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PubMed2001

Oncolytic virotherapy for cancer with the adenovirus dl1520 (Onyx-015): results of phase I and II trials.

Replication-selective viral agents hold promise as a novel cancer treatment platform (virotherapy). dl1520 (Onyx-015, now CI-1042, Pfizer Corp., Groton, CT, USA), an E1B-55kD gene-deleted adenovirus, was the first such genetically engineered agent to be tested in humans. Over 250 cancer patients have now been treated on approximately ten clinical trials (phase I-III). The virus was generally well tolerated at doses of up to 2 x 10(12) particles by intratumoural, intraperitoneal, hepatic arterial and i.v. administration; no maximally-tolerated doses were identified following intra-vascular administration. Viral replication was tumour-selective and was documented after administration by all routes; however, viral replication was variable depending on tumour histology. Single agent efficacy has been relatively limited to date (0-14% local tumour regression rates). In combination with chemotherapy, however, encouraging antitumoural activity has been demonstrated. These clinical research results demonstrate the potential of this novel treatment platform, as well as the hurdles to be overcome. Novel replication-selective agents with improved potency are being developed.

Adenoviridae↗

PubMed1988

Studies on the role of the phi X174 gene A protein in phi X174 viral strand synthesis. III. Replication of DNA containing two viral replication origins.

Supercoiled plasmid bearing two wild-type phi X origin sequences on the same strand supported the phi X A protein-dependent in vitro formation of two smaller single-stranded circles, the lengths of which were equivalent to the distance between the two origins. Additional double origin plasmids were utilized to determine whether origins defective in the initial nicking event (initiation) could support circularization (termination). In all cases tested, the presence of a mutant origin on the same strand with a wild-type origin affected the level of replication in a manner consistent with the previously determined activity of the mutant origin. When a functional mutant origin was present on the same strand as a wild-type origin, the efficiency of replication and the DNA products formed were almost identical to those of the plasmid containing two wild-type origins. Plasmid DNA bearing both a wild-type origin and a mutant origin that did not support phi X A protein binding or nicking activity, on the other hand, supported efficient DNA synthesis of only full-length circular products, indicating that the origin defective for initiation was incapable of supporting termination. In contrast, the presence of a wild-type origin and an origin that did bind the phi X A protein but was not cleaved resulted in a marked decrease in DNA synthesis along with the production of only full-length products. This suggests that the phi X A protein stalls when it encounters a sequence to which it can bind but cannot cleave. Replication of double origin plasmids containing one functional phi X origin on each strand of the supercoiled DNA was also examined. With such templates, synthesis from the wild-type origin predominated, indicating preferential cleavage of the intact origin sequence. Replication of such substrates also produced a number of aberrant structures, the properties of which suggested that interstrand exchange of the phi X A protein had occurred.

Bacteriophage phi X 174↗

PubMed1992

Characterization of the mammary hyperplasia, dysplasia and neoplasia induced in athymic female adult mice by polyomavirus.

We have characterized mammary oncogenesis induced after polyomavirus infection of adult female nude mice regarding histopathogenesis, viral replication and viral and cellular oncogene expression. A unique transient generalized epithelial hyperplasia was observed (starting at 2 weeks post infection), preceding the development of dysplasias (onset 6 weeks post infection) and multiple neoplasias (onset 6 weeks post infection) in all glands. The ductal epithelium was the target for neoplastic transformation, and the occurrence of numerous ductal dysplasias coincided with the appearance of frank tumors. Stromal abnormalities were also seen. Tumor growth was not dependent upon ovarian hormones, and new tumors continued to develop in ovariectomized mice. Viral replication, high although variable, preceded but did not correlate with oncogenesis. Most but not all tumors contained high levels of unintegrated viral DNA. Tumors produced very low levels of live virus. Viral gene expression was markedly increased in the tumors compared with the infected but morphologically normal glands. The expression of c-myc was moderately increased (fourfold); changes in c-int-2 and c-Ha-ras expression were slight and inconsistent, while expression of c-neu and c-int-1 was unchanged.

Animals↗

PubMed1995

Isolation of caliciviruses from skunks that are antigenically and genotypically related to San Miguel sea lion virus.

Caliciviruses were isolated from feces of skunks imported from the north central United States to Canada. Virus isolation was accomplished using adenovirus-transformed human kidney (293) cells, swine testes and Vero cells. Plaque size variants were presented, but there was no apparent difference in virus morphology by negative stain or immune electron microscopy. Pigs infected with skunk calicivirus had a slightly elevated body temperature at 3 days postinfection. Although the infected animals seroconverted, no overt clinical signs were observed. Purified infectious genomic skunk calicivirus RNA behaved exactly as San Miguel sea lion virus (SMSV) 1 and 4 genomic RNA in cell culture transfection studies. Of the cell types examined, only primary porcine kidney, 293 and Vero cells supported viral replication. No viral replication was detected in cells of bovine, equine, ovine, caprine or feline origin. The skunk caliciviruses contained a single capsid protein with a relative mobility similar to SMSV virus 1 and 4 capsid proteins. The capsid protein was positive by Western blot analysis with SMSV and vesicular exanthema of swine virus (VESV) antisera. Purified RNA from skunk calicivirus infected cells was subjected to reverse transcription followed by polymerase chain reaction. Nucleotide sequences were identified that had greater than 85% similarity to the 2C and RNA polymerase gene regions of SMSV 1 and 4 and VESV A48. Predicted amino acid sequences of these regions were greater than 95% similar and the partial coding sequence of the polymerase gene contained the YGDD sequence common to positive-strand RNA virus polymerases.

Amino Acid Sequence↗

PubMed1984

Identification of intranuclear structures containing the 72K DNA-binding protein of human adenovirus type 5.

Immunocytochemical techniques have been applied to cells infected with human adenovirus type 5 in order to study the distribution of antigens related to the virus-encoded Mr = 72000 (72K) DNA-binding protein. In embedded cells 72K antigen was found to be associated with 'clear' fibrillar inclusions, in which viral DNA replication is known to take place. "Dense" fibrillar inclusions, which are thought to contain previously replicated viral DNA, do not contain detectable levels of 72K antigen, suggesting that the conversion of clear inclusions to dense inclusions is accompanied by loss of the 72K protein. In cells examined by the spreading technique of Miller, 72K antigen is associated with the 10 nm "thick filaments" previously described by several laboratories, consistent with the established role of the 72K protein in viral replication complexes.

Adenovirus E2 Proteins↗

PubMed1984

The intranuclear location of a herpes simplex virus DNA-binding protein is determined by the status of viral DNA replication.

The herpes simplex viral DNA-binding protein, ICP8, is targeted to two different locations in the cell nucleus as part of its maturation pathway. Prior to viral DNA synthesis ICP8 was found at discrete pre-replicative sites throughout the nucleus, where it exhibited a high salt-labile association with the nuclear matrix. During viral DNA replication ICP8 was localized in randomly distributed replication compartments, where it is bound to viral DNA. Initiation of viral DNA replication caused the protein to move from the prereplicative sites to the replication compartments, while inhibition of replication caused movement in the opposite direction. In cells where viral DNA synthesis was proceeding, a minor population of ICP8 may also have been associated with the prereplicative sites. The prereplicative sites may serve as a nuclear reservoir for ICP8 not bound to replicating or progeny DNA.

Animals↗

PubMed1993

Altered properties of the herpes simplex virus ICP8 DNA-binding protein in cells infected with ICP27 mutant viruses.

Herpes simplex virus mutant strains that fail to encode a functional ICP27 polypeptide show a reduction in their ability to replicate viral DNA compared to wild-type virus. To define the nature of this defect, we examined the formation of viral DNA replication structures in the nucleus of cells infected with these mutant viruses. In cells infected with wild type HSV, viral DNA replication occurs in replication compartments. If viral DNA replication is blocked during infection, a subset of these proteins are found in a punctate staining pattern termed prereplicative sites. ICP8, an essential DNA replication protein, is required for formation of these structures and for localization of polymerase and other proteins to these structures. To study the assembly of replication proteins in cells infected with ICP27 defective mutants, we examined the intranuclear localization of ICP8. Our results suggest that assembly of replication proteins into active sites is defective in a majority of cells infected with mutant virus encoding altered ICP27 proteins. Furthermore, this defect in assembly occurs at an early step in localization of replication proteins, possibly due to an observed change in the conformation of ICP8. Last, only actively dividing cells at a certain stage of the cell cycle are able to form replication compartments when infected with ICP27 defective mutants. The residual DNA synthesis seen with these mutants occurs in a defined subpopulation of infected cells. Thus, ICP27 may promote the proper folding and/or localization of ICP8.

Animals↗

PubMed2000

Functional and structural defects in HIV type 1 nef genes derived from pediatric long-term survivors.

DNA sequences and three distinct in vitro functions of Nef were evaluated in a group of seven perinatally infected children. nef gene sequences obtained before and after virus culture showed that one of the five non-/slow progressors harbored a virus with large deletions. nef genes from the remaining four children were full length but contained discrete changes at a higher frequency than the rapid progressors. In functional studies, 40 of 44 Nef proteins derived from the whole study group were capable of binding the cellular serine kinase p62, indicating that this function is well conserved among naturally occurring viruses. In contrast, representative Nef proteins derived from the long-term non-/slow progressors were found to be defective or far less capable of enhancing viral replication and/or viral infectivity in herpesvirus saimiri-transformed human T cells and peripheral blood mononuclear cells. On reversion of highly prevalent point mutations in the defective proteins, viral replication could be restored to wild-type levels. Our results suggest that nef genes derived from pediatric long-term nonprogressors have gross deletions in isolated cases but a higher prevalence of discrete changes that may impair Nef function in primary T cell assays, but not all functions reported for Nef.

Adolescent↗

PubMed1993

Hepatitis delta virus cDNA monomer can be used in transfection experiments to initiate viral RNA replication.

The complementary DNA (cDNA) of hepatitis delta virus (HDV), constructed as tandem repeats under appropriate exogenous promoters, has been used to initiate viral replication in transfection experiments. Whether the structure of tandem repeats is essential has not yet been systematically examined. In this study, expression vectors containing only an HDV cDNA monomer, permutated at unique position of the genome, were shown still able to initiate viral replication. Furthermore, HDV cDNA monomer, separated from plasmid sequences by restriction enzyme digestion, also could be used to initiate viral RNA replication. The competence of HDV cDNA alone to direct viral RNA production suggested the presence of cryptic internal promoter-like elements. Such elements actually demonstrated by the chloramphenicol acetyltransferase assay. Therefore, HDV cDNA, in contrast to that of viroids, could be used to initiate viral RNA replication in monomeric form. This observation simplified the use of HDV cDNA for studying viral biology in transfection systems.

Cell Line↗

PubMed2006

Nucleocytoplasmic shuttling of bovine papillomavirus E1 helicase downregulates viral DNA replication in S phase.

The papillomavirus E1 protein is essential for the initiation of viral replication. We previously showed that the bovine papillomavirus E1 protein is unstable and becomes resistant to ubiquitin-mediated degradation when tightly bound to cyclin E-cyclin-dependent kinase 2 (Cdk2) before the start of DNA synthesis. However, neither the protection nor the targeted degradation of E1 appears to depend on its phosphorylation by Cdk. Here, we report that Cdk phosphorylation of E1 is also not a prerequisite for the initiation of viral DNA replication either in vitro or in vivo. Nevertheless, we found that phosphorylation of one Cdk site, Ser283, abrogates E1 replicative activity only in a cellular context. We show that this site-specific phosphorylation of E1 drives its export from the nucleus and promotes its continuous nucleocytoplasmic shuttling. In addition, we find that E1 shuttling occurs in S phase, when cyclin A-Cdk2 is activated. E1 interacts with the active cyclin A-Cdk2 complex and is phosphorylated on Ser283 by this kinase. These data suggest that the phosphorylation of E1 on Ser283 is a negative regulatory event that is involved in preventing the amplification of viral DNA during S phase. This finding reveals a novel facet of E1 regulation that could account for the variations of the viral replication capacity during different cell cycle phases, as well as in different stages of the viral cycle.

Active Transport, Cell Nucleus↗

PubMed2005

Interaction between coat protein and replication initiation protein of Mung bean yellow mosaic India virus might lead to control of viral DNA replication.

In addition to their encapsidation function, viral coat proteins (CP) contribute to viral life cycle in many different ways. The CPs of the geminiviruses are responsible for intra- as well as inter-plant virus transmission and might determine the yield of viral DNA inside the infected tissues by either packaging the viral DNA or interfering with the viral replicative machinery. Since the cognate Rep largely controls the rolling circle replication of geminiviral DNA, the interaction between Rep and CP might be worthwhile to examine for elucidation of CP-mediated control of the viral DNA copy number. Here a reasonably strong interaction between Rep and CP of the geminivirus Mung bean yellow mosaic India virus is reported. The domain of interaction has been mapped to a central region of Rep. The replication initiation activity of Rep, i.e., its nicking and closing function, is down regulated by CP. This report highlights how CP could be important in controlling geminiviral DNA replication.

Capsid Proteins↗

PubMed1984

Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication.

We have examined the activation of the SV40 late promoter by inserting the late promoter and the viral origin of replication into chloramphenicol acetyltransferase (CAT) transient expression vectors. Very little late promoter activity was detected in CV-1 cells, compared with high activity in COS cells, in which replication occurs due to endogenous T antigen. Nonreplicative counterparts of these plasmids, containing a mutated origin of replication, produced significantly more late promoter activity in COS cells than any of the plasmids in CV-1 cells. When plasmids were cotransfected into CV-1 cells with a plasmid that supplies T antigen, the nonreplicative plasmid displayed 30% of the activity of the replicative plasmid. Using mutant T antigens unable to replicate viral DNA, late promoter activation occurred only with mutant T antigens that retain DNA binding activity. These results demonstrate that T antigen can substantially stimulate late promoter activity directly and independent of viral DNA replication.

Acetyltransferases↗

PubMed1976

Multiplication of parvovirus LuIII in a synchronized culture system. III. Replication of viral DNA.

The replication of the single-stranded DNA (ssDNA) of parvovirus LuIII was studied in synchronized HeLa cells. After infection of the cells in early S phase, synthesis of a replicative form (RF) DNA became detectable as early as 9 h postinfection, i.e., after display of the cellular helper function(s) indispensable for the replication of LuIII virus. According to digestion with nuclease S1, hybridization studies, and electron microscopy, RF DNA is a linear, double-stranded molecule comparable in length to mature ssDNA. It sedimented around 15S in neutral solution and banded at 1.714 g/ml in CsCl. Moreover, replication of LuIII DNA obviously includes a further replicative intermediate DNA which sedimented in front of RF DNA and bore single-stranded side-chains. Newly synthesized DNA disappeared from pools containing both RF DNA and replicative intermediate DNA within 5 min and reappeared in progeny virions only after 15 min. Intranuclear accumulation of significant amounts of progeny ssDNA could not be detected. It was postulated, therefore, that newly synthesized ssDNA is immediately enclosed in a stable maturation complex and resists extraction by the method of Hirt (1967).

Cell Division↗

PubMed1993

Binding sites for the herpes simplex virus immediate-early protein ICP4 impose an increased dependence on viral DNA replication on simple model promoters located in the viral genome.

We examined the ability of binding sites for the herpes simplex virus immediate-early protein ICP4 to alter the regulation of closely linked promoters by placing strong ICP4 binding sites upstream or downstream of simple TATA promoters in the intact viral genome. We found that binding sites strongly reduced the levels of expression at early times postinfection and that this effect was partially overcome after the onset of viral DNA replication. These data confirm that DNA-bound ICP4 can inhibit the activity of a closely linked promoter and raise the possibility that ICP4 binding sites contribute to temporal regulation during infection.

Animals↗

PubMed1989

A promoter of Epstein-Barr virus that can function during latent infection can be transactivated by EBNA-1, a viral protein required for viral DNA replication during latent infection.

A viral promoter that functions on recombinant plasmids in cells immortalized by Epstein-Barr virus was identified and characterized. It is identical to that mapped on the viral genome by Bodescot et al. (M. Bodescot, M. Perricaudet, and P.J. Farrell, J. Virol. 61:3424-3430, 1987) which functions during the latent phase of the viral life cycle in some but not all cells to encode several latent viral gene products. Experiments with these plasmids indicated that this promoter requires the enhancer within oriP of Epstein-Barr virus in cis to function efficiently. They also indicated that it requires the EBNA-1 gene in trans to function efficiently. The EBNA-1 gene therefore positively affects both viral DNA replication (J.L. Yates, N. Warren, and B. Sugden, Nature [London] 313:812-815, 1985) and viral transcription.

Antigens, Viral↗

PubMedDate not supplied

[Diversity and unity of methods of replication of viral DNA].

A systematization of the modes of viral DNA genomes replication was attempted. The key problem of replication, the complete copying of the genome, has several fundamentally different ("strategic") solutions, which utilize different mechanisms of initiation of a round of replication. Noticeable diversity may exist also within a given strategy, since consecutive replication steps may be accomplished by several (but not many) distinct means. There appears to exist a significant similarity between the viral and cellular replication systems. The approach used permits an assessment of the actual diversity in DNA replication systems.

Base Sequence↗

PubMed2005

Effect of hypoxia on Ad5 infection, transgene expression and replication.

Oxygen deprivation (hypoxia) is a common feature of various human maladies, including cardiovascular diseases and cancer; however, the effect of hypoxia on Ad-based gene therapies has not been described. In this study, we evaluated how hypoxia (1% pO(2)) affects different aspects of Ad-based therapies, including attachment and uptake, transgene expression, and replication, in a series of cancer cell lines and primary normal cells. We found that hypoxia had no significant effect on the expression or function of the Ad5 attachment (Coxsackievirus and Adenovirus Receptor) and internalization (alpha(v) integrins) proteins, nor on the human cytomegalovirus-driven expression of an exogenous gene carried by a replication-incompetent Ad. Viral replication, however, was compromised by hypoxic conditions. Our studies revealed hypoxia-induced reductions in E1A levels that were mediated at the post-transcriptional level. E1A drives cells into the viral replication optimal S phase of the cell cycle; consequently, the combination of reduced E1A protein and hypoxia-induced G1 arrest of cells may be responsible for the lack of efficient viral replication under hypoxic conditions. Consequently, while traditional replication-incompetent Ad-based vectors appear to be viable delivery systems for hypoxia-associated disease indications, our studies suggest that Oncolytic Ads may need additional factors to efficiently treat hypoxic regions of human tumors.

Adenovirus E1A Proteins↗

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↗