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PubMed1981

Measles virus: evolution of a persistent infection in BGM cells.

An African green monkey kidney cell line (BGM) persistently infected with measles virus (BGM/Hallé cells) has been studied during 3 years in culture. The early cell passages were characterized by slow growing cultures producing high yields of infections virus (10(6)--10(7) PFU/ml). These cells were gradually replaced by a population of cells multiplying at a similar rate as non-infected cells. During this evolution, the virus released from the cells changed from a large plaque variant, to a small plaque and eventually unlysed foci. Despite greater than or equal to 95 per cent of the cells being infected, the virus yield fell to the limits of detection. [35S]-methionine labelling of BGM/Hallé cultures at the 20th and 140th passage showed that in both cases all the measles virus structural proteins were synthesized. There were no changes in the apparent molecular sizes of the viral proteins during passage. Cell surface [125I]-labelling of BGM/Hallé cells indicates that viral envelope antigens are inserted into the membrane despite the diminution in virus yield. Several cell proteins not labelled in non-infected BGM cells are also labelled. These proteins could also be [125I]-labelled in clones of BGM/Hallé cells with had been "cured" of virus. Chase experiments of [125I]-pre-labelled BGM/Hallé cultures showed the radiolabelled antigen to be incorporated into virus particles. Non-infections virus particles released from the cells contained the same polypeptides as those released from a lytic infection.

Animals↗
PubMed2006

Human cytomegalovirus disrupts both ataxia telangiectasia mutated protein (ATM)- and ATM-Rad3-related kinase-mediated DNA damage responses during lytic infection.

Many viruses (herpes simplex virus type 1, polyomavirus, and human immunodeficiency virus type 1) require the activation of ataxia telangiectasia mutated protein (ATM) and/or Mre11 for a fully permissive infection. However, the longer life cycle of human cytomegalovirus (HCMV) may require more specific interactions with the DNA repair machinery to maximize viral replication. A prototypical damage response to the double-stranded ends of the incoming linear viral DNA was not observed in fibroblasts at early times postinfection (p.i.). Apparently, a constant low level of phosphorylated ATM was enough to phosphorylate its downstream targets, p53 and Nbs1. p53 was the only cellular protein observed to relocate at early times, forming foci in infected cell nuclei between 3.5 and 5.5 h p.i. Approximately half of these foci localized with input viral DNA, and all localized with viral UL112/113 prereplication site foci. No other DNA repair proteins localized with the virus or prereplication foci in the first 24 h p.i. When viral replication began in earnest, between 24 and 48 h p.i., there were large increases in steady-state levels and phosphorylation of many proteins involved in the damage response, presumably triggered by ATM-Rad3-related kinase activation. However, a sieving process occurred in which only certain proteins were specifically sequestered into viral replication centers and others were particularly excluded. In contrast to other viruses, activation of a damage response is neither necessary nor detrimental to infection, as neither ATM nor Mre11 was required for full virus replication and production. Thus, by preventing simultaneous relocalization of all the necessary repair components to the replication centers, HCMV subverts full activation and completion of both double-stranded break and S-phase checkpoints that should arrest all replication within the cell and likely lead to apoptosis.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1980

Regulation of integrated adenovirus sequences during adenovirus infection of transformed cells.

A human cell line (293) transformed by adenovirus type 5 encodes mRNA's and proteins from the early region 1 (E1) of the viral genome. These products correspond to those synthesized early after adenovirus infection of normal cells. This pattern of expression is different from that observed at later times in the lytic cycle. We have determined whether integrated sequences can undergo the early-late transition during infection of transformed cells. Cultures of 293 cells were infected with mutants of adenovirus type 5 that have deletions in EI genes. In such infections, the integrated sequence complements the deletion mutants so that viral DNA replication, late mRNA and protein synthesis, and viral assembly occur. Because the infecting genomes lack EI sequences, the products synthesized from the integrated DNA could be analyzed. In contrast to the early-late transition that occurs with EI DNA in free viral genomes, the pattern of mRNAs and proteins made from the integrated sequences was restricted to the early pattern. Assuming that the viral sequences in 293 cells have not become altered during the history of the cells, our results suggest that regulation of integrated adenovirus genes may not be determined exclusively by nucleotide sequence recognition. Apparently, during infection certain factors prevent the integrated viral genes from responding to the regulatory signals which control late expression from free EI DNA. The distinction between integrated and free viral sequences might reflect the different fates of viral and host transcripts during the lytic cycle of adenovirus.

Adenoviruses, Human↗
PubMed1993

In vivo and in vitro analysis of transcription of the L region from the Halobacterium salinarium phage phi H: definition of a repressor-enhancing gene.

The 12-kb L region of the Halobacterium salinarium phage phi H is able to replicate as a plasmid, conferring a certain immunity to the host cell. We show here that the whole region is utilised for transcription at one stage or another in the phage life cycle. The DNA segment between the lytic transcripts T4 and T1 is shown to be constitutively transcribed. The effects of transcription on immunity were investigated using transformation experiments with H. salinarium. In this way, we show in vivo that the immune transcript T9, which on its own has no influence on phage growth or immunity, has a co-operative effect on the phi H repressor. The immunity effects analysed are sufficient to account for the degree of immunity conferred by the L region.

Amino Acid Sequence↗
PubMed1995

Biochemical characterization and localization of JC virus large T antigen phosphorylation domains.

Large T antigen (T Ag), the major regulatory protein produced by the primate polyomaviruses, is a multifunctional phosphoprotein expressed early in the viral life cycle. T Ag performs many functions essential to viral DNA replication, and studies with SV40 T Ag indicate that the regulation of these functions is modulated, in part, by the phosphorylation status of this oncoprotein. In this study, we demonstrate that JC virus (JCV) T Ag obtained from lytically infected and transformed cells is phosphorylated at serine and threonine residues. Analysis of JCV T Ag via two-dimensional tryptic peptide mapping generates 14 phosphopeptides. Additional mapping studies of intact, hybrid, mutant, and truncated forms of JCV T Ag have aided the localization of phosphorylation sites to the N- or C-terminal region of the protein; both serine and threonine residues are modified at each terminus. The data indicate that, unlike the corresponding regulatory phosphorylation site Ser677 in SV40, Thr664 is not phosphorylated in JCV T Ag. The phosphorylation sites utilized for JCV T Ag, and the regulatory role of these sites, are predicted to contribute to the unique biology of this human virus.

Animals↗
PubMed1990

Constraints in simian virus 40 (SV40) encapsidation, as determined by SV40-based shuttle viruses.

Simian virus 40 (SV40)-based shuttle vectors, containing the SV40 late genes, can be packaged as infectious pseudovirions. In terms of their function as bacterial plasmids, modifications in the overall size of these plasmids can be tolerated within a very wide range, which has allowed us to determine the requirements for SV40 encapsidation, free of the more stringent limitations of SV40 virus. Monkey COS7 cells were transfected with over- and undersized SV40-based shuttle virus plasmids and their progeny have been analysed to follow the stability and evolution of these genomes. Two of the three plasmids analysed undergo recombination, generating molecules with sizes of between 4.0 and to 4.8 kb which were selected after multiple lytic cycles. This size range may correspond to the DNA lengths preferentially packaged in SV40 capsids. The structure of the rearranged plasmids indicates that there is a strong selective pressure for genomes that retain the functions necessary for replication and virus production. Depending on the parent DNA, two main classes of rearrangements were generated: duplications in tandem with the SV40 origin of replication and deletions. Both classes are probably a result of selective size and replicative advantages, which are then biologically amplified during plasmid transmission as virus particles.

Animals↗
PubMed1988

Trypanosoma cruzi: cell biological behavior of epimastigote and amastigote forms in axenic culture.

A simple protocol to maintain Trypanosoma cruzi amastigote stocks indefinitely in axenic culture is described. The growth characteristics of amastigotes differ markedly from epimastigotes cultured under identical conditions. The amastigotes replicate for two generations, followed by a transformation to epimastigotes and resumption of growth. By changing the culture medium at the end of the second amastigote generation, transformation to epimastigotes is inhibited. Therefore, the protocol used to maintain amastigotes in culture is based upon changing the culture medium at preselected intervals. Flow cytometric analyses indicate that at the end of the exponential phase of growth the amastigote population consists of predominately G1 cells; changing the medium induces the amastigotes to begin a para-synchronous round of DNA synthesis without a pre-replicative lag phase. In contrast, when exponentially growing or stationary-phase epimastigotes are transferred to fresh culture medium, they grow asynchronously until reaching a limiting cell density. Amastigotes also differ from epimastigotes in being resistant to the lytic activity of human complement. These data demonstrate that marked differences in phenotypic expression exist between developmental stages of T. cruzi even when cultured under identical conditions.

Animals↗
PubMed1985

Phasmid vectors for identification of genes by complementation of Escherichia coli mutants.

A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon. A general method for transferring cloned DNA segments onto bacteriophage lambda was developed. The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4. Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.

Bacteriophage lambda↗
PubMed1977

Persistent adenovirus infections of nonpermissive monkey cells.

Persisten infections of monkey cells have been established by using two serotypes of human adenovirus. The persistently infected cells show no morpho logical changes, but continue ot produce low titers of infectious adenovirus. The inapparent infection can, at any time, be converted to a cytolytic productive one by superinfection with simian virus 40. Persistence in this system does not appear to result from multiple rounds of lytic infection, nor is it mediated by production of defective interfering particles. The persistently infected cells do not possess the characteristics of oncogenic transformation. Results of these studies also whow that the nonpermissiveness of monkey cells to adenovirus replication can be partially overcome by infection at high multiplicity.

Adenoviruses, Human↗
PubMed1989

Complement and infectious agents: a tale of disguise and deception.

The primary functions of the immune system center on the recognition and elimination of infectious agents, foreign substances, and altered or transformed host cells. Bacterial and viral infections represent major challenges to the immune system because of the ability of these agents to replicate and injure host cells and tissues, and to impair recognition and elude destruction by humoral or cellular defense mechanisms. Complement functions at multiple levels in the control of bacterial infections: recognition, in vivo clearance, opsonization, killing and lytic destruction. Although bacteria frequently evade destruction and cause disease, the ultimate control of bacterial infections is largely dependent on the actions of antibody and complement functioning synergistically with phagocytic cells. The crucial role of complement in these actions is apparent in the striking susceptibility to life-threatening bacterial infections of most individuals who are genetically deficient in complement components, particularly C3, C3 regulatory proteins and C5, C6, C7 or C8. The complement system also interacts with viruses and virus-infected cells at multiple levels and, as in the case of bacteria, viruses have evolved various mechanisms to avoid destruction. As obligate intracellular parasites which reside in host cells; however, the elimination of viruses is largely dependent on cellular immune responses. Complement also interacts with parasites and fungi; however, the physiologic importance of these reactions has yet to be elucidated. The various types of interactions of infectious agents with the complement system and the mechanisms which these pathogens have evolved to evade destruction and, in some cases, to potentiate and facilitate infection are summarized here.

Bacterial Infections↗
PubMed1973

Mixed infections of Bacillus subtilis involving bacteriophage SPO2c 1 .

The synthesis of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) was studied in Bacillus subtilis cells mixedly infected with phages SPO2c(1) and either SP82 or beta22. It was found that cells preinfected 5 min with either beta22 or SP82 could not support significant amounts of RNA or DNA synthesis from the genome of superinfecting SPO2c(1). Conversely, cells preinfected with SPO2c(1) remained susceptible to superinfection even at the midpoint of the latent period. When SP82 was added to cells preinfected 5 min with SPO2c(1), the former greatly inhibited the replication and transcription of the SPO2c(1), DNA, but if superinfection with SP82 was delayed until the 15th min of the SPO2c(1) latent period, RNA and DNA specific for both phages were synthesized. Both viral genomes were transcribed throughout the remainder of the lytic cycle when beta22 was added to cells preinfected either 5 or 15 min with SPO2c(1). In both the SPO2c(1)-SP82 and the SPO2c(1)-beta22 mixed infections, the types of phage-specific RNAs synthesized at a given time in the latent period were similar to those synthesized in single infections with each phage. The association of SPO2c(1) DNA with the host cell membrane in certain of the doubly infected cells was different from that observed in singly infected cells. In the SPO2c(1)-SP82 infection, SPO2c(1) DNA was not released from the membrane; in the SPO2c(1)-beta22 mixed infections, significantly less SPO2c(1) DNA was membrane-bound when beta22 was added before SPO2c(1). However, binding and release of SPO2c(1) DNA was normal in cells infected first with SPO2c(1) and then with beta22. The synthesis of phage-specific nucleic acids and the binding of phage DNA to the membrane are discussed with respect to dominance relationships among these phages.

Bacillus subtilis↗
PubMed1996

Inflammatory infiltration of the trigeminal ganglion after herpes simplex virus type 1 corneal infection.

Following herpes simplex virus type 1 (HSV-1) infection of the cornea, the virus is transmitted to the trigeminal ganglion, where a brief period of virus replication is followed by establishment of a latent infection in neurons. A possible role of the immune system in regulating virus replication and maintaining latency in the sensory neurons has been suggested. We have investigated the phenotype and cytokine pattern of cells that infiltrate the A/J mouse trigeminal ganglion at various times after HSV-1 corneal infection. HSV antigen expression in the trigeminal ganglion (indicative of the viral lytic cycle) increased until day 3 postinfection (p.i.) and then diminished to undetectable levels by day 7 p.i. The period of declining HSV antigen expression. was associated with a marked increase in Mac-1+ cells. These cells did not appear to coexpress the F4/80+ (macrophage) or the CD8+ (T cell) markers, and none showed polymorphonuclear leukocyte morphology, suggesting a possible early infiltration of natural killer cells. There was also a significant increase in the trigeminal ganglion of cells expressing the gamma delta T-cell receptor, and these cells were found almost exclusively in very close association with neurons. This period was also characterized by a rapid and equivalent increase in cells expressing gamma interferon and interleukin-4. The density of the inflammatory infiltrate in the trigeminal ganglion increased until days 12 to 21 p.i., when it was predominated by CD8+, Mac-1+, and tumor necrosis factor-expressing cells, which surrounded many neurons. By day 92 p.i., the inflammatory infiltrate diminished but was heaviest in mice with active periocular skin disease. Our data are consistent with the notion that gamma interferon produced by natural killer cells and/or gamma delta T cells may play an important role in limiting HSV-1 replication in the trigeminal ganglion during the acute stage of infection. In addition, tumor necrosis factor produced by CD8+ T cells and macrophages may function to maintain the virus in a latent state.

Animals↗
PubMed1987

SV40-based Escherichia coli shuttle vectors infectious for monkey cells.

We describe SV40-based Escherichia coli shuttle vectors which can be packaged as pseudovirions without excision of plasmid sequences and which can be rescued in bacteria. These vectors replicate and are transmitted as virus in monkey COS cells without requiring a helper virus. Extrachromosomal vector DNA isolated from infected cells can be rescued in E. coli, so that DNA alterations can be easily screened. Indeed, some of the constructions give rise to very stable plasmids with no detectable rearrangements after multiple lytic cycles in COS cells. The spontaneous mutation frequency measured in bacteria, on the lacO target, is smaller than those usually found with shuttle vectors. We also constructed an expression vector derived from one of our infectious viruses by inserting the chloramphenicol acetyl transferase gene, expressed from the SV40 early promoter, which is efficiently transduced to cells by infection. In this system, the shuttle virus combines the convenience of plasmid rescue and analysis in bacteria, with the advantages of infectious virus.

Acetyltransferases↗
PubMed1988

Legionella pneumophila immunity and immunomodulation: nature and mechanisms.

L. pneumophila is a facultative intracellular opportunistic pathogen ubiquitously present in the environment. Much is now known concerning the ecological niche of this organism as well as many other characteristics of these bacteria, including physiology and biochemistry. However, much less is known about immune mechanisms responsible for host resistance vs susceptibility. Not only outer membrane protein rich fractions but also LPS-rich components are potent immunogens, both in experimental animals such as susceptible guinea pigs and more resistant rodent species like rats and mice. Immunity to these organisms can be readily observed by a variety of serologic techniques. Antibody titers increase rapidly after exposure of individuals to these bacteria either by infection or immunization. However, such antibody does not appear to play an important role in host resistance. Serum antibody plus complement is not lytic for the bacteria in vitro. Furthermore, antibody appears to promote the phagocytosis of the bacteria by monocytes and/or macrophages in culture but such phagocytosis does not result in killing of the bacteria, merely an enhanced uptake and subsequent replication of the organisms. Studies on cellular immunity have focused attention on the role of T lymphocytes, monocytes and macrophages. In addition, cutaneous hypersensitivity is readily induced by infection or immunization of experimental animals with Legionella or antigenic components. In vitro correlates of hypersensitivity is also readily evident after infection or immunization. Although lymphoid cells from guinea pigs only show evidence of responsiveness to Legionella antigens by the lymphocyte blastogenic reaction after animals have been sensitized, peripheral blood monocytes from man as well as splenocytes from mice show evidence of responsiveness to Legionella even before known infection or sensitization. However, higher blastogenic responses become evident after sensitization or infection. In addition, interleukins, such as interleukin 1 and 2, as well as interferon and tumor necrotizing factor, appear in response to Legionella antigens and seem to play a role in resistance mechanisms. Cellular replication of Legionella in monocytes from man as well as macrophages from susceptible animals seems related to susceptibility or resistance to these organisms. Further analyses of the nature and mechanism of humoral vs cellular immune responses to Legionella antigens will provide valuable information about immunity and resistance to these intracellular pathogens in susceptible individuals.

Animals↗
PubMed1992

Interaction between bacteriophage lambda and its Escherichia coli host.

Bacteriophage lambda relies to a large extent on processes requiring interactions between viral- and host-encoded proteins for its lytic growth, establishment of lysogeny, and release from the prophage state. Both biochemical and genetic studies of these interactions have yielded new information about important host and lambda functions. In particular, mutations in Escherichia coli that compromise lambda DNA replication, genome packaging, transcription elongation, and site-specific recombination have led to the identification of bacterial genes whose products are chaperones, transcription factors, or DNA-binding proteins.

Bacterial Proteins↗
PubMed1998

The mutation rate and cancer.

The stability of the human genome requires that mutations in the germ line be exceptionally rare events. While most mutations are neutral or have deleterious effects, a limited number of mutations are required for adaptation to environmental changes. Drake has provided evidence that DNA-based microbes have evolved a mechanism to yield a common spontaneous mutation rate of approximately 0.003 mutations per genome per replication (Drake 1991). In contrast, mutation rates of RNA viruses are much larger (Holland et al. 1982) and can approach the maximum tolerable deleterious mutation rate of one per genome (Eigen and Schuster 1977; Eigen 1993). Drake calculates that lytic RNA viruses display spontaneous mutation rates of approximately one per genome while most have mutation rates that are approximately 0.1 per genome (Drake 1993). This constancy of germline mutation rates among microbial species need not necessarily mean constancy of the somatic mutation rates. Furthermore, there need not be a constant rate for somatic mutations during development. In this review, we consider mutations in cancer, a pathology in which there appears to be an increase in the rate of somatic mutations throughout the genome. Moreover, within the eukaryotic genome, as in microbes, there are "hot-spots" that exhibit unusually high mutation frequencies. It seems conceivable to us that many tumors contain thousands of changes in DNA sequence. The major question is: how do these mutations arise, and how many are rate-limiting for tumor progression?

Animals↗