Beatriz G. T. Pogo

Biology of poxviruses.

I. Introduction.- A. Scope of the Presentation.- B. Historical Development.- C. Natural Distribution and Classification.- II. The Virus Particles-Elementary Bodies.- A. Isolation and Purification.- B. Structure and Physical Properties.- C. Composition and Physical Properties.- D. Estimating Infectiousness of EB.- E. Virion-Associated Antigens.- F. Virion-Associated Polypeptides.- G. Virion-Associated Enzymatic Activities.- i) Enzymes Related to DNA Transcription and Modifications of the Polyribonucleotide Messengers (mRNA).- ii) Enzymes Related to DNA of the Virion.- iii) Nucleoside Triphosphate Phosphohydrolase.- iv) Kinases.- v) Alkaline Protease.- H. Relationship of Virus Proteins and Functions.- III. Organization and Replication of the Genome.- A. Organization.- B. Replication.- IV. Events During Penetration.- A. Adsorption.- B. Penetration from the Surface.- C. The Phenomenon of Uncoating.- V. Transcription and Translation.- A. Early Transcription.- B. Early vs Late Transcription.- C. Translation.- VI. Assembly and Morphogenesis.- A. The Envelope.- B. Differentiation into Mature Virions.- VII. Dissemination from Host Cells.- A. Involvement of Cell Membranes.- B. Occlusion in Proteinaceous Capsules.- VIII. Pathology and Disease.- A. Events at the Cell Surface.- B. Hyperplastic Response.- C. Metabolic Derangements.- D. Systemic Infections.- E. Infection and the Immune System.- IX. Genetics.- A. Introduction.- B. Spontaneous Genetic Variability.- C. Induced Genetic Variability.- D. Rescue and Reactivation Phenomena.- E. Concerning a Natural Reservoir of Poxviruses: Can Genetic Variability Lead to Reemergence of Smallpox?.- Acknowledgments.- References.

Increasing evidence for a human breast carcinoma virus with geographic differences

An early immunologic study suggesting that a virus similar to the mouse mammary tumor virus (MMTV) was associated highly with breast carcinoma in Tunisian patients, compared with patients in the United States, led the authors to examine different breast carcinoma populations by using more current molecular techniques.

Possibilities of a viral etiology for human breast cancer. A review.

Previous studies related mouse mammary tumor virus (MMTV) to human breast cancer. However, the presence of human endogenous retroviruses (HERs) confounded these results. We selected a 660-bp sequence of the MMTVenv gene with low homology to HER (or any other known gene) and searched for a sequence homologous to it, using the polymerase chain reaction (PCR). The 660-bp sequence was detected in 131 (39%) of 335 unselected breast cancers, in 2 (6.9%) of 29 fibroadenomas, and in 2 (1.65%) of 121 normal breast specimens. The sequence was not present in normal tissues, or in other human cancers or cell lines.Cloning and sequencing of the 660-bp sequence revealed that it is 95–98% homologous to MMTVenv gene, but not the known HERs or other viral or human gene. Southern blot hybridization using labeled cloned sequences demonstrated that the 660-bp sequence was present in very low copy number as a 6–8 kbEcoRI fragment only in breast cancer samples and in some of the human breast cancer cell lines that were positive by PCR. Preliminary experiments using reverse transcriptase (RT)-PCR indicated that expression of the 660-bp sequence can be detected in 65% of the positive tumors. We were also able to identify in breast cancer DNA a segment of 1.6 kb comprising LTR andenv gene sequences, which are homologous to MMTV, but not to the HERs. The origin of the MMTV-like sequences in tumor DNA could be the result of integrated MMTV-like sequences derived from a human mammary virus.

MMTV-like env gene sequences in human breast cancer.

Summary. We have previously detected an MMTV env gene-like 660 bp sequence in 38% of human breast cancers, but not in normal tissues or other tumors. In this communication we report the sequences from eleven tumors and three breast cancer cell lines, and compare them to four strains of MMTV and to the known endogenous retroviral sequences. The breast cancer sequences were highly homogenous to the MMTV’s, but not to the endogenous sequences suggesting an exogenous origin.

High prevalence of MMTV-like env gene sequences in gestational breast cancer.

Gestational breast cancer (BC) is generally associated with rapid growth and increased mortality. Because the presence of MMTV-like sequences in BC has been associated with laminin receptor expression, a marker of poor prognosis, gestational BCs were analyzed for MMTV env gene-like sequences to explore whether MMTV-like sequences were also associated with its adverse outcome. Whereas 30–38% of sporadic BC have the sequences, in gestational BC the prevalence is 62%. We suggest that hormonal response elements present in the MMTV-like LTR may play a role in promoting cell growth, as they do in the mouse system.

Human mammary tumor virus in inflammatory breast cancer.

The authors have found that retroviral sequences with 85% to 95% homology to the mouse mammary tumor virus were present in 40% of the sporadic breast cancers of American women. These sequences were not found in normal breasts or other tumors. A whole proviral structure was detected in 2 tumors. Breast cancer cells in culture were shown to contain and shed betaretroviral particles. This virus was designated human mammary tumor virus (HMTV). The authors have investigated the presence of HMTV sequences in a variety of breast conditions and geographic locations. Here they report that inflammatory breast cancer from American women shows a higher incidence of viral sequences (71%) than sporadic breast cancers. Similar incidence has been found in inflammatory breast cancers from Tunisia, and in gestational breast cancers. Because these conditions represent highly invasive malignancies, it is concluded that HMTV is sometimes associated with a particularly malignant phenotype. Cancer 2010;116(11 suppl):2741–4.

Detection of human mammary tumor virus proteins in human breast cancer cells.

Mouse mammary tumor virus (MMTV) has been proven to induce mammary cancer in mice. MMTV-like env gene sequences have been detected in one-third of the human breast tumors studied. The whole proviral structure with 95% homology to MMTV was found in two human breast tumors and was designated as human mammary tumor virus (HMTV). HMTV viral particles with betaretroviral features have been isolated. In addition, a retrovirus called human betaretrovirus (HBRV), homologous to the mentioned retroviruses, has been isolated from tissues of patients with primary biliary cirrhosis. In this report, the expression of HMTV envelope (Env) and capsid (Ca) was detected in 10 primary cultures of human breast cancer containing HMTV sequences (MSSM) by Western blot and fluorescence activated cell sorting (FACS), using a panel of antibodies against HMTV Env, HBRV Env and Ca and the MMTV Env Gp36 and Ca P27 proteins. By contrast, HMTV proteins did not react with antibody against the MMTV Env Gp52 protein. All the antibodies detected MMTV proteins with exception of two out of four monoclonal antibodies against HMTV Env. Approximately 13% of the MSSM cells showed HMTV protein expression by FACS analysis. This report shows the expression of HMTV proteins for the first time in human breast cancer cells using a panel of antibodies against HMTV, HBRV and MMTV proteins. This should be taken into consideration when MMTV antibodies are used to detect HMTV proteins in human tissues.

Initiation and termination of vaccinia virus DNA replication

Abstract The initiation and termination sites of replication of vaccinia DNA has been studied by determining the radioactivity in restriction fragments of the pulse-labeled newly synthesized molecules. The results indicate a bimodal distribution of radioactivity in the molecules completed during a pulse shorter than the completion time of synthesis, implying termination at both ends. The symmetry of replication was studied by hybridization of an intermediate in replication (34 S) to the isolated strands of virion DNA followed by restriction analysis. The frequency of hybridization of the fragments to the light or heavy strand shows a unimodal distribution of radioactivity indicating asymmetric replication. Together, these observations suggest that initiation and termination sites are located at both ends of the chromosome. Changes observed in the conformation of parental DNA at the time of replication, such as high degree of single-strandness, are compatible with strand displacement during synthesis.

Effects of pyrethroid insecticides and estrogen on WNT10B proto-oncogene expression.

Breast cancer is a serious illness affecting approximately one in nine women in the United States. Although an actual cause for breast cancer is unknown, genetic and environmental factors have been associated with its onset. Elevated levels of estrogen and heightened expression of the WNT10B proto-oncogene have been implicated in the development of human malignant breast tumors because they enhance the proliferation of mammary tissue. Two pyrethroid insecticides, sumithrin and fenvalerate, have been shown to mimic estrogenic activity in MCF-7 human breast carcinoma cells by inducing pS2 expression whereas two other pyrethroids, permethrin and d-trans allethrin do not have the same capability. To investigate if estrogen and these four pyrethroid insecticides could affect the expression of a gene related to mammary gland development, WNT10B expression in pyrethroid-treated MCF-7 cells was examined. MCF-7 cells under normal growth conditions do not express WNT10B. Reverse-transcriptase polymerase chain reaction (RT-PCR), nested PCR and Southern hybridization were employed to detect WNT10B expression. As controls, cells were treated with either ethanol, corn oil, or Vista LPA solvent. When compared to the solvent-treated controls, sumithrin, fenvalerate and estrogen treated MCF-7 cells all had increased levels of WNT10B expression. The non-estrogenic pyrethroids, d-trans allethrin and permethrin, demonstrated a similar elevation of WNT10B expression at a lower concentration, but not at the higher concentration. The results suggest that pyrethroid insecticides and estrogen can enhance the expression of the WNT10B proto-oncogene. However, since both the estrogenic and non-estrogenic substances amplified Wnt10B expression, the mechanism likely involves multiple distinct pathways.

Investigation of vaccinia virus DNA replication employing a conditional lethal mutant defective in DNA.

After infection of L cells with the DNA-defective temperature-sensitive (ts) mutant 6389 of vaccinia virus, [3H]thymidine incorporation into cytoplasmic DNA is inhibited at 39 degrees, but resumes upon shiftdown to 32 degrees, the permissive temperature. Following a 30-min lag period DNA synthesis is linear and contingent upon continuous protein synthesis. Sedimentation analysis of nascent DNA labeled during 10 to 60-min pulses revealed that the mutant molecules are produced at a slower rate, but are approximately the same size as those of wild-type vaccinia, synthesized under the same circumstances. During more prolonged incubation beyond 60 min, labeled DNA molecules sedimenting more rapidly than mature, full-length virus genomes are observed. The integration of mutant DNA into mature virions is less rapid than that of the wide-type DNA. Upon extraction from the virosomes, the ts6389 DNA sediments as both genome-size and larger, faster sedimenting DNA. Upon treatment with restriction endonucleases, the ts6389 virosomal DNA exhibited an additional fragment after separation on agarose gels, perhaps as a consequence of fusion between the terminal fragments of the molecule. Taken together these observations suggest that concatemeric intermediates are formed during vaccinia DNA replication. By measuring the radioactivity incorporated into the fragments and subfragments of the molecules labeled during the first round of replication, the initiation site of replication can be localized to a region within the terminal 150 bp.