Parvovirus variation for disease: a difference with RNA viruses?

The Parvoviridae, a family of viruses with single-stranded DNA genomes widely spread from invertebrates to mammal and human hosts, display a remarkable evolutionary capacity uncommon in DNA genomes. Parvovirus populations show high genetic heterogeneity and large population sizes resembling the quasispecies found in RNA viruses. These viruses multiply in proliferating cells, causing acute, persistent or latent infections relying in the immunocompetence and developmental stage of the hosts. Some parvovirus populations in natural settings, such as carnivore autonomous parvoviruses or primate adeno associated virus, show a high degree of genetic heterogeneity. However, other parvoviruses such as the pathogenic B19 human erythrovirus or the porcine parvovirus, show little genetic variation, indicating different virus-host relationships. The Parvoviridae evolutionary potential in mammal infections has been modeled in the experimental system formed by the immunodeficient scid mouse infected by the minute virus of mice (MVM) under distinct immune and adaptive pressures. The sequence of viral genomes (close to 10(5) nucleotides) in emerging MVM pathogenic populations present in the organs of 26 mice showed consensus sequences not representing the complex distribution of viral clones and a high genetic heterogeneity (average mutation frequency 8.3 x 10(-4) substitutions/nt accumulated over 2-3 months). Specific amino acid changes, selected at a rate up to 1% in the capsid and in the NS2 nonstructural protein, endowed these viruses with new tropism and increased fitness. Further molecular analysis supported the notion that, in addition to immune pressures, the affinity of molecular interactions with cellular targets, as the Crml nuclear export receptor or the primary capsid receptor, as well as the adaptation to tissues enriched in proliferating cells, are major selective factors in the rapid parvovirus evolutionary dynamics.

Prevention of cervical cancer with vaccines.

Worldwide, cervical cancer is one of the most common cancers in women. This is especially true in developing countries, where Papanicolaou smear screening, an effective preventive measure against cervical cancer, is insufficiently implemented. With growing evidence for human papillomavirus as a central etiologic factor in cervical neoplasia, development of a vaccine against this virus has emerged as an important objective in prevention of cervical cancer. International efforts in vaccine development have culminated in advancement of various vaccine strategies and initiation of human clinical trials. Reports from animal vaccine trials and early phase I human trials indicate markedly enhanced immune response through vaccination. However, the clinical significance of these results requires confirmation from long-term human trials.

Natural biology of polyomavirus middle T antigen.

"It has been commented by someone that 'polyoma' is an adjective composed of a prefix and suffix, with no root between--a meatless linguistic sandwich" (C. J. Dawe). The very name "polyomavirus" is a vague mantel: a name given before our understanding of these viral agents was clear but implying a clear tumor life-style, as noted by the late C. J. Dawe. However, polyomavirus are not by nature tumor-inducing agents. Since it is the purpose of this review to consider the natural function of middle T antigen (MT), encoded by one of the seemingly crucial transforming genes of polyomavirus, we will reconsider and redefine the virus and its MT gene in the context of its natural biology and function. This review was motivated by our recent in vivo analysis of MT function. Using intranasal inoculation of adult SCID mice, we have shown that polyomavirus can replicate with an MT lacking all functions associated with transformation to similar levels to wild-type virus. These observations, along with an almost indistinguishable replication of all MT mutants with respect to wild-type viruses in adult competent mice, illustrate that MT can have a play subtle role in acute replication and persistence. The most notable effect of MT mutants was in infections of newborns, indicating that polyomavirus may be highly adapted to replication in newborn lungs. It is from this context that our current understanding of this well-studied virus and gene is presented.

Parvovirus-mediated antineoplastic activity exploits genome instability.

The generation and accumulation of genetic mutations have been associated with carcinogenesis. Epidemiological and experimental evidence implicate parvoviruses in growth inhibition (oncosuppression) and selective destruction (oncolysis) of tumor cells. It is proposed here that parvoviruses can preferentially target genetically unstable tumor cells, which are deficient in DNA repair mechanisms. This selective strategy may serve as a virus-based therapeutic approach against cancer.

T-Ag inhibits implantation by EC cell derived embryoid bodies.

When introduced into EC cells of a blastocyst, polyomavirus (Py) T-Ag results in mice mosaic for T-Ag but otherwise essentially normal. It had been reported that SV40 T-Ag does not inhibit differentiation of F9 EC cells, but did inhibit endogenous retrovirus (ERV) production. We therefore sought to determine if Py T-Ag had any affect on EC derived embryoid body implantation onto mouse placenta. F9 EC cells were selected for T-Ag maintenance. Like the SV40 transformed cells, we show that these Py T-Ag selected EC cells no longer express IAP transcripts following differentiation into embryoid bodies. Normal and Py T-Ag selected F9 cells were differentiated into embryoid bodies then implanted into pseudopregnant mice. We observe, that normal F9 derived embryoid bodies underwent the initial stages of implantation whereas the Py T-Ag selected embryoid bodied did not implant. The implications of this observation with respect to trophectoderm and ERV function are discussed. We examine the idea that ERVs may be a required element for normal embryo implantation.

n-Butyrate mediated inhibition of papovavirus DNA replication in vivo and in cell culture: a mechanistic approach.

n-Butyrate, an inhibitor of G1-to-S transition inhibits papovavirus DNA replication in cell culture. To explore the efficacy of n-butyrate in vivo and to better understand its mechanism, we studied the effect of n-butyrate on viral DNA replication in mice acutely infected with polyomavirus and in the papovavirus-infected cells in culture. Newborn mice treated with n-butyrate stop growing and become runted. When infected with polyomavirus, these mice show a strong overall inhibition of viral DNA. However, a notable exception to this was the continued viral DNA replication in the differentiated mouse keratinocytes and renal epithelial cells as determined by in situ hybridization. n-Butyrate significantly inhibited viral DNA replication in the cultured IDL cells, and in polyomavirus-infected C2C12 myoblasts based on Southern blot analysis and in situ hybridization. DNA polymerase alpha (but not DNA polymerase beta) and the characteristic nuclear expression of PCNA were both inhibited in the n-butyrate treated IDL and C2C12 cells. n-Butyrate, therefore, inhibited host and viral DNA synthesis in the undifferentiated cells.

A hypothesis for DNA viruses as the origin of eukaryotic replication proteins.

The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction.

The use of oligonucleotide directed cleavage of DNA and homologous recombination in the production of large recombinant adenoviral vectors.

The genetic manipulation of large plasmid DNA often requires the fortuitous presence of convenient restriction enzyme sites. For large plasmids, such as those containing full length recombinant adenovirus, it is desirable to direct the cloning or sequence alterations without having to depend on such convenient restriction sites. We report a general and efficient method to modify or clone large covalently closed circular DNA molecules at any predetermined sequence. This procedure involves two main steps. First, supercoiled DNA is hybridized to a short pre-selected synthetic oligonucleotide to form a D-loop. This hybrid is then linearized in vitro at that target site by digestion with S1 nuclease. Second, D-loop/S1 linearized DNA is transformed into E. coli with a second linear DNA fragment carrying a foreign gene flanked by sequences homologous to the target site. In vivo recombination results in the desired recombinant construct. We demonstrate the use of this method by cloning the SV40 origin of replication into the E3 transcription unit of human adenovirus type 5.

Prediction of gene therapy-induced tumor size changes by the vascularity changes measured using dynamic contrast-enhanced MRI.

We studied the changes of tumor size after gene therapy treatment and its relationship with the changes of vascular volume as measured by dynamic contrast-enhanced magnetic resonance imaging (MRI), to investigate whether the vascular changes is predictive of tumor regression. The study was carried out using a spontaneously regressing rat tumor model (C6 Glioma grown subcutaneously in rats). Three rats were treated with recombinant adenoviruses expressing three genes, mouse interleukin 1-alpha (IL1-alpha), mouse interferon gamma (IFN-gamma), and human transforming growth factor beta (TGF-beta), one from each kind. Two rats were treated with saline as controls. Longitudinal studies were performed to monitor the changes of tumor volume (based on T(2)-weighted images) and the vascular volume (based on dynamic contrast enhanced images). In untreated animals, tumor regression was preceded by several days with a decrease in vascular volume. When the tumor growth was perturbed by expression of mouse IL-1alpha, the increase in vascular volume was correlated with the continuing growth in size, and the decrease in vascular volume was predictive of the onset of tumor regression. As new advances in immunotherapy in cancer treatment emerge, the ability to determine the efficacy of therapy as early as possible will enable optimization of treatment regiments. The vascularity changes measured by dynamic MRI may provide a means to serve for this purpose.

Development and assessment of a general theory of cervical carcinogenesis utilizing a severe combined immunodeficiency murine-human xenograft model.

OBJECTIVE: Currently, we lack a theoretical explanation for why squamous cell cervical cancer develops predominantly in specific sites (i.e., along the squamocolumnar junction). We therefore implanted human cervical tissues containing the transformation zone in severe combined immunodeficiency (SCID) mice and studied morphology, steroid effects, gene expression, and human papillomavirus (HPV) factors. METHODS: Normal and dysplastic human cervical tissues (3 x 2 mm) were placed subcutaneously in SCID-beige mice and later assessed by in situ hybridization for HPV 16/18 DNA and by immunohistochemistry for expression of CD31, keratin, proliferating-cell nuclear antigen, HPV 16 E6, p53, and Notch-1 (a binary cell fate determination protein). Some normal tissues were implanted with either a 90-day release 1.7-mg 17beta-estradiol pellet or a 5-mg tamoxifen pellet; others were infected prior to implantation with human recombinant adenovirus 5 vector containing a human cytomegalovirus promoter-driven beta-galactosidase gene and later assessed by X-gal staining. RESULTS: Murine and human vessels formed anastomoses by 3 weeks. For at least 11 weeks, normal tissue retained the transformation zone and normal cell-type-specific keratin expression and exhibited normal proliferation; Notch-1 was present only in the basal cell layer. Dysplastic tissues exhibited koilocytosis, increased levels of cellular proliferation, and aberrant keratin, p53, and Notch-1 expression; HPV 16/18 DNA and HPV 16 E6 protein were detected for at least 6 weeks. Squamous metaplasia of normal cervical epithelium resulted from estrogen exposure, and a predominant columnar differentiation pattern was associated with tamoxifen administration. Through stable adenovirus infection, beta-galactosidase was expressed for at least 6 weeks. CONCLUSIONS: This small manipulatable xenograft model maintains normal and dysplastic human cervical epithelium through neovascularization. Neoplastic tissue retains HPV 16/18 DNA and a premalignant phenotype, including elevated levels of cellular proliferation and aberrant keratin, p53, and Notch-1 expression. These attributes constitute essential features of a biologic model through which one may study HPV-mediated human disease and may be superior to cell culture and transgenic murine systems. Furthermore, this may serve as a model for gene therapy. Finally, we suggest that the normal cervical epithelium is maintained through putative interactions between the Notch locus and cell cycle growth regulators such as p53 and pRb. Neoplastic cervical epithelium may arise through disruption of this pathway. This theory may be testable in our animal model.

The distribution and kinetics of polyomavirus in lungs of intranasally infected newborn mice.

The primary cell types that sustain polyomavirus (Py) replication following intranasal infection as well as the nature of the host cellular response to Py were unknown. As this is an essential and specific site for virus entry, it seems likely that viral gene function must be adapted to these mucosal tissues. Using immunohistochemistry and in situ hybridization, we determined the cell types in the lung that support Py gene expression and replication following intranasal inoculation of newborn mice within 24 h of birth. Lungs were collected daily from days 1 to 10 postinfection for Py DNA and early T antigen analysis and for histological examination by H&E staining, using methods that preserve the delicate newborn lung architecture. Viral DNA was present in increasing quantities from 2 to 6 dpi in a subset of the Clara cells lining the inner lumen of the bronchi and bronchioles, while T antigen expression was present in a majority of the cells in the bronchi and bronchiole lumen. A distinct and transient pattern of hyperplasia was observed among the cells expressing T antigen and was present from 3 through 6 dpi. Py DNA-containing cells exfoliated into the bronchiole lumen and alveolar ducts, but Py T antigen was not detected in these cells. Py DNA was first detected at 2 dpi, increased through 6 dpi, and abruptly declined through 9 dpi at which time there was no sign of viral DNA in the lungs by in situ hybridization. An unusual infiltration of neutrophils began before the presence of exfoliated cells or Py replication and continued for 2-3 days and was followed by a lymphocytic infiltration at 8-10 dpi lasting 2-3 days. Neither the hyperplasia nor the neutrophil infiltration occurred following infection with the MOP1033 MT-Ag or RB1 LT-Ag mutants of Py. In addition, both the neutrophil infiltration and the transient hyperplasia are in stark contrast to the heavy macrophage infiltration that follows infection of lungs with mouse adenovirus. Thus it appears that Py elicits a distinct host response pattern not seen with other DNA viral infections.

Immunohistochemical analysis, human papillomavirus DNA detection, hormonal manipulation, and exogenous gene expression of normal and dysplastic human cervical epithelium in severe combined immunodeficiency mice.

The cervical squamocolumnar junction of normal and dysplastic human xenografts was maintained in SCID-beige mice. Dysplastic tissue maintained a dysplastic morphology, irregular pattern of keratin expression, elevated levels of cellular proliferation, and human papillomavirus type 16 and/or type 18 DNA. Hyperplastic changes of normal xenografts occurred via high-dose estrogen exposure, and through recombinant adenovirus infection, the introduction and stable expression of an exogenous gene was accomplished.

Approaches to study interactions between small DNA viruses and differentiated tissue.

Polyomavirus (Py) derives its name from the early observation of multiple tumors that develop in newborn mice following inoculation with this family of viruses. In nature, however, tumor development is rare in the virus life cycle, rather a two-phase infection occurs, acute and persistent, resulting in a final latent infection in the kidneys. The acute phase induces an antiviral immune response, although no recognizable inflammation, which can last the lifetime of the mouse, even passing on antibodies to its offspring. The structure, replication, and expression of the Py viral genome in permissive and nonpermissive infections has been studied extensively in various cell culture systems. However, the nature of Py expression, replication, and immunopathogenesis in mice has not been thoroughly researched.

Transduction of hippocampal CA1 by adenovirus in vivo.

Replication-deficient adenoviral recombinants were assessed for in vivo transduction of rat hippocampal CA1 cells. Results show that efficient widespread transduction of CA1 in vivo was rapidly achievable and was sustained for more than 5 weeks. Assessment of electrophysiological properties in acute hippocampal slices showed that synaptic functioning and mechanisms involved in long-term potentiation (LTP) were preserved for minimally 5 weeks postinfection. Hence, adenovirus-mediated gene transfer in vivo promises to be a valuable tool for dissecting molecular mechanisms of synaptic plasticity, such as LTP and long-term depression (LTD).

Potential role of DNA polymerase beta in gene therapy against cancer: a case for colorectal cancer.

Genetic instability characterized by the accumulation of mutations of tumor suppressor genes and oncogenes appears to be associated with carcinogenesis in colorectal and other cancers. Mutations of DNA polymerase beta (pol beta) and related chromosomal alterations appear to be consistent with the causal role of a "mutator phenotype' in carcinogenesis. However, homozygous knockout pol beta mutations appear to interfere with embryogenesis. Increased pol beta activity (i.e. relative to pol alpha activity) has been associated with cell cycle arrest. The related aphidicolin-resistant DNA replication has been observed primarily in differentiating cells, including the mammalian blastocyst, adrenal cortex, thyroid, anterior pituitary, and the mechanism of endoreduplication (amitotic over-replication of DNA) can be traced to lower eukaryotes. This increased activity in relation to terminal commitment is inconsistent with a simple "DNA repair' view of pol beta. It is therefore proposed that pol beta may play a more fundamental role in cellular differentiation through involvement in a putative subgenomic DNA replication-based model of terminal gene expression. Thus genetic instability, loss of differentiation, and carcinogenesis may result from aberration(s) or "derailment' of such replication-based mechanism of terminal gene expression. It is suggested to examine the relationship of DNA pol beta to genomic instability and carcinogenesis using genetic analyses and antisense technology with possible applications for gene therapy against colorectal cancer.

Mapping of polyomavirus DNA replicative intermediates by two-dimensional gel analysis using chemiluminescent detection.

Py DNA replicative intermediates (RIs) were mapped using a neutral/neutral two-dimensional (N/N 2D) technique by both chemiluminescent and radioactive detection. The nonradioactive method provides similar sensitivity to radioactive methods while possessing the advantages of stability of labeled probes, faster processing, multiple exposures, easy disposal and safety associated with nonradioactive detection. Application of this method to 2D gel analysis requires some technical modification to reduce background and save the substrate. The patterns of Py RIs with Afl II digestion suggest that bidirectional replication occurs during Py DNA synthesis in cell culture, but other unexpected structures of DNA replication are also seen.

A model for mixed virus disease: co-infection with Moloney murine leukemia virus potentiates runting induced by polyomavirus (A2 strain) in Balb/c and NIH Swiss mice.

Polyomavirus was originally isolated by Ludwick Gross from a mixture that also contained a murine retrovirus. A possible pathogenic interaction between polyomavirus and an endogenous mouse retrovirus locus (mtv-7) in polyomavirus-induced cancer has also been reported. To study potential interactive effects of polyomavirus (Py) and Moloney murine leukemia retrovirus (M-MuLV), newborn Balb/c and NIH Swiss mice were infected with high titer wild-type Py (A2 strain) and M-MuLV. Dramatically stunted growth (runting) occurred in 100% of the doubly inoculated mice, while much lower frequency of runting occurred in animals infected with Py alone and not at all with M-MuLV-infected mice. In situ hybridization for Py DNA showed ongoing Py replication and inflammation in kidneys (atypical of most mice singly infected by Py) of runted doubly inoculated mice. In addition, high Py viral replication continued well past the usual acute stage termination. M-MuLV replication was also initially inhibited in bone marrow by simultaneous Py infection. No M-MuLV replication was seen in singly or doubly infected mouse kidneys. Runting was very rapid, observable within 2 days after co-infection, arguing against an adaptive or antigen-specific immunological mechanism. One possibility was that a cytokine-driven acute response mechanism was involved. Supporting this view, RNAse protection assays for various cytokine RNAs showed that several were specifically elevated in kidneys of doubly infected mice. Three patterns were observed: (1) IL-6 was elevated in doubly infected mice early after infection (7 days), but it declined at later times (19 days); (2) IFN-gamma, IL-1 beta, and IL-10 were elevated at both early and late times; and (3) TNF-alpha, IL-12p40, and possibly TNF-beta were elevated only at late times. While the cytokines in the third category might be indicative of infiltrating inflammatory cells, it seems possible that cytokines in the first or second categories might be involved in establishing runting and ongoing polyoma DNA replication in the doubly infected mice.

The evolution of small DNA viruses of eukaryotes: past and present considerations.

Historically, viral evolution has often been considered from the perspective of the ability of the virus to maintain viral pathogenic fitness by causing disease. A predator-prey model has been successfully applied to explain genetically variable quasi-species of viruses, such as influenza virus and human immunodeficiency virus (HIV), which evolve much faster rates than the host. In contrast, small DNA viruses (polyomaviruses, papillomaviruses, and parvoviruses) are species specific but are stable genetically, and appear to have co-evolved with their host species. Genetic stability is attributable primarily to the ability to establish and maintain a benign persistent state in vivo and not to the host DNA proofreading mechanisms. The persistent state often involves a cell cycle-regulated episomal state and a tight linkage of DNA amplification mechanisms to cellular differentiation. This linkage requires conserved features among viral regulatory proteins, with characteristic host-interactive domains needed to recruit and utilize host machinery, thus imposing mechanistic constrains on possible evolutionary options. Sequence similarities within these domains are seen amongst all small mammalian DNA viruses and most of the parvo-like viruses, including those that span the entire spectrum of evolution of organisms from E. coli to humans that replicate via a rolling circle-like mechanism among the entire spectrum of organisms throughout evolution from E. coli to humans. To achieve benign inapparent viral persistence, small DNA viruses are proposed to circumvent the host acute phase reaction (characterized by minimal inflammation) by mechanisms that are evolutionarily adapted to the immune system and the related cytokine communication networks. A striking example of this is the relationship of hymenoptera to polydnaviruses, in which the crucial to the recognition of self, development, and maintenance of genetic identity of both the host and virus. These observations in aggregate suggest that viral replicons are not recent "escapies" of host replication, but rather provide relentless pressure in driving the evolution of the host through cospeciation.