovirus↗

PubMed2005

Circularization of the herpes simplex virus type 1 genome upon lytic infection.

For many years, the generally accepted model for the replication of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1) incorporated initial circularization of linear molecules in the cell nucleus. Ensuing DNA synthesis resulted in the generation of head-to-tail concatemers which were subsequently cleaved into monomeric units and packaged into the nascent viral capsid. Recently, however, it has been proposed that circularization of HSV-1 genomes does not occur at the onset of lytic infection and moreover that this event is specifically inhibited by the HSV-1 transcriptional transactivator, ICP0 (S.A. Jackson and N.A. DeLuca, Proc. Natl. Acad. Sci. USA 100:7871-7876, 2003). To further investigate genome circularization, we have generated HSV-1 derivatives in which the viral a sequences, which contain the cleavage-packaging signals, have been replaced by a minimal packaging element located in the thymidine kinase gene. In contrast to wild-type HSV-1, fusion of the genomic termini of these viruses produces a novel fragment in circular or concatemeric DNA which can be detected by Southern blot hybridization. Utilizing these viruses, we demonstrate that fusion of the genomic termini occurred rapidly upon infection and in the presence of inhibitors of viral DNA or protein synthesis. We provide evidence indicating that the end joining represented circularization rather than concatemerization of input molecules and that circularized molecules functioned as templates for replication. Since the termini of these viruses lack direct repeats, our findings indicate that circularization can be mediated by direct end-to-end ligation of linear input genomes.

Animals↗

PubMed2002

The lytic enzyme of bacteriophage PRD1 is associated with the viral membrane.

Bacteriophage PRD1 encodes two proteins (P7 and P15) that are associated with a muralytic activity. Protein P15 is a soluble beta-1,4-N-acetylmuramidase that causes phage-induced host cell lysis. We demonstrate here that P15 is also a structural component of the PRD1 virion and that it is connected to the phage membrane. Small viral membrane proteins P20 and P22 modulate incorporation of P15 into the virion and may connect it to the phage membrane. The principal muralytic protein involved in PRD1 DNA entry seems to be the putative lytic transglycosylase protein P7, as the absence of protein P15 did not delay initiation of phage DNA replication in the virus-host system used. The incorporation of two different lytic enzymes into virions may reflect the broad host range of bacteriophage PRD1.

Bacteriolysis↗

PubMed2001

Epstein-Barr virus latently infected cells are selectively deleted in simulated-microgravity cultures.

Rotating-wall vessels (RWVs) allow for the cultivation of cells in simulated microgravity. Previously, we showed that the cultivation of lymphoblastoid cells in simulated microgravity results in the suppression of Epstein-Barr virus (EBV) reactivation. To determine if the suppression generated by simulated microgravity could be reversed by changing to static culture conditions, cells were cultured in an RRWV for 5 d, and then switched to static conditions. Following the switch to static conditions, viral reactivation remained suppressed (significantly lower) relative to static control cultures over a 4-d period. Additionally, experiments were conducted to determine if chemical treatment could induce viral reactivation in cells from simulated-microgravity cultures. Cells were cultured in static flask cultures and in simulated microgravity in RWVs for 4-7 d. The cells were then transferred to 50-cm3 tubes, and treated with 3 mM n-butyrate for 48 h, or 18 ng/ml of phorbol ester, viz., 12-0-tetradecanoylphorbol-13 acetate (TPA) for either 2 or 48 h, under static conditions. Although EBV was inducible, the cells from simulated-microgravity cultures treated with n-butyrate displayed significantly lower levels of viral-antigen expression compared with the treated cells from static cultures. Also, incubation with TPA for 2-3 h, but not for 48 h, reactivated EBV in cells from RWV cultures. In contrast, EBV was inducible in cells from static cultures treated for either 2-3 or 48 h with TPA. TPA reactivation of EBV following a 2-3-h period of treatment indicates that the protein kinase C signal-transduction pathway is not impaired in lymphoblastoid cells cultured in simulated microgravity. However, the exposure of B-lymphoblastoid cells from simulated-microgravity cultures to TPA for more than 3-4 h triggered a lytic event (apoptosis or necrosis), which prevented replication of the virus. Thus, EBV-infected cells in simulated microgravity were negatively selected in the absence of any cytotoxic cells.

B-Lymphocytes↗

PubMed1974

Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA.

Explanted mouse blastocysts were microinjected in the blastocoel cavity with simian virus 40 (SV40) viral DNA. After surgical transfer to the uteri of pseudopregnant surrogate mothers, approximately 40% of the blastocysts developed to term and became healthy adults without apparent tumors at 1 year of age. Molecular hybridization tests for the presence of SV40-specific DNA sequences were conducted on DNA extracted from various organs of these animals. Between 0.5 and 13 SV40 genome equivalents per diploid mouse DNA value were found in some organs of approximately 40% of the adult survivors; this represents a substantial augmentation of the amount administered per embryo. The results are consistent with the working hypothesis that the SV40 DNA may have been integrated into the host genome; alternatively, the viral DNA may have replicated as an extrachromosomal entity or by lytic infection in a few permissive cells. Persistence of the viral DNA from preimplantation stages to adult life may thus provide a new tool for experimental investigation of vertical transmission and expression of tumor viruses.

Animals↗

PubMed1990

The enhancer factor R of Epstein-Barr virus (EBV) is a sequence-specific DNA binding protein.

In cells latently infected with EBV, the switch from latency to productive infection is linked to the expression of two EBV transcription factors called EB1 (or Z) and R. EB1 is an upstream element factor which has partial homology to the AP1/ATF family, whereas R is an enhancer factor. In the R-responsive enhancer of the replication origin only active during the EBV lytic cycle (ORIIyt), R-responsive elements are located in a region of about 70 bp (RRE-DR). Here we show that R, produced either by in vitro translation, or present in nuclear extracts from HeLa cells constitutively producing R, binds directly to and protects against DNAase I digestion, two regions in RRE-DR. Using mobility shift assay and DMS interference, we have characterized the contact-points between R and the DNA. Two binding sites, RRE-DR1 and RRE-DR2, were characterized and are contiguous in RRE-DR. R binds to these two sites probably by simultaneously contacting two sequences within the sites, which are separated by 7 bp in RRE-DR1, cctGTGCCttgtcccGTGGACaatgtccc, and by 6bp in RRE-DR2, caatGTCCCtccagcGTGGTGgctg. Direct interaction of R with its cognate sequences is conferred by its N-terminal 355 amino-acids. Directed mutagenesis in RRE-DR, of either R-binding site, impaired binding of R in vitro and, as assayed by transient expression in HeLa cells, impaired R-activation by a factor of two. This suggests that RRE-DR1 and RRE-DR2 do not respond cooperatively to R.

Base Sequence↗

PubMed2005

Real-time PCR measures Epstein-Barr Virus DNA in archival breast adenocarcinomas.

The role of Epstein-Barr Virus (EBV) in breast cancer pathogenesis remains controversial. Fifty-five cases of paraffin-embedded, formalin-fixed invasive breast cancer were screened for the presence of EBV using quantitative polymerase chain reaction (PCR) directed at five different targets within the EBV genome (BamH1W, LMP1, EBNA1, LMP2, and BZLF1 regions). In four tumors (7%), low level EBV DNA was detected by at least one of the assays, with levels of up to 11 copies of EBV DNA per 100,000 cells. Immunohistochemisty for viral BMRF1 and BZLF1 and in situ hybridization for lytic gene transcripts showed no evidence of replicative EBV gene expression. Lymphocytes and malignant cells were also negative for latent infection by EBER in situ hybridization. Laser capture microdissection followed by quantitative real-time PCR was not useful in localizing EBV DNA to malignant cells or bystander lymphocytes. In conclusion, EBV DNA is detectable in a fraction of breast cancer specimens using real-time PCR as a screening tool, albeit at quite low levels, which suggests that only rare cells are infected. The low levels probably confounded our ability to localize the virus to particular cell types or to characterize viral gene expression.

Adenocarcinoma↗

PubMed2006

Pharmacodynamics of non-replicating viruses, bacteriocins and lysins.

The pharmacodynamics of antibiotics and many other chemotherapeutic agents is often governed by a 'multi-hit' kinetics, which requires the binding of several molecules of the therapeutic agent for the killing of their targets. In contrast, the pharmacodynamics of novel alternative therapeutic agents, such as phages and bacteriocins against bacterial infections or viruses engineered to target tumour cells, is governed by a 'single-hit' kinetics according to which the agent will kill once it is bound to its target. In addition to requiring only a single molecule for killing, these agents bind irreversibly to their targets. Here, we explore the pharmacodynamics of such 'irreversible, single-hit inhibitors' using mathematical models. We focus on agents that do not replicate, i.e. in the case of phage therapy, we deal only with non-lytic phages and in the case of cancer treatment, we restrict our analysis to replication of incompetent viruses. We study the impact of adsorption on dead cells, heterogeneity in adsorption rates and spatial compartmentalization.

Anti-Bacterial Agents↗

PubMed1997

Resistance to herpes simplex virus type 1 and its latent infection of human T cell lymphotropic virus type I-transformed T cell lines of rabbits.

Fourteen T cell lines of rabbits were infected with herpes simplex virus type 1 (HSV-1) and examined for their susceptibility to lytic infection and ability to support virus replication. T cell lines of CD4+ 8- phenotype were more vulnerable to lysis and supported higher levels of virus replication than those of other phenotypes. Cell lines of CD4+ 8+ and CD4- 8+ phenotypes continued to proliferate, while remaining productively infected, for more than a month. These latently infected cell lines could be established by treatment with anti-HSV-1 serum and complement. Viral genes were only partially expressed and the CD8 membrane antigen was down-regulated. Infected cell lines, as well as peripheral blood lymphocytes, were shown to induce meningoencephalitis when inoculated intravenously into syngeneic hosts, suggesting a possible role for infected lymphocytes in HSV-1 transport in vivo.

Animals↗

PubMed1992

Latent infection of SCID mice with herpes simplex virus 1 and lethal cutaneous lesions in pregnancy.

Some SCID mice survived primary infection with herpes simplex virus 1 without the development of peripheral lesions but established coculture-positive ganglionic latency when a low dose of a wild-type strain was inoculated intracutaneously. The latency was also evidenced by the development of the fatal zosteriform skin lesions and the isolation of the virus during pregnancy. We consider that the viral entry into neurons without successive replication, rather than the arrest of the lytic infection within the cells, is an important mechanism in the establishment of latency.

Animals↗

PubMed1995

Structure and role of the terminal repeats of Epstein-Barr virus in processing and packaging of virion DNA.

The linear virion Epstein-Barr virus (EBV) DNA is terminated at both ends by a variable number of direct, tandemly arranged terminal repeats (TRs) which are approximately 500 bp in size The number of TRs at each terminus can vary. After infection of host cells, the EBV DNA circularizes via the TRs by an unknown mechanism, and replication of the viral DNA during the lytic phase of the EBV life cycle leads to large DNA concatemers which need to be cleaved into virion DNA units, eventually. This cleavage event occurs at an unknown locus within the TRs of EBV, which are the cis-acting elements essential for cleavage of the concatemers and encapsidation of the virion DNA. To investigate the mechanism of DNA processing during genome circularization and cleavage of concatemeric DNA, the genomic termini of EBV were cloned, sequenced, and analyzed by direct labeling of the virion DNA. Both termini ended with identical 11-bp elements; the right end has acquired an additional 9-bp stretch that seemed to originate from the leftmost unique sequences. The left terminus is blunt, whereas the right terminus appears to have a 3' single-base extension. In a transient packaging assay, a single terminal repeat was found to be sufficient for encapsidation of plasmid DNA, and mutagenesis of the TR element defined a region of 159 bp, including the 11-bp element, which is essential for packaging. These results indicate that the genomic termini of EBV are not generated by a simple cut of a hypothetical terminase. The mechanism for cleavage of concatemers seems to involve recombination events.

Base Sequence↗

PubMed1999

Primate herpesviral oncogenes.

Gammaherpesviruses are the most rapidly growing members of the herpesviridae family. Gamma herpesviruses share similarity in their genome organizations and in early and late lytic genes that are required for viral replication. A distinct characteristic of gamma herpesviruses is their ability to establish latent infection in lymphoid cells, and some of these viruses are closely associated with abnormal proliferation and cancer in primates. The first open reading frame of the primate gamma herpesviruses has been shown to directly contribute to virus-associated pathogenesis. This open reading frame encodes latent membrane protein-1 (LMP1) in Epstein-Barr virus, Saimiri transformation protein (STP) in Herpesvirus Saimiri, K1 in Kaposi's sarcoma-associated herpesvirus, and R1 in Rhesus monkey Rhadinovirus. All of these gene products are capable of eliciting cellular signal transduction events, resulting in cell growth transformation. This review briefly summarizes the current view on the transforming mechanisms utilized by primate herpesviral oncogenes.

Animals↗

PubMed1998

Excess production of phage lambda delayed early proteins under conditions supporting high Escherichia coli growth rates.

Bacteriophage lambda is unable to lysogenize Escherichia coli hosts harbouring the rpoA341 mutation due to a drastic reduction in transcription from CII-activated lysogenic promoters (pE, pI and paQ). In addition, the level of early transcripts involved in the lytic pathway of lambda development is also decreased in this genetic background due to impaired N-dependent antitermination. Here, it is demonstrated that despite the reduced level of early lytic pL- and pR-derived transcripts, lytic growth of bacteriophage lambda is not affected in rich media. The level of the late lytic, pR-derived transcripts also remains unaffected by the rpoA341 mutation under these conditions. However, it was found that whilst there is no significant difference in the phage burst size in rpoA+ and rpoA341 hosts growing in rich media, phage lambda is not able to produce progeny in the rpoA341 mutant growing in minimal medium, in contrast to otherwise isogenic rpoA+ bacteria. Provision of an excess of the phage replication proteins O and P in trans or overproduction of the antitermination protein N restore the ability of phage lambda to produce progeny in the rpoA341 mutant under the latter conditions. These results suggest that in rich media phage lambda produces some early proteins in excess of that needed for its effective propagation and indicate that replication proteins may be limiting factors for phage lytic growth in poor media.

Bacteriophage lambda↗

PubMed1988

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells.

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.

Antibodies, Monoclonal↗