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Multiple regulatory mechanisms controlling phage-plasmid P4 propagation.

Bacteriophage P4 autonomous replication may result in the lytic cycle or in plasmid maintenance, depending, respectively, on the presence or absence of the helper phage P2 genome in the Escherichia coli host cell. Alternatively, P4 may lysogenize the bacterial host and be maintained in an immune-integrated condition. A key step in the choice between the lytic/plasmid vs. the lysogenic condition is the regulation of P4 alpha operon. This operon may be transcribed from two promoters, PLE and PLL, and encodes both immunity (promoter proximal) and replication (promoter distal) functions. PLE is a constitutive promoter and transcription of the downstream replication genes is regulated by transcription termination. The trans-acting immunity factor that controls premature transcription termination is a short RNA encoded in the PLE proximal part of the operon. Expression of the replication functions in the lytic/plasmid condition is achieved by activation of the PLL promoter. Transcription from PLL is insensitive to the termination mechanism that acts on transcription starting from PLE.PLL is also negatively regulated by P4 orf88, the first gene downstream of PLL. An additional control on P4 DNA replication is exerted by the P4 cnr gene product.

Bacteriophage P1↗

A gammaherpesvirus 68 gene 50 null mutant establishes long-term latency in the lung but fails to vaccinate against a wild-type virus challenge.

The gammaherpesvirus immediate-early genes are critical regulators of virus replication and reactivation from latency. Rta, encoded by gene 50, serves as the major transactivator of the lytic program and is highly conserved among all the gammaherpesviruses, including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (gammaHV68). Introduction of a translation stop codon in gammaHV68 gene 50 (gene 50.stop gammaHV68) demonstrated that Rta is essential for virus replication in vitro. To investigate the role that virus replication plays in the establishment and maintenance of latency, we infected mice with gene 50.stop gammaHV68. Notably, the gene 50.stop virus established a long-term infection in lung B cells following intranasal infection of mice but was unable to establish latency in the spleen. This complete block in the establishment of latency in the spleen was also seen when lytic virus production was inhibited by treating mice infected with wild-type virus with the antiviral drug cidofovir, implicating virus replication and not an independent function of Rta in the establishment of splenic latency. Furthermore, we showed that gene 50.stop gammaHV68 was unable to prime the immune system and was unable to protect against a challenge with wild-type gammaHV68, despite its ability to chronically infect lung B cells. These data indicate gammaherpesviruses that are unable to undergo lytic replication in vivo may not be viable vaccine candidates despite the detection of cells harboring viral genome at late times postinfection.

Animals↗

Perturbations in simian virus 40 DNA synthesis by ultraviolet light.

Perturbations of Simian Virus 40 (SV40) DNA replication by ultraviolet (UV) light during the lytic cycle in permissive monkey CV-1 cells resemble those seen in host cell DNA replication. Formation of Form I DNA molecules (i.e. completion of SV40 DNA synthesis) was more sensitive to UV irradiation than synthesis of replicative intermediates or Form II molecules, consistent with inhibition of DNA chain elongation. The observed amounts of [3H]thymidine incorporated in UV-irradiated molecules could be predicted on the assumption that pyrimidine dimers are responsible for blocking nascent DNA strand growth. The relative proportion of labeled Form I molecules in UV-irradiated cultures rapidly increased to near-control values with incubation after 20 or 40 J/m2 of light (0.9--1.0 or 1.8--2.0 dimers per SV40 genome, respectively). This rapid increase and the failure of Form II molecules to accumulate suggest that SV40 growing forks can rapidly bypass many dimers. Form II molecules formed after UV irradiation were not converted to linear (Form III) molecules by the dimer-specific T4 endonuclease V, suggesting either that there are no gaps opposite dimers in these molecules or that T4 endonuclease V cannot use Form II molecules as substrates.

Cell Line↗

Replication of mycoplasmavirus MVL51. III. Identification of a progeny viral DNA-protein intermediate.

Intracellular replication of the non-lytic single stranded circular DNA mycoplasmavirus MVL51 has been shown to involve three DNA intermediates: RFI, RFII and SSI. Growth in Eagle's basal medium, rather than richer tryptose medium, has allowed the identification of an intermediate between nascent progeny chromosomes (SSI) and mature virus. This intermediate is a protein associated form of SSI.

Acholeplasma laidlawii↗

The control of SV40 transcription during a lytic infection: late RNA synthesis in the presence of inhibitors of DNA replication.

The transition from early to late transcription of SV40 DNA in productively infected BSC-1 cells was analyzed using both inhibitors of DNA replication, and early (Group A) temperature sensitive (ts) mutants of SV40. Late virus specific cytoplasmic RNA sedimenting at 16S in neutral sucrose gradients and complementary to the plus (L) DNA strand of SV40 was detected in cultures infected in the presence of three inhibitors of DNA replication (Ara-C, FdU, and chloroquine), even though the inhibition of viral DNA replication appeared to be essentially complete. After infection with the early SV40 mutant tsA58, no DNA replication was detected at the restrictive temperature (41 degrees C) and no significant late RNA complementary to the plus (L) strand was found, in either the cytoplasm or nuclei of infected cells. These data support the concept that expression of late viral functions requires the initiation of viral DNA synthesis or a functional gene A protein, or both.

Cell Line↗

Genomic sequence and evolution of marine cyanophage P60: a new insight on lytic and lysogenic phages.

The genome of cyanophage P60, a lytic virus which infects marine Synechococcus WH7803, was completely sequenced. The P60 genome contained 47,872 bp with 80 potential open reading frames that were mostly similar to the genes found in lytic phages like T7, phi-YeO3-12, and SIO1. The DNA replication system, consisting of primase-helicase and DNA polymerase, appeared to be more conserved in podoviruses than in siphoviruses and myoviruses, suggesting that DNA replication genes could be the critical elements for lytic phages. Strikingly high sequence similarities in the regions coding for nucleotide metabolism were found between cyanophage P60 and marine unicellular cyanobacteria.

Bacteriophages↗

Deoxyribonucleic acid replication in simian virus 40-infected cells. 3. Comparison of simian virus 40 lytic infection in three different monkey kidney cell lines.

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.

Animals↗

Distinct patterns of viral antigen expression in Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus coinfected body-cavity-based lymphoma cell lines: potential switches in latent gene expression due to coinfection.

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), are human gammaherpesviruses associated with numerous lymphomas and proliferative diseases in humans. We were interested in the protein expression patterns of specific latent and lytic proteins from the EBV genome in two body-cavity-based lymphoma cell lines, BC-1 and BC-2, which are coinfected with EBV and KSHV. BC-1 and BC-2 were analyzed using specific antibodies to latent proteins known to be essential for EBV immortalization of human primary B-lymphocytes in vitro and lytic antigens important for EBV replication and production of viral progeny. The coinfected cell lines are compared with two singly infected KSHV cell lines to determine whether antibodies against EBV-specific proteins cross-reacted against KSHV antigens. All the KSHV-infected cell lines express the KSHV-specific latency-associated nuclear antigen (LANA) with a specific pattern in the nucleus. This staining was distinct from that seen for EBNA1 in the EBV coinfected lines BC-1 and BC-2 staining the nucleus as a diffused pattern throughout the nucleus with denser staining in some regions. The coinfected cell lines all express EBNA1 and LMP1 at lower levels compared with singly infected EBV lymphoblastoid cell lines (LCLs). However, the essential latent antigens EBNA2, EBNA3A, and EBNA3C are not expressed in BC-1 and BC-2. This indicates potential regulation of EBV latent gene expression by KSHV-encoded viral or KSHV-induced cellular gene products. Additionally, lytic gene expression analysis demonstrated that BZLF1 and BMRF1 are expressed along with other early antigens (EA-D). A specific protein is detected in a singly infected KSHV cell line with cross-reactivity to antibodies that detected the EA-D complex. Moreover, in all the cell lines infected with EBV, KSHV, or EBV and KSHV, human serum with antibodies against KSHV antigens recognizes specific viral antigens approximately 110 and 41-42 kDa, suggesting that human antibodies against KSHV-specific antigens can cross-react with similar EBV antigens. Therefore these data suggest that the EBV pattern of gene expression in the coinfected cell lines is a type II pattern of latency also seen in other human tumors including nasopharyngeal carcinoma and Hodgkin's lymphoma. This distinct pattern of latent and lytic gene expression in these cell lines may provide clues as to the selection for coinfection in these body cavity based lymphomas in immunocompromised hosts.

Antigens, Viral↗

Unimpaired histone synthesis in human fibroblasts infected by human cytomegalovirus.

Serum-starved human foreskin fibroblasts were infected by human cytomegalovirus (Towne strain) that is thought to induce DNA replication in host cells during lytic infection. At various times postinfection, the cultures were pulse labeled with either 3H-thymidine or 14C-thymidine and 3H-lysine to examine DNA synthesis and histone synthesis, respectively. Isopycnic centrifugation of labeled DNA in CsCl revealed that precursor incorporation into host-cell DNA was enhanced over the control around 24 h postinfection and decreased after onset of viral DNA synthesis which reached a peak around 72 h postinfection. For analysis of histones 3H-lysine-labeled proteins of lysates of unfractionated cells and of chromatin preparations were subjected to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and subsequent fluorography. Comparison of the fluorograms from the various pulses postinfection suggested that 3H-lysine incorporation into histones exhibited no major variations concurrent with the changes of host-cell DNA synthesis. In contrast, herpes simplex virus type 1 was found progressively to extinguish histone synthesis in the course of the cellular infection. Furthermore, histone synthesis in phosphonoacetic acid-treated cytomegalovirus-infected cultures was not enhanced over that in mock-infected controls. These observations do not support the view that human cytomegalovirus induces host-cell DNA replication under the conditions used.

Cell Line↗

Epstein-Barr virus-specific CD8(+) T cells that re-express CD45RA are apoptosis-resistant memory cells that retain replicative potential.

During acute infection, latent and lytic Epstein-Barr virus (EBV) epitope-specific CD8(+) T cells have a CD45RO(+) CD45RA(-) phenotype. However, after resolution of the infection, a large proportion of these cells, particularly those specific for lytic viral epitopes, re-express the CD45RA molecule. The role of CD8(+) CD45RA(+) T cells in ongoing immunity to EBV and other viruses is unknown. We now demonstrate that, relative to their CD45RO(+) counterparts, the EBV-specific CD8(+) T cells that revert to CD45RA expression after acute infectious mononucleosis are not in cell cycle, have longer telomeres, and are more resistant to apoptosis partly because of increased Bcl-2 expression. However, the EBV-specific CD8(+) CD45RA(+) T cells have shorter telomeres than the total CD8(+) CD45RA(+) T-cell pool and predominantly express low levels of the CCR7 chemokine receptor, indicating that they are not naive cells. In addition, EBV-specific CD8(+) CD45RA(+) T cells can be induced to proliferate and exhibit potent cytotoxic activity against target cells loaded with specific peptide. Our results strongly suggest, therefore, that EBV-specific CD8(+) CD45RA(+) T cells represent a stabilized virus-specific memory pool and not terminally differentiated effector cells. The identification of mechanisms that enable stable virus-specific CD8(+) T cells to persist after acute infection may lead to the enhancement of antiviral immunity in immunocompromised and elderly persons.

Acute Disease↗

Mechanism of action of the antileukemic xanthone psorospermin: DNA strand breaks, abasic sites, and protein-DNA cross-links.

Psorospermin, a cytotoxic dihydrofuranoxanthone isolated from Psorospermum febrifugum, produced aberrant simian virus 40 DNA replication intermediates when added to lytically infected CV-1 monkey kidney cells. The aberrant viral intermediates showed dose-dependent DNA strand breaks and protein-DNA cross-links, as well as decreased electrophoretic mobility. Simian virus 40 DNA from psorospermin-treated cells was shown to contain numerous abasic (apyrimidinic/apurinic) sites. The density of abasic sites was a function of the psorospermin dose. We conclude that psorospermin causes extensive loss of DNA bases in vivo. Primary amine groups of cellular proteins are known to react with abasic sites to form covalent protein-DNA cross-links and DNA strand breaks. Cytochrome c cross-linked spontaneously to viral DNA prepared from psorospermin-treated cells but not to DNA from untreated cells. This suggests that the protein-DNA cross-links and many of the DNA strand breaks observed in vivo result from reactions between abasic sites and chromosomal proteins. It is likely that the protein-DNA cross-links and DNA strand breaks contribute to the cytotoxicity and antineoplastic activity of psorospermin.

Aldehydes↗

The importance of lytic and nonlytic immune responses in viral infections.

Antiviral immune effector mechanisms can be divided broadly into lytic and nonlytic components. We use mathematical models to investigate the fundamental question of which type of response is required to combat different types of viral infection. According to our model, the relative roles of the two types of component depend on the cytopathicity of the virus relative to its rate of replication. If the viral cytopathicity is low relative to the rate of viral replication, the model predicts that a combination of lytic and nonlytic effector mechanisms is likely to be required to resolve the disease, particularly if the virus replicates at a fast rate. By contrast, if viral cytopathicity is high relative to the replication rate of the virus, then lytic and nonlytic mechanisms can, in principle, resolve the infection independently. We discuss our findings in the context of specific viral infections and use our model to interpret empirical data.

Animals↗

Short-term assays for detection of conditional cancerogens. I. Construction of DR-CAT Raji cells and some of their characteristics as tester cells.

A number of agents including the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) (TPA) can induce an abortive virus cycle in the EBV-non-producer Burkitt's-lymphoma line Raji. Two distant regions, DL and DR, of the EBV genome with almost complete homology carry strong promoters which are induced in an abortive or lytic cycle and additionally function as lytic origins of viral DNA replication. To set up a system in which the activity of EBV-inducing agents can be measured in a quantitative and reproducible fashion, we generated a cell line which carries multiple copies of a DR-promoter chloramphenicol-acetyltransferase (CAT) construct on an episomal vector. CAT activity is low in untreated cells, but high upon treatment of the cells with various EBV-inducing agents. Combinations of different agents can produce an over-additive effect. The Raji-DR-CAT cell line may provide a simple quantitative and reproducible test system for EBV-inducing agents, especially for tumor promoters which activate protein kinases C.

Carcinogens↗

The limits of protection by "memory" T cells in Ig-/- mice persistently infected with a gamma-herpesvirus.

Can CD4(+) and CD8(+) "memory" T cells that are generated and maintained in the context of low-level virus persistence protect, in the absence of antibody, against a repeat challenge with the same pathogen? Although immune T cells exert effective, long-term control of a persistent gamma-herpesvirus (gammaHV68) in Ig(-/-) microMT mice, subsequent exposure to a high dose of the same virus leads to further low-level replication in the lung. This lytic phase in the respiratory tract is dealt with effectively by the recall of memory T cells induced by a gammaHV68 recombinant (M3LacZ) that does not express the viral M3 chemokine binding protein. At least for the CD8(+) response, greater numbers of memory T cells confer enhanced protection in the M3LacZ-immune mice. However, neither WT gammaHV68 nor the minimally persistent M3LacZ primes the T cell response to the extent that a WT gammaHV68 challenge fails to establish latency in the microMT mice. Memory CD4(+) and CD8(+) T cells thus act together to limit gammaHV68 infection but are unable to provide absolute protection against a high-dose, homologous challenge.

Animals↗

Revisiting the lysogenization control of bacteriophage lambda. Identification and characterization of a new host component, HflD.

Upon infection to the Escherichia coli cell, the genome of bacteriophage lambda either replicates to form new progenies (lytic growth) or integrates into the host chromosome (lysogenization). The lambda CII protein is a key determinant in the lysis-lysogeny decision. It is a short-lived transcription activator for the lambda genes essential for lysogeny establishment. In this study, we isolated a new class of hfl (high frequency lysogenization) mutants of E. coli, using a new selection for enhancement of CII-stimulated transcription. The gene affected was termed hflD, which encodes a protein of 213 amino acids. An hflD-disrupted mutant indeed showed an Hfl phenotype, indicating that HflD acts to down-regulate lysogenization. HflD is associated peripherally with the cytoplasmic membrane. Its interaction with CII was demonstrated in vitro using purified proteins as well as in vivo using the bacterial two-hybrid system. Pulse-chase examinations demonstrated that the HflD function is required for the rapid in vivo degradation of CII, although it interfered with FtsH-mediated CII proteolysis in an in vitro reaction system using detergent-solubilized components. We suggest that HflD is a factor that sequesters CII from the target promoters and recruits it to the membrane where the FtsH protease is localized.

Bacteriophage lambda↗

Assembly of the epstein-barr virus BBLF4, BSLF1 and BBLF2/3 proteins and their interactive properties.

Epstein-Barr virus (EBV) encodes putative helicase-primase proteins BBLF4, BSLF1 and BBLF2/3, which are essential for the lytic phase of viral DNA replication. The BSLF1, BBLF4 and BBLF2/3 proteins were expressed in B95-8 cells after induction of a virus productive cycle, possessing apparent molecular masses of 89 kDa, 90 kDa and 80 kDa, respectively. The anti-BSLF1 or anti-BBLF2/3 protein-specific antibody, which recognizes its target protein in both Western blotting and immunoprecipitation analyses, immunoprecipitated all of the BSLF1, BBLF4 and BBLF2/3 proteins from the extract of the cells with a virus productive cycle, indicating that these viral proteins are assembled together in vivo. To characterize their protein-protein interactions in detail, recombinant baculoviruses capable of expressing each of these viral gene products in insect cells were constructed. The assembly of the three virus replication proteins was reproduced in insect cells co- infected with the three recombinant baculoviruses, indicating that complex formation does not require other EBV replication proteins. Furthermore, experiments performed by using the extracts from insect cells co-infected with each pair of the recombinant viruses demonstrated that the BSLF1 protein could interact separately with both the BBLF4 and BBLF2/3 proteins and that the BBLF2/3 protein also interacted with the BBLF4 protein. These observations strongly suggest that within the BBLF4-BSLF1-BBLF2/3 complex each component interacts directly with the other two, resulting in helicase-primase enzyme activity.

Animals↗

Intracellular survival of Shigella.

Bacterial invasion of eukaryotic cells and host recognition and killing of the invading bacteria are a key issue in determining the fate of bacterial infection. Once inside host cells, pathogenic bacteria often modify the phagosomal compartment or enter the host cytosol to escape from the lytic compartment and gain a replicative niche. Cytosolic invaders, however, are monitored by host innate immune systems, such as mediated by Nod/CARD family proteins, which induce inflammatory responses via activation of NF-kappaB. Furthermore, recent studies indicate that autophagy, a major cytoplasmic degradation system that eliminates cytosolic protein and organelles, also recognizes invading bacteria. Indeed, unless they are able to circumvent entrapping by autophagic membranes, bacteria targeted by autophagy ultimately undergo degradation by delivery into autolysosomes. In this article, we review recent advances in understanding of Shigella strategies to infect epithelial cells, and then focus on recent studies of an intriguing bacterial survival strategy against autophagic degradation.

Animals↗

Characterization of the temperate bacteriophage phi adh and plasmid transduction in Lactobacillus acidophilus ADH.

Lactobacillus acidophilus ADH is lysogenic and harbors an inducible prophage, phi adh. Bacteriophage were detected in cell lysates induced by treatment with mitomycin C or UV light. Electron microscopy of lysates revealed phage particles with a hexagonal head (62 nm) and a long, noncontractile, flexible tail (398 nm) ending in at last five short fibers. Phage phi adh was classified within Bradley's B1 phage group and the Siphoviridae family. The phi adh genome is a linear double-stranded DNA molecule of 41.7 kilobase pairs with cohesive ends: a physical map of the phi adh genome was constructed. A prophage-cured derivative of strain ADH, designated NCK102, was isolated from cells that survived UV exposure. NCK102 did not exhibit mitomycin C-induced lysis, but broth cultures lysed upon addition of phage. Phage phi adh produced clear plaques on NCK102 in media containing 10 mM CaCl2 at pH values between 5.2 and 5.5. A relysogenized derivative (NCK103) of NCK102 was isolated that exhibited mitomycin C-induced lysis and superinfection immunity to phage phi adh. Hybridization experiments showed that the phi adh genome was present in the ADH and NCK103 chromosomes, but absent in NCK102. These results demonstrated classic lytic and lysogenic cycles of replication for the temperate phage phi adh induced from L. acidophilus ADH. Phage phi adh also mediates transduction of plasmid DNA. Transductants of strain ADH containing pC194, pGK12, pGB354, and pVA797 were detected at frequencies in the range of 3.6 x 10(-8) to 8.3 x 10(-10) per PFU. Rearrangements or deletions were not detected in these plasmids as a consequence of transduction. This is the first description of plasmid transduction in the genus Lactobacillus.

Bacteriophages↗