The human papillomavirus type 11 E1/\E4 protein is not essential for viral genome amplification.

An abundant human papillomavirus (HPV) protein E1/\E4 is expressed late in the virus life cycle in the terminally differentiated layers of epithelia. The expression of E1/\E4 usually coincides with the onset of viral DNA amplification. However, the function of E1/\E4 in viral life cycle is not completely understood. To examine the role of E1/\E4 in the virus life cycle, we introduced a single nucleotide change in the HPV-11 genome to result in a truncation of E1/\E4 protein without affecting the E2 amino acid sequence. This mutated HPV-11 genome was introduced into a human foreskin keratinocyte cell line immortalized by the catalytic subunit of human telomerase, deficient in p16(INK4a) expression, and previously shown to support the HPV-11 life cycle when grown in organotypic raft culture. We have demonstrated that E1/\E4 is dispensable for HPV-11 viral DNA amplification in the late stages of the viral life cycle.

Induction of productive human papillomavirus type 11 life cycle in epithelial cells grown in organotypic raft cultures.

The study of the human papillomavirus (HPV) life cycle was hampered for more than 50 years by the lack of a conventional cell culture system for propagating HPV. Considerable progress has been made in the production of several HPV types using either organotypic rafts or human epithelial xenografts in immunocompromised mice. In this study, we demonstrated episomal maintenance of HPV-11 DNA in N-Tert cells. HPV-11 episomal DNA containing cell populations grown in raft culture showed induction of the productive viral life cycle. HPV-11 DNA amplification and viral capsid antigen synthesis were detected in differentiated layers of epithelia. The viruses generated were able to infect keratinocytes in vitro, which indicate that viruses generated were infectious. The demonstration of the productive HPV-11 life cycle in raft culture from cloned HPV-11 DNA will facilitate genetic analyses of viral gene functions that was not possible using the human xenograft athymic mouse model.

Suppression of human papillomavirus gene expression in vitro and in vivo by herpes simplex virus type 2 infection.

Recent epidemiological studies have found that women infected with both herpes simplex virus type 2 (HSV-2) and human papillomavirus (HPV) type 16 or HPV-18 are at greater risk of developing cervical carcinoma compared to women infected with only one virus. However, it remains unclear if HSV-2 is a cofactor for cervical cancer or if HPV and HSV-2 interact in any way. We have studied the effect of HSV-2 infection on HPV-11 gene expression in an in vitro double-infection assay. HPV transcripts were down-regulated in response to HSV-2 infection. Two HSV-2 vhs mutants failed to reduce HPV-16 E1;E4 transcripts. We also studied the effect of HSV-2 infection on preexisting experimental papillomas in a vaginal epithelial xenograft model. Doubly infected grafts demonstrated papillomatous transformation and the classical cytopathic effect from HSV-2 infection. HPV and HSV DNA signals were mutually exclusive. These studies may have therapeutic applications for HPV infections and related neoplasms.

Immunological characterization of human vaginal xenografts in immunocompromised mice: development of a small animal model for the study of human immunodeficiency virus-1 infection.

A small animal model for the in vivo study of human immunodeficiency virus-1 and other fastidious infectious agents in human host target tissues is critical for the advancement of therapeutic and preventative strategies. Our laboratory has developed a human vaginal xenograft model that histologically recapitulates features of the human vaginal epithelial barrier. Vaginal xenografts were surgically implanted into C.B.-Igh-1(b)/IcrTac-Prkdc(scid) (SCID) and NOD/LtSz-scid/scid (NOD/SCID) mice, with and without human peripheral blood mononuclear cell reconstitution. Immunohistochemical staining of vaginal xenografts demonstrated that in the SCID strain healed vaginal xenografts did not retain intrinsic human immune cells at baseline levels, whereas the NOD/SCID strain supported retention of intrinsic human immune cell populations within the xenografts for at least 2 months after engraftment. In peripheral blood mononuclear cell-reconstituted NOD/SCID mice with vaginal xenografts, flow cytometric analyses detected human immune cell populations in the peripheral blood and immunohistochemical methods detected infiltration of human CD45+ cells in the mouse spleens and vaginal xenografts for at least 2 months after reconstitution. This optimized NOD/SCID human vaginal xenograft model may provide a unique small animal in vivo system for the study of human immunodeficiency virus-1 transmission and infection.

Papillomavirus microbicidal activities of high-molecular-weight cellulose sulfate, dextran sulfate, and polystyrene sulfonate.

The high-molecular-weight sulfated or sulfonated polysaccharides or polymers cellulose sulfate, dextran sulfate, and polystyrene sulfonate were tested for microbicidal activity against bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 11 (HPV-11) and type 40 (HPV-40). In vitro assays included the BPV-1-induced focus-forming assay and transient infection of human A431 cells with HPVs. The compounds were tested for microbicidal activity directly by preincubation with virus prior to addition to cell cultures and indirectly by addition of virus to compound-treated cells and to virus-coated cells to test inactivation of the virus after virus-cell binding. The data indicated that all three compounds showed direct microbicidal activity with 50% effective concentrations between 10 to 100 microg/ml. These concentrations were nontoxic to cell cultures for both assays. When a clone of C127 cells was tested for microbicidal activity, approximately 10-fold-less compound was required to achieve a 50% reduction in BPV-1-induced foci than for the uncloned parental C127 cells. Pretreatment of cells with compound prior to addition of virus also demonstrated strong microbicidal activity with dextran sulfate and polystyrene sulfonate, but cellulose sulfate required several orders of magnitude more compound for virus inactivation. Polystyrene sulfonate prevented subsequent infection of HPV-11 after virus-cell binding, and this inactivation was observed up to 4 h after addition of virus. These data indicate that the polysulfated and polysulfonated compounds may be useful nontoxic microbicidal compounds that are active against a variety of sexually transmitted disease agents including papillomaviruses.

Preventing cervical cancer.

The Pap smear has been the classic screening strategy for preventing cervical cancer for 50 years. The finding that infection with human papillomavirus is associated with an increased risk of cervical cancer has prompted the development of new strategies for cervical cancer screening and prevention. In their Policy Forum, Cain and Howett discuss the introduction of HPV testing, anti-HPV microbicidal agents and vaccination against HPV. They point out the benefits but also the potential for over and under treatment and the need for considerable improvements in public education.

Sodium dodecyl sulfate and C31G as microbicidal alternatives to nonoxynol 9: comparative sensitivity of primary human vaginal keratinocytes.

A broad-spectrum vaginal microbicide must be effective against a variety of sexually transmitted disease pathogens and be minimally toxic to the cell types found within the vaginal epithelium, including vaginal keratinocytes. We assessed the sensitivity of primary human vaginal keratinocytes to potential topical vaginal microbicides nonoxynol-9 (N-9), C31G, and sodium dodecyl sulfate (SDS). Direct immunofluorescence and fluorescence-activated cell sorting analyses demonstrated that primary vaginal keratinocytes expressed epithelial cell-specific keratin proteins. Experiments that compared vaginal keratinocyte sensitivity to each agent during a continuous, 48-h exposure demonstrated that primary vaginal keratinocytes were almost five times more sensitive to N-9 than to either C31G or SDS. To evaluate the effect of multiple microbicide exposures on cell viability, primary vaginal keratinocytes were exposed to N-9, C31G, or SDS three times during a 78-h period. In these experiments, cells were considerably more sensitive to C31G than to N-9 or SDS at lower concentrations within the range tested. When agent concentrations were chosen to result in an endpoint of 25% viability after three daily exposures, each exposure decreased cell viability at the same constant rate. When time-dependent sensitivity during a continuous 48-h exposure was examined, exposure to C31G for 18 h resulted in losses in cell viability not caused by either N-9 or SDS until at least 24 to 48 h. Cumulatively, these results reveal important variations in time- and concentration-dependent sensitivity to N-9, C31G, or SDS within populations of primary human vaginal keratinocytes cultured in vitro. These investigations represent initial steps toward both in vitro modeling of the vaginal microenvironment and studies of factors that impact the in vivo efficacy of vaginal topical microbicides.

Inactivation of human immunodeficiency virus type 1 by nonoxynol-9, C31G, or an alkyl sulfate, sodium dodecyl sulfate.

A highly desirable approach to prevention of human immunodeficiency virus type 1 (HIV-1) transmission during sexual intercourse is the development of nontoxic, topical, broad spectrum microbicides effective against transmission of cell-associated and cell-free virus. Toward this end, the HIV-1 inactivation potential of surface active agents C31G and an alkyl sulfate, sodium dodecyl sulfate (SDS) was assessed. Because of its extensive use as a microbicidal agent, nonoxynol-9 (N-9) was used as a reference against which C31G and SDS were compared. Viral inactivation was measured using HIV-1 LTR-beta-galactosidase indicator cells (expressing CD4 or CD4/CCR5) derived from HeLa cells, a cell line of human cervical adenocarcinoma origin. In experiments which examined inactivation of cell-free HIV-1, C31G was generally more effective than N-9. Viral inactivation by SDS occurred at twice the concentration necessary to achieve similar levels of inactivation using either N-9 or C31G. Using HeLa and HeLa-derived cells in cytotoxicity studies, it was demonstrated that SDS is as much as 11 and five times less cytotoxic than N-9 or C31G, respectively, during 48 h of continuous exposure. SDS (unlike C31G and N-9) can inactivate non-enveloped viruses such as human papillomavirus (HPV) [Howett, M.K., Neely, E.B., Christensen, N.D., Wigdahl, B., Krebs, F.C., Malamud, D., Patrick, S.D., Pickel, M.D., Welsh, P.A., Reed, C.A., Ward, M.G., Budgeon, L.R., Kreider, J.W., 1999. A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses. Antimicrob. Agents Chemother. 43(2), 314-321]. Since addition of SDS to C31G or N-9 may make the resulting microbicidal mixtures broadly effective against both enveloped and non-enveloped viruses, several surface active agent combinations were evaluated for their abilities to inactivate HIV-1. Addition of SDS to either C31G or N-9 resulted in mixtures that were only slightly less effective than equivalent concentrations of C31G or N-9 alone. To investigate inactivation of cell-associated infectivity, HIV-1 IIIB-infected SupT1 cells were treated with N-9, C31G, or SDS. Inactivation of cell-associated infectivity required higher microbicide concentrations than were needed for inactivation of cell-free virus. However, the relative activities of N-9, C31G, or SDS were similar to those seen in assays of inactivation using cell-free virus. These studies suggest that C31G and SDS may be attractive candidates for human trials as topical microbicides effective against HIV-1 transmission since both function at concentrations that provide effective viral inactivation with low levels of cytotoxicity. SDS microbicides (used alone or with other microbicides) may provide the added advantage of protection from HPV infection.

A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses.

Sodium dodecyl sulfate (SDS), an alkyl sulfate surfactant derived from an organic alcohol, possesses surfactant properties but also denatures and unfolds both monomeric and subunit proteins. In preliminary experiments, we demonstrated that SDS is a potent inactivator of herpes simplex virus type 2 and human immunodeficiency virus type 1 at concentrations comparable to those used for the surfactant nonoxynol-9. We hypothesized that SDS might be capable of denaturing the capsid proteins of nonenveloped viruses. In this report, we demonstrate inactivation of rabbit, bovine, and human papillomaviruses after brief treatment with dilute solutions of SDS. Effective concentrations were nontoxic to rabbit skin and to split-thickness grafts of human foreskin epithelium. This is the first report of a microbicidal surfactant that will inactivate papillomaviruses. We propose that SDS is now a candidate microbicide for formulation and testing with humans.

Correlation of TGF beta 1 overexpression with down-regulation of proliferation-inducing molecules in HPV-11 transformed human tissue xenografts.

The epidemiologic association of human papillomavirus (HPV) infection with dysplasia and cervical cancer is well established. Transforming growth factor beta 1 (TGF beta 1) has regulatory effects on a broad spectrum of cell types and is a growth inhibitory protein for epithelial cells. To examine the phenotype of experimentally generated, HPV-11 transformed human tissues, we looked at expression of TGF beta 1 and a number of proliferation-enhancing molecules which are known to be regulated by TGF beta 1, including bcl-2, c-myc, c-Ha-ras, c-jun and NFkB. HPV-11 transformed xenografts showed up-regulation of TGF beta 1 expression and down-regulation of the expression levels of bcl-2, c-myc, c-Ha-ras, c-jun and NFkB. These results suggest that TGF beta 1 may exert antiproliferative effects on HPV-11 transformed papillomas by down-regulating different proliferation-enhancing molecules.

Transforming growth factor beta 1 (TGF beta 1) down-regulates expression and function of proliferation-inducing molecules in HPV-transformed cells.

The epidemiologic association of human papillomavirus (HPV) infection with dysplasia and cervical cancer is well established. Transforming growth factor beta 1 (TGF beta 1) is a growth inhibitory protein for epithelial cells. To examine the phenotype of HPV-transformed cells, we examined expression of TGF beta 1 and a number of cellular proliferation-enhancing molecules which are known to be regulated by TGF beta 1, including bcl-2, c-jun and NFkB. Previous studies had identified significant induction of TGF beta 1 and concomitant down-regulation of other growth stimulatory molecules in experimental papillomas. We used HPV-16 and -18 transformed cell lines. The HPV-16 transformed cells showed down-regulation of bcl-2 and NFkB as well as NFkB function upon TGF beta 1 treatment. The results suggest that TGF beta 1 may exert antiproliferative effects on some HPV-transformed cells by down-regulating expression and function of different proliferation-enhancing molecules. It is uncertain if this function is virus type specific and/or related to state of tumor cell progression.

Localization of protein kinase C alpha isoform expression in the human gastrointestinal tract.

Protein kinase C (PKC) includes a family of related proteins which constitutes a major signal transduction pathway. The aim of this study was to determine the localization of the PKC-alpha isoform throughout the human gastrointestinal tract. PKC-alpha expression was also measured and compared between normal and neoplastic colorectal tissue. PKC-alpha mRNA expression was detected in normal human gastrointestinal tract tissue using Northern blot analyses and in situ hybridization. PKC-alpha protein expression was detected in normal gastrointestinal tissue and colorectal neoplasia using Western blot and immunohistochemical analyses. PKC-alpha was expressed throughout the human gastrointestinal tract. Distinct organ and cellular localization was characterized. PKC-alpha mRNA and protein localization were most prevalent in the deep basal layer of the esophageal mucosa. In the stomach, PKC-alpha expression was detected predominately in the cells of the deep glands and surface epithelial cells but less in the mucous neck cells of the gastric pits. In the duodenum and ileum, PKC-alpha mRNA expression was greater in the deeper crypt cells than in the differentiated cells that line the villi. However, immunohistochemistry showed greater expression in the cells of the villi compared to crypt cells. In normal colonic tissue, PKC-alpha mRNA and protein predominated in the cells of the upper crypt and surface epithelial cells. PKC-alpha protein was also prominently expressed in the glands of colorectal adenocarcinoma. There was no quantitative difference in the level of PKC-alpha protein expression between normal and neoplastic colorectal tissue. The specific organ and cellular expression of PKC-alpha suggests separate and distinct functional roles for this PKC isoform throughout the gastrointestinal tissues.

Expression of an oncogenic rasHa gene in murine keratinocytes induces tyrosine phosphorylation and reduced activity of protein kinase C delta.

Murine keratinocytes expressing an oncogenic rasHa gene produce benign tumors in vivo and demonstrate altered responses to phorbol esters in vitro. Cultured keratinocytes transduced with the v-rasHa gene (v-rasHa keratinocytes) are resistant to Ca(2+)-induced terminal differentiation, a process that is dependent on protein kinase C (PKC) activation in normal keratinocytes. Five PKC isoforms expressed in keratinocytes (alpha, delta, epsilon, zeta, and eta) were examined for quantitative or qualitative changes in v-rasHa-transformed cells. No quantitative changes were detected, but PKC delta was tyrosine-phosphorylated in v-rasHa keratinocytes and in benign neoplastic keratinocyte cell lines expressing an activated allele of the c-rasHa gene. Analysis of phosphorylated and non-phosphorylated forms of PKC delta from keratinocytes indicated that phosphorylated PKC delta was not stimulated by phorbol ester treatment. The protein kinase inhibitor staurosporine was able to induce differentiation in v-rasHa keratinocytes and benign tumor cell lines, and concomitantly tyrosine phosphorylation of PKC delta decreased. This interaction between tyrosine kinases and PKC delta in cells expressing an oncogenic rasHa gene may represent a molecular block to differentiation in neoplastic keratinocytes.

Infectious cycle of human papillomavirus type 11 in human foreskin xenografts in nude mice.

We have performed the first molecular analysis of a time course of infection by a papillomavirus. The Hershey isolate of the human papillomavirus type 11 was used to infect human foreskin tissues, which were then implanted under the renal capsules of nude mice. The xenografts were recovered every 2 weeks for 14 weeks, fixed in formalin, and embedded in paraffin. Four-micrometer serial sections were examined by light microscopy for morphological changes, by immunocytochemistry for virion antigen production, and by in situ hybridization with 3H-labeled RNA probes for viral DNA replication and expression of the major mRNA species. After a lag period, probes spanning the E4 and E5 open reading frames, which are present in all E region viral mRNAs, generated the first detectable signals at week 4. Signals of other E region probes were minimally detected at week 6. Between weeks 6 and 8, there was an abrupt change in the implant such that cellular proliferation, viral DNA replication, and E and L region mRNA transcription were robust and reached a plateau. By weeks 10 to 12, the experimental condylomata were morphologically and histologically indistinguishable from naturally occurring condylomata acuminata. These findings suggest that cellular hyperproliferation and the morphologic features of condylomata are direct results of viral genetic activities. Unlike other DNA viruses, the E region transcripts increased with cell age and cellular differentiation and persisted throughout the entire experiment. In particular, the mRNA encoding the E1iE4 and perhaps E5 proteins remained overwhelmingly abundant. In contrast, viral DNA replication, L region mRNA synthesis, and virion antigen production were restricted to the most differentiated, superficial cells.

Human xenografts. A model system for human papillomavirus infection.

Epithelial chips of human foreskin or cervix infected in vitro with one strain of human papillomavirus type 11 (HPV-11) and subsequently transplanted to the renal capsule of athymic mice will yield, after 3 months of in vivo incubation, epithelial cysts that are morphologically transformed and appear identical in every way to human condylomata. These cysts synthesize virus RNA, DNA, proteins, and infectious virions. The cysts can be utilized as a source of virus for continued passage of infection. The original HPV-11 infecting material was a cell-free saline extract of pooled human vulvar condylomata. DNAs of other HPV types were not detected in this material by either dot blot or Southern blot analyses. The copy number of HPV-11 virus genomes per cell genome in experimental condylomata ranged from about 200 to 1,000, a range expected for episomal papillomavirus DNA. Analyses of cloned HPV-11 DNA (pBT-1) from experimental lesions demonstrated that the size and restriction endonuclease map of the HPV-11-Hershey isolate closely matched that of the prototype. A few nucleotide changes that were detected during analyses of pBT-1 DNA resulted either in no amino acid change or a conservative change of amino acid. Physical characterization of the cloned experimental HPV-11 DNA as well as HPV typing in clinical lesions and experimental cysts are presented.

Nucleocapsid, nuclear association, and genome location of the herpes simplex virus type 2 38-kD DNA-binding protein.

A previously-described herpes simplex virus type 2 DNA-binding protein with a molecular size of 38 kD has been further characterized. Using purified nucleocapsids, a DNase release assay, and intertypic recombinants, this protein was found to be a component of the nucleocapsid, intimately associated with the nuclear matrix, and encoded between 0.605 and 0.720 on the herpes simplex virus type 2 genome.

Phenotypic properties of herpesvirus-transformed cells with high tumorigenic and metastatic ability.

Herpes simplex virus type 2-transformed hamster embryo fibroblasts (333-8-9 cells) produce increased plasminogen activator (PA) compared with normal hamster cells. These cells produce undifferentiated fibrosarcomas at the inoculation site in newborn hamsters, and metastasize to the lungs. Using a direct PA assay, in which 125I-labeled plasminogen is cleaved, the optimum pH and osmolarity for detection of the 333-8-9 extracellular PA were pH 8.9 and approximately 150 mOsmol. Secretion of enzyme did not vary significantly on a per cell basis over cell densities from 0.1 to 8.0 X 10(7) cells/T-75 cm2 flask. This assay demonstrates that the 333-8-9 cells produce at least 20-fold greater levels of PA than normal cell counterparts. Based on the molecular weight (50-58 kDa) of secreted 333-8-9 cells PA and lack of fibrin stimulation, we conclude that it is a urokinase type PA. Subclonal lines of the 333-8-9 cells, selected for an increased PA phenotype were stable in culture, more tumorigenic and probably more metastatic. Correlation of these two events was examined by passaging 333-8-9 cells in vivo to select for greater tumorigenic potential and then determining the production of PA by the in vivo-derived sublines. The metastatic potential of the resulting cells was heterogeneous. Increased PA production upon increased passage in vivo did not always occur, whether the cells were passaged as subcutaneous tumors or as ascites tumors. Thus, while enzyme production correlated with tumorigenicity when selecting cells for an increased protease phenotype, this correlation was not observed when selecting for in vivo tumorigenicity. The results suggest that increased ability to make PA represents only one of multiple selective advantages for tumor growth.