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PubMed1987

Intracellular reovirus survives cytotoxic T lymphocyte-mediated lysis of its host cell.

Cytotoxic T lymphocytes (CTLs) induce rapid, extensive internal disintegration in target cells and this is unique among immune lytic mechanisms studied. This raises the question of whether CTLs are uniquely capable of halting virus infections by inducing damage within the target cell causing inactivation of intracellular virus. Reovirus infection of mouse P815 cells provided a suitable system for evaluating this question. An increase in cell-associated infectious virions began 8 h after infection and increased until 20 h post-infection, at which time the titre levelled off at about 100- to 1000-fold higher than the initial value. The infectious activity was compared between host cells killed by CTLs and those killed by sonication at various points in the infection cycle. The presence of reovirus within the target cell did not inhibit the usual internal disintegration events associated with the death of a target killed by CTLs. Nevertheless, the results indicated that CTLs were incapable of inactivating intracellular reovirus at any point in the life cycle of the virus: CTL-induced cytolysis simply released the infectious virions into the medium. Thus, at least in the case of reovirus, the utility of direct killing by CTLs would appear to be limited to reduction of the virus yield by lysis of the host cell before virus replication and assembly is completed.

Animals↗
PubMed2000

Distinct transcriptional and functional properties of the R transactivator gene orf50 of the transforming herpesvirus saimiri strain C488.

The transformation-associated region of herpesvirus saimiri strains is variable, whereas other parts of the virus genome are highly conserved. However, we observed considerable interstrain sequence divergence of the early viral regulatory orf50 gene, which encodes the R transactivator, a homolog of Epstein-Barr virus BRLF1. The orf50 gene of strain C488 was transcribed at low abundance during lytic infection, whereas antisense transcripts were simultaneously expressed at high levels. A spliced variant, orf50a, was detectable by RT-PCR and RNase protection assays in stimulated C488-transformed, nonpermissive human T cells. In contrast to strain A11, the short, unspliced orf50b form of C488 displayed complete transactivation capability on the orf6 and orf57 promoters. In summary, there are unexpected structural and functional differences between the orf50 genes of herpesvirus saimiri strains, which differ in their capability to transform human T lymphocytes.

Amino Acid Sequence↗
PubMed1978

Genetic characterization of Mu-like bacteriophage D108.

Infection of Escherichia coli by bacteriophage D108 was shown to result in the generation of apparently random chromosomal mutations. Approximately 1% of the cells lysogenized by D108, as with Mu, acquired new auxotrophic mutations. D108-induced mutations were nonreverting and were most probably the result of insertion of the D108 genome into regions of genetic function. D108 and Mu shared many similar properties but were heteroimmune and had different host ranges. Lytic infections of Mu lysogens with D108 and D108 lysogens with Mu resulted in 100-fold increases in release of phage with prophage markers over those due to spontaneous induction. Phenotypic mixing was common, with most phage carrying the prophage immunity being packaged in particles with the host range of the superinfecting phage. A fraction of the superinfecting phage genomes were, however, packaged in particles with the prophage-specified host range. Although 10% of the prophage progeny were D108-Mu genetic hybrids, superinfecting phage-induced release of the prophage with reciprocal phenotypic mixing occurred in recA hosts, in which the frequency of D108-Mu genetic hybrids was reduced 100-fold.

Coliphages↗
PubMed1985

Endpoint distribution for deletions into imm lambda region forming p lambda CM replicons: phage lambda gene rex affects plasmid establishment.

For the p lambda CM family of lambda-derived self-encapsidating plasmids, the rexB gene product facilitates plasmid establishment following injection into a new host cell. Temperature-stable chloramphenicol resistance (CmR at 40 degrees C) conferred by low-multiplicity infection with lambda::Tn9 cI857 lysates (Tn9 sites tested: 22.60 or 24.08 kb, in the b region, or 28.41 kb, in int) is usually due to a lambda::Tn9 plasmid (p lambda CM) formed by a deletion penetrating the lambda immunity region. These grow either as plasmids in the absence of, or lytic phages in the presence of N function supplied by a host such as lambda cI857 delta H1 lysogen MS1449. The 'groplaque' (plaque-shaped growth spot) assay, which selects for CmR growth in an MS1449 lawn at 32 degrees C after an initial plaquing period at 37 degrees C, reveals two distinguishable classes of p lambda CM isolates. All variants whose deletions extend into or beyond rexB give rise to visible CmR growth only after the temperature shift to 32 degrees C, and thus produce a hollow-centered 'donut' type of groplaque. In contrast, 16 out of 17 variants whose deletions fall short of rexB produce 'solid' groplaques which appear before the temperature shift. Tests of T4rII phage exclusion show the exceptional 17th variant to be Rex-, confirming the identification of rex as the lambda component whose loss results in the 'donut' groplaque morphology. More specific physiological tests showed that in the absence of Rex the establishment of a newly injected p lambda CM plasmid becomes temperature-sensitive (ts), while plasmid maintenance remains unaffected. This indicates that the role of Rex in plasmid survival is confined to the early stages of transduction, where it might either assist plasmid replication or retard host replication, to help the plasmid replicon achieve a copy number sufficient for stable transmission.

Bacteriophage lambda↗
PubMed2003

ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties.

Herpes simplex virus type-1 (HSV1) in which the neurovirulence factor ICP34.5 is inactivated has been shown to direct tumour-specific cell lysis in several tumour models. Such viruses have also been shown to be safe in Phase I clinical trials by intra-tumoral injection in glioma and melanoma patients. Previous work has used serially passaged laboratory isolates of HSV1 which we hypothesized may be attenuated in their lytic capability in human tumour cells as compared to more recent clinical isolates. To produce ICP34.5 deleted HSV with enhanced oncolytic potential, we tested two clinical isolates. Both showed improved cell killing in all human tumour cell lines tested compared to a laboratory strain (strain 17+). ICP34.5 was then deleted from one of the clinical isolate strains (strain JS1). Enhanced tumour cell killing with ICP34.5 deleted HSV has also been reported by the deletion of ICP47 by the up-regulation of US11 which occurs following this mutation. Thus to further improve oncolytic properties, ICP47 was removed from JS1/ICP34.5-. As ICP47 also functions to block antigen processing in HSV infected cells, this mutation was also anticipated to improve the immune stimulating properties of the virus. Finally, to provide viruses with maximum oncolytic and immune stimulating properties, the gene for human or mouse GM-CSF was inserted into the JS1/34.5-/47- vector backbone. GM-CSF is a potent immune stimulator promoting the differentiation of progenitor cells into dendritic cells and has shown promise in clinical trials when delivered by a number of means. Combination of GM-CSF with oncolytic therapy may be particularly effective as the necrotic cell death accompanying virus replication should serve to effectively release tumour antigens to then induce a GM-CSF-enhanced immune response. This would, in effect, provide an in situ, patient-specific, anti-tumour vaccine. The viruses constructed were tested in vitro in human tumour cell lines and in vivo in mice demonstrating significant anti-tumour effects. These were greatly improved compared to viruses not containing each of the modifications described. In vivo, both injected and non-injected tumours showed significant shrinkage or clearance and mice were protected against re-challenge with tumour cells. The data presented indicate that JS1/ICP34.5-/ICP47-/GM-CSF acts as a powerful oncolytic agent which may be appropriate for the treatment of a number of solid tumour types in man.

Animals↗
PubMed2005

Recombination in the alphaherpesvirus bovine herpesvirus 1.

Herpesviruses are DNA viruses characterized by a low rate of nucleotide substitution. Therefore, other mechanisms must be involved to their evolution, like recombination that can be seen as an essential evolutionary driving force of these viruses. Recombination contributes to the long-term evolution of alphaherpesviruses. It acts also to continuously create new alphaherpesvirus strains. We have used bovine herpesvirus 1 to investigate recombination both within DNA concatemers in infected cells and in vitro and in vivo at the end of the lytic cycle. The following results have been obtained: (i) intramolecular recombination occurs at the level of concatemers and gives rise to genomic segment inversions; (ii) intraspecific recombination occurs frequently both in vitro and in vivo; (iii) interspecific recombination is possible and requires two highly genetically related viruses; (iv) only simultaneous or closely separated infections lead to the production of recombinant viruses; (v) recombination between wild-type and glycoprotein defective vaccine virus can produce a glycoprotein defective virus keeping part of the virulence of parental wild-type virus. Recombination, by exchanging genomic segments, may modify the virulence of alphaherpesviruses. It must be carefully assessed for the biosafety of antiviral therapy, alphaherpesvirus-based vectors and live attenuated vaccines.

Alphaherpesvirinae↗
PubMed1977

Maintenance of bacteriophage P1 plasmid.

Three mutants of bacteriophage P1 affected in their ability to maintain the lysogenic state stably are described here. These mutants were normal in lytic growth, but lysogenic derivatives segregated nonlysogens at abnormally high rates (1 to 30% per division). Cells harboring these mutant prophages were elongated or filamentous. The mutations responsible for this prophage instability fell into two classes on the bases of their genetic location, their effect on the ability to lysogenize recA bacteria, and their suppressibility by ant mutations eliminating antirepressor activity. The two mutants that were able to form recA lysogens showed the same prophage instability and partial inhibition of cell division in recA as in rec+ lysogens. The fact that plasmid-linked mutations can cause prophage instability suggests that P1 codes for at least some of the functions determining its own autonomy and segregation.

Chloramphenicol↗
PubMed2000

Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice.

During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.

Animals↗
PubMed1980

Association of polyoma T antigen and DNA with the nuclear matrix from lytically infected 3T6 cells.

Nuclear matrix prepared from mouse 3T6 cells lytically infected with polyoma virus retained significant amounts of the 100K T antigen and intact viral genomes. Bound T antigen was resistant to the extraction by high salt (2 M NaCl), detergent (1% Triton X-100) and exhaustive DNAase treatment. Only conditions sufficient to disrupt the integrity of the matrix itself solubilized the matrix T antigen. During the time period of 16-30 hr after infection, both the accumulation (in microgram) and the incorporation of 35S-methionine into T antigen increased steadily in cell extracts to a peak at 26 hr and then declined. In contrast, the amount of labeled T antigen retained by the matrix was relatively constant over the same time period. Matrix-bound T antigen was more highly phosphorylated and newly synthesized compared with the extractable T antigen. Viral DNA steadily accumulates in nuclei and on the matrix from 18 to 30 hr after infection. The fraction of viral DNA retained by the matrix was greatest early in infection (25% at 16 hr), declining to less than 10% by 24 hr. These data are consistent with the existence of a fixed (and limited) number of sites for T antigen (more highly phosphorylated) on the matrix and implicate the nuclear matrix as a site of viral DNA replication and possibly encapsidation.

Animals↗
PubMed1977

Histone synthesis during infection of monkey kidney cells with Simian Virus 40.

The synthesis of histones during lytic infection of BSC-1 (African Green Monkey kidney) cells with SV40 has been investigated. The synthesis of all five classes of histones was stimulated, and all classes appeared to be stimulated to the same extent. The increase in rate of histone synthesis in response to SV40 infection was detectable several hours before SV40 DNA synthesis was measureable, and the rate of histone synthesis decreased at a time when SV40 DNA synthesis was occuring at a maximal or relatively high rate. In addition, the changes in rates of histone synthesis did not correlate well with the rates of host DNA synthesis during infection. Thus it appears that DNA synthesis and histone synthesis may not be strictly coupled in SV40 infected cells.

Cell Line↗
PubMed1982

Changes in the topography of early region transcription during polyoma virus lytic infection.

We have studied the pattern of transcription of the early region of polyoma virus DNA after the onset of the late phase of lytic infection of mouse cells. Following initiation of viral DNA synthesis, the early/late switch is accompanied not only by efficient production of late mRNAs but also by the appearance of previously unidentified early-strand RNAs which have certain structural features in common with the classical early mRNAs. Stable poly(A)+RNAs have been identified by blot analysis and S1 nuclease mapping that are not detected early during infection or in polyoma virus-transformed cells. One group consists of transcripts whose 5' ends map 150-200 nucleotides upstream from the major early 5' ends (at positions 148 and 153 on the polyoma virus genome) but whose splicing pattern and poly(A) addition sites are indistinguishable from those of mRNAs produced early in infection. The 5' exons of these early region transcripts contain an open translational reading frame that extends from nucleotide positions 5,255 to 124 and is capable of encoding a basic protein of 53 amino acids. Transcription of these RNAs does not appear to be negatively regulated by large tumor antigen. A transcript of 1,800 nucleotides appears to map predominantly between 93 and 26 map units and does not contain sequences present in the early mRNA 5' exons. These data suggest that, after the onset of polyoma DNA replication, the activation of new early-strand promoters leads to the expression of previously untranscribed viral DNA sequences.

Antigens, Viral↗
PubMed1988

Host range mutants of adenovirus type 12 E1 defective for lytic infection, transformation, and oncogenicity.

The human adenovirus type 12 (H12) E1A region encodes two early proteins of 266 amino acid residues (266R) and 235R whilst the H12 E1B promoter directs the synthesis of two major proteins of 163R and 482R. To determine the functions of E1A and E1B in lytic infection and oncogenic transformation we have isolated and characterized a series of H12 E1 mutants. Mutant H12 hr 700 contains a point mutation in exon 1 that alters a single amino acid common to both the 266 and 235R proteins. This mutant synthesized reduced levels of E1 and structural proteins at delayed times in HEK cells, transformed BRK cells, and induced tumors in newborn rats at reduced efficiency compared to wild-type virus. The mutation in H12 in 600 truncates the 266R protein in its unique sequences but this mutant synthesized the 235R, E1B, and structural proteins at delayed times in HEK cells. H12 in 600 was nontransforming but induced rare tumors in newborn rats. A third E1A mutant H12 in 601 synthesized no E1A proteins, reduced levels of E1B and structural proteins at delayed times in lytic infections, and was not a transforming or oncogenic virus. Three E1B mutants were studied in detail. Both H12 hr 703 and H12 in 602 encode N-terminal truncated 482R proteins whereas H12 del 620 encodes an in-frame internally deleted 482R protein. All three synthesized reduced amounts of E1A proteins and the E1B 163R protein, identifying a regulatory function for the 482R protein. None of the E1B mutants could transform and only H12 del 620 could induce rare tumors in newborn rats. These results show that H12 oncogenesis requires the coordinated expression of the E1 proteins.

Adenovirus Early Proteins↗
PubMed1996

Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter.

The adenovirus major late promoter is strongly activated after the onset of viral DNA replication. Sequence elements located downstream of the major later promoter start site have previously been shown to be essential for this activation. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (C. Tribouley, P. Lutz, A. Staub, and C. Kedinger, J. Virol. 68:4450-4457, 1994). Here we show that pIVa2, while monomeric in solution, binds to its recognition sequence as a dimer and that two 20-residue amphipathic alpha helices play an essential role in this DNA-binding activity. Attempts to purify DEF-A have failed, but its chromatographic behavior, together with its immunological properties, established that pIVa2 is also a component of this heteromeric protein. In addition, the time course of pIVa2 synthesis during infection correlated with simultaneous detection of the binding of both DEF-A and DEF-B complexes to the downstream elements. Finally, as revealed by immunomicroscopy, pIVa2 is targeted to the nucleus, where it distributes to restricted locations in the nucleoplasm, as well as to the nucleoli. Altogether, these results demonstrate that pIVa2 plays a critical role in the transition from the early to the late phase of the lytic cycle. Furthermore, pIVa2 may serve additional functions yet to be uncovered, as suggested by its presence within the cell nucleolus.

Adenoviruses, Human↗
PubMed1997

Significance of macrophage tropism of SIV in the macaque model of HIV disease.

Microglia, alveolar macrophages, and Langerhans cells are representatives of cells of macrophage lineage that are susceptible to infection with HIV-1 and they play important roles in the pathogenesis of AIDS dementia, lymphoid interstitial pneumonia, and systemic viral invasion from mucosal surfaces, respectively. In contrast, elimination of CD4+ T cells with resultant development of immunosuppression and AIDS is thought to be reflective of the exclusive tropism of the virus for CD4+ T cells. Examination of these concepts in macaques infected with molecularly cloned strains of SIVmac suggested that all strains of the virus are both macrophage- and lymphocyte-tropic and that all aspects of pathogenesis including loss of CD4+ T cells are dependent on infection in both cell types. However, viral clones that caused productive lytic infection in macrophages were less virulent than those which caused persistent nonproductive infection. The former caused subclinical and even immunizing infections, whereas the latter caused activation and productive infection in CD4+ T cells, AIDS, and systemic infection, even after inoculation of the virus on mucosal surfaces. If these findings on SIVmac are relevant to HIV-1 disease, then demonstration that HIV-1 isolates are macrophage-tropic probably does not necessarily correlate with their pathogenic potential.

Acquired Immunodeficiency Syndrome↗
PubMed2002

HF2: a double-stranded DNA tailed haloarchaeal virus with a mosaic genome.

HF2 is a haloarchaeal virus infecting two Halorubrum species (Family Halobacteriaceae). It is lytic, has a head-and-tail morphology and belongs to the Myoviridae (contractile tails). The linear double-stranded DNA genome was sequenced and found to be 77 670 bp in length, with a mol% G+C of 55.8. A total of 121 likely open reading frames (ORFs) were identified, of which 37 overlapped at start and stop codons. The predicted proteins were usually acidic (average pI of 4.8), and less than about 12% of them had homologues in the sequence databases. Four complete tRNA-like sequences (tRNA-Arg, -Asx, -Pro and -Tyr) and an incomplete tRNA-Thr were detected. A transcription map showed that most of the genome was transcribed and that the synthesis of transcripts occurred in a highly organized and reproducible pattern over a 5 h infection cycle. Transcripts often spanned multiple ORFs, suggesting that viral genes were organized into operons. The predicted ORF and observed transcript directions matched well and showed that transcription is mainly directed inwards from the genome termini, meeting at about 45-48 kb, and this was also a turning point in a cumulative GC-skew plot. The low point in cumulative GC-skew, near the left end, was a region rich in short repeats and lacking ORFs, which is likely to be an origin of replication. The HF2 genome is a mosaic of components from widely different sources, demonstrating clearly that viruses of haloarchaea, like their bacteriophage counterparts, are vectors for the exchange and transmission of genetic material between wide taxonomic distances, even across domains.

Archaea↗
PubMed1985

Infection of cultured human muscle cells by influenza virus.

In a search for myotropic viruses with a potential to initiate muscle autoimmunity, we found that two strains of influenza A virus, A/England/863/78 (H3N2) and the reassortant virus X-47 (H3N2), could infect human syncytial myotubes lytically. The X-47 strain could, in addition, infect unicellular precursor myoblasts. Intracellular viral protein synthesis was demonstrated by pulse-labelling studies in both cell types with both virus strains. By immunofluorescence and immunoelectron microscopy, viral antigens were demonstrated in infected muscle cells specifically identified by double staining with monoclonal antibodies to either of two independent muscle-specific antigens. However, using 'co-capping' techniques in conjunction with electron microscopy, there was no evidence of association between viral haemagglutinin and the acetylcholine receptor (one major target of autoimmunity to muscle cells) on the infected cell membrane.

Antigens, Viral↗
PubMed2006

KSHV/HHV-8 infection of human hematopoietic progenitor (CD34+) cells: persistence of infection during hematopoiesis in vitro and in vivo.

The cellular reservoir for Kaposi sarcoma-associated herpesvirus (KSHV) infection in the hematopoietic compartment and mechanisms governing latent infection and reactivation remain undefined. To determine susceptibility of human CD34+ hematopoietic progenitor cells (HPCs) to infection with KSHV, purified HPCs were exposed to KSHV, and cells were differentiated in vitro and in vivo. Clonogenic colony-forming activity was significantly suppressed in KSHV-infected CD34+ cells, and viral DNA was predominantly localized to granulocyte-macrophage colonies differentiated in vitro. rKSHV.219 is a recombinant KSHV construct that expresses green fluorescent protein from a cellular promoter active during latency and red fluorescent protein from a viral lytic promoter. Infection of CD34+ HPCs with rKSHV.219 showed similar patterns of infection, persistence, and hematopoietic suppression in vitro in comparison with KSHV. rKSHV.219 infection was detected in human CD14+ and CD19+ cells recovered from NOD/SCID mouse bone marrow and spleen following reconstitution with rKSHV.219-infected CD34+ HPCs. These results suggest that rKSHV.219 establishes persistent infection in NOD/SCID mice and that virus may be disseminated following differentiation of infected HPCs into the B-cell and monocyte lineages. CD34+ HPCs may be a reservoir for KSHV infection and may provide a continuous source of virally infected cells in vivo.

Animals↗
PubMed2001

Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells.

Exploiting the lytic life cycle of viruses has gained recent attention as an anticancer strategy (oncolysis). To explore the utility of adenovirus (Ad)-mediated oncolysis for rhabdomyosarcoma (RMS), we tested RMS cell lines for Ad gene transduction and infection. RMS cells were variably transduced by Ad. Compared with control cells, RMS cells were less sensitive or even resistant to oncolysis by wild-type virus. RMS cells expressed the Ad internalization receptors, alpha(v) integrins, but had low or undetectable expression of the major attachment receptor, coxsackievirus-Ad receptor (CAR). Mutant Ads with ablated CAR binding exhibited only 5-20% of transgene expression in RMS cells seen with a wild-type vector, suggesting that residual or heterogeneous CAR expression mediated the little transduction that was detectable. Immunohistochemical analysis of archived clinical specimens showed little detectable CAR expression in five embryonal and eight alveolar RMS tumors. Stable transduction of the cDNA for CAR enabled both efficient Ad gene transfer and oncolysis for otherwise resistant RMS cells, suggesting that poor CAR expression is the limiting feature. Gene transfer to RMS cells was increased >2 logs using Ads engineered with modified fiber knobs containing either an integrin-binding RGD peptide or a polylysine peptide in the exposed HI loop. The RGD modification enabled increased oncolysis for RMS cells by a conditionally replicative Ad, Ad delta24RGD, harboring a retinoblastoma-binding mutation in the E1A gene. Thus, the development of replication-competent vectors targeted to cell surface receptors other than CAR is critical to advance the use of Ad for treating RMS.

Adenoviridae↗