Sulfated K5 Escherichia coli polysaccharide derivatives as wide-range inhibitors of genital types of human papillomavirus.

Genital human papillomaviruses (HPV) represent the most common sexually transmitted agents and are classified into low or high risk by their propensity to cause genital warts or cervical cancer, respectively. Topical microbicides against HPV may be a useful adjunct to the newly licensed HPV vaccine. A main objective in the development of novel microbicides is to block HPV entry into epithelial cells through cell surface heparan sulfate proteoglycans. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was integrated with innovative biochemical and biological assays to prepare a collection of sulfated K5 derivatives with a backbone structure resembling the heparin/heparan biosynthetic precursor and to test them for their anti-HPV activity. Surface plasmon resonance assays revealed that O-sulfated K5 with a high degree of sulfation [K5-OS(H)] and N,O-sulfated K5 with a high [K5-N,OS(H)] or low [K5-N,OS(L)] sulfation degree, but not unmodified K5, N-sulfated K5, and O-sulfated K5 with low levels of sulfation, prevented the interaction between HPV-16 pseudovirions and immobilized heparin. In cell-based assays, K5-OS(H), K5-N,OS(H), and K5-N,OS(L) inhibited HPV-16, HPV-18, and HPV-6 pseudovirion infection. Their 50% inhibitory concentration was between 0.1 and 0.9 mug/ml, without evidence of cytotoxicity. These findings provide insights into the design of novel, safe, and broad-spectrum microbicides against genital HPV infections.

TGF-beta1 and IL-4 downregulate human papillomavirus-16 oncogene expression but have differential effects on the malignant phenotype of cervical carcinoma cells.

Host immune response to human papillomavirus (HPV) is a crucial factor in viral clearance and control of persistent infections. The existence of an intercellular control mechanism mediated by cytokines to suppress HPV-gene transcription and to prevent malignant conversion of HPV-infected cells, has been postulated. In a previous study, we demonstrated the inhibitory activity of several cytokines on the HPV-16 long control region (LCR)-driven transcription; among these, IL-4 was reported as a LCR inhibitor for the first time and proposed as a candidate for further studies. Here, we addressed the question of whether IL-4 represses HPV-16 oncogene transcription and exerts antitumor activity in HPV-16 positive cervical carcinoma cell lines. Results indicated that downregulation of E6 and E7 levels by IL-4 in CaSki cells is weaker than that exerted by TGF-beta1, a known LCR inhibitor, although both cytokines are equally active in suppressing LCR-driven transcriptional activity in a reporter cell line. Moreover, only TGF-beta rescued p53 expression, Rb response pathway, and induced cellular senescence. SiHa cells were unresponsive to both cytokines. These findings suggest that the two cytokines may play a role in the control of HPV infections, however, cervical carcinoma cells developed a partial or a total resistance to their inhibitory activity.

Preparation and in vitro evaluation of the antiviral activity of the Acyclovir complex of a beta-cyclodextrin/poly(amidoamine) copolymer.

A poly(amidoamine) (PAA) copolymer with beta-cyclodextrin was obtained by polyaddition reaction of 6-deoxy-6-amino-beta-cyclodextrin (beta-CD-NH(2)) and 2-methylpiperazine to 2,2-bis(acrylamido)acetic acid in aqueous medium. This beta-CD/PAA copolymer bears beta-CD units along the macromolecular chain, is water-soluble and non-cytotoxic. The complexing capacity of beta-CD/PAA was determined using an antiviral drug, Acyclovir, as a model of poorly water-soluble drug. Complex formation was confirmed by means of DSC and FTIR analyses. beta-CD/PAA can solubilize up to 11% w/w of Acyclovir notably increasing the aqueous solubility of the drug. The in vitro release studies showed the dependence of Acyclovir release rate on the solution pH. The antiviral activity of Acyclovir beta-CD/PAA complex was evaluated against herpes simplex virus type I in cell cultures. The Acyclovir beta-CD/PAA complex exhibited a higher antiviral activity than the free drug.

Effect of high-risk human papillomavirus oncoproteins on p53R2 gene expression after DNA damage.

The p53R2 protein is a p53-inducible small subunit of ribonucleotide reductase. It plays a crucial role in p53-dependent cellular response to DNA damage and oxidative stress by providing deoxyribonucleotides (dNTPs) to the DNA repair machinery and by scavenging reactive oxygen species (ROS). To investigate the effects of high-risk human papillomavirus (HPV) oncoproteins on p53R2 expression after DNA damage, we analyzed the p53R2 protein levels in human cells ectopically expressing the HPV-16 E6 and E7 genes, and in the HPV-positive cancer cell lines SiHa, CaSki and HeLa, exposed to adriamycin or to H(2)O(2). We found that in normal cells, p53R2 expression is efficiently induced by both H(2)O(2) and adriamycin, supporting the role of p53R2 in cellular response to oxidative stress. Ectopic expression of E6 impaired p53 and p53R2 induction after DNA damage in human fibroblasts. Moreover, SiHa, CaSki and HeLa cells were unresponsive to H(2)O(2) exposure, and adriamycin induced p53R2 levels only in SiHa cells. Our results imply that high-risk HPV infection may suppress the p53R2-dependent dNTPs supply to the DNA repair system and the ROS scavenging activity; they also suggest that an altered p53R2 response to genotoxins and to oxidative stress may contribute to HPV-induced genetic instability and carcinogenesis.

A cell-based high-throughput assay for screening inhibitors of human papillomavirus-16 long control region activity.

Cervical carcinomas express human papillomavirus (HPV) E6 and E7 oncoproteins, which are required to maintain the proliferative state of cancer cells. Repression of E6 and E7 expression results in acquisition of senescent phenotype and in the rescue of functional p53 and p105(Rb) pathways; therefore, therapies directed against either viral protein may be beneficial. However, the systems to study HPV in vitro are technically difficult and not convenient for screening of antiviral compounds. This has hampered the discovery of drugs against HPV. Here we describe the generation and use of a high-throughput screening system based on keratinocytes stably transfected with a reporter construct containing the regulatory sequence of E6 and E7 gene transcription (LCR) that allows easy detection of inhibitors of E6 and E7 expression in libraries of synthetic or biological compounds. The assay was used to screen a wide panel of cytokines: among them, IL-4, IL-13, TGF-beta1, TGF-beta2, TGF-beta3, TNF-alpha, IFN-alpha, and IFN-beta were found to induce a strong inhibition of the LCR activity. Our assay provides a validated tool in the search for drugs against HPV-associated cervical carcinomas and allowed the first systematic analysis of the effect of cytokines on the HPV-16 LCR transcriptional activity.

The ribonucleotide reductase R1 homolog of murine cytomegalovirus is not a functional enzyme subunit but is required for pathogenesis.

Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Alpha- and gammaherpesviruses express a functional enzyme, since they code for both the R1 and the R2 subunits. By contrast, betaherpesviruses contain an open reading frame (ORF) with homology to R1, but an ORF for R2 is absent, suggesting that they do not express a functional RNR. The M45 protein of murine cytomegalovirus (MCMV) exhibits the sequence features of a class Ia RNR R1 subunit but lacks certain amino acid residues believed to be critical for enzymatic function. It starts to be expressed independently upon the onset of viral DNA synthesis at 12 h after infection and accumulates at later times in the cytoplasm of the infected cells. Moreover, it is associated with the virion particle. To investigate direct involvement of the virally encoded R1 subunit in ribonucleotide reduction, recombinant M45 was tested in enzyme activity assays together with cellular R1 and R2. The results indicate that M45 neither is a functional equivalent of an R1 subunit nor affects the activity or the allosteric control of the mouse enzyme. To replicate in quiescent cells, MCMV induces the expression and activity of the cellular RNR. Mutant viruses in which the M45 gene has been inactivated are avirulent in immunodeficient SCID mice and fail to replicate in their target organs. These results suggest that M45 has evolved a new function that is indispensable for virus replication and pathogenesis in vivo.