Mutations in HPV18 E1^E4 Impact Virus Capsid Assembly, Infectivity Competence, and Maturation.

The most highly expressed protein during the productive phase of the human papillomavirus (HPV) life cycle is E1^E4. Its full role during infection remains to be established. HPV E1^E4 is expressed during both the early and late stages of the virus life cycle and contributes to viral genome amplification. In an attempt to further outline the functions of E1^E4, and determine whether it plays a role in viral capsid assembly and viral infectivity, we examined wild-type E1^E4 as well as four E1^E4 truncation mutants. Our study revealed that HPV18 genomes containing the shortest truncated form of E1^E4, the 17/18 mutant, produced viral titers that were similar to wild-type virus and significantly higher compared to virions containing the three longer E1^E4 mutants. Additionally, the infectivity of virus containing the shortest E1^E4 mutation was equivalent to wild-type and significantly higher than the other three mutants. In contrast, infectivity was completely abrogated for virus containing the longer E1^E4 mutants, regardless of virion maturity. Taken together, our results indicate for the first time that HPV18 E1^E4 impacts capsid assembly and viral infectivity as well as virus maturation

UVC radiation as an effective disinfectant method to inactivate human papillomaviruses

<div><p>Endocavitary ultrasound probes are part of a commonly used procedure in the clinical arena. The cavities examined, vaginal canal and cervix, anal canal, and oral cavity are all areas commonly infected with the human papillomavirus (HPV), thus making them susceptible to contamination by HPV. It has been demonstrated that these probes can remain contaminated with high-risk HPV even when approved disinfection protocols have been performed. we have previously shown that HPV is resistant to some high-level disinfectant (HLD). In our present study we analyzed efficacy of using high-level ultra-violet C (UVC) radiation against HPV16 and HPV18 using a hard-surface carrier test. Stocks of infectious authentic HPV16 and HPV18 virions were dried onto carriers with a 5% (v/v) protein soil or 4ppm hard water. Efficacy testing were performed with the automated device, Antigermix S1 device (UVC radiation at 253.7nm) and 0.55% OPA in quadruplicate with matched input, neutralization, and cytotoxicity controls. Hypochlorite was included as a positive control for viral deactivation. Infectivity was determined by the abundance (qRT-PCR) of the spliced E1^E4 transcript in infected recipient cells. The automated Antigermix S1 device showed excellent efficacy against HPV16 and HPV18 whereas OPA showed minimal efficacy. While HPV is highly resistant to OPA, high-level UVC radiation offers an effective disinfection practice for ultrasound probes. Our results suggest that healthcare facilities using endocavitary ultrasound probes need to strongly consider disinfection methods that are effective against HPV.</p></div

Differing efficacy profiles of disinfectants against HPV.

<p>HPV16 (A) or HPV18 (B) virions were subjected to hard surface carrier tests based on the ASTM E1053-11 standard test method against the disinfectants indicated. Virus films were dried onto 50mm diameter ABS carriers in the presence of a 5% fetal bovine serum (soil) and 4ppm CaCO₃ (hard water) before being disinfected according to the disinfectant or device manufacturer’s instructions. Viral films were assayed for infectivity using a quantitative RT-PCR based method detecting the spliced E1^E4 transcript in infected recipient cells. Post-disinfection infectivity was compared to input to determine log10 reductions. Each efficacy test was conducted in quadruplicate and was paired with a matched neutralization control. Data is expressed as an average of n = 3; error bars indicate standard deviation.</p

Average C_t values obtained in this study.

<p>Average C_t values obtained in this study.</p

Anti-Retroviral Protease Inhibitors Regulate Human Papillomavirus 16 Infection of Primary Oral and Cervical Epithelium

Epidemiology studies suggest that Human Immunodeficiency Virus (HIV)-infected patients on highly active anti-retroviral therapy (HAART) may be at increased risk of acquiring opportunistic Human Papillomavirus (HPV) infections and developing oral and cervical cancers. Effective HAART usage has improved survival but increased the risk for HPV-associated cancers. In this manuscript, we report that Protease Inhibitors (PI) treatment of three-dimensional tissues derived from primary human gingiva and cervical epithelial cells compromised cell-cell junctions within stratified epithelium and enhanced paracellular permeability of HPV16 to the basal layer for infection, culminating in de novo biosynthesis of progeny HPV16 as determined using 5-Bromo-2&prime;-deoxyuridine (BrdU) labeling of newly synthesized genomes. We propose that HAART/PI represent a novel class of co-factors that modulate HPV infection of the target epithelium. Our in vitro tissue culture model is an important tool to study the mechanistic role of anti-retroviral drugs in promoting HPV infections in HAART-na&iuml;ve primary epithelium. Changes in subsequent viral load could promote new infections, create HPV reservoirs that increase virus persistence, and increase the risk of oral and cervical cancer development in HIV-positive patients undergoing long-term HAART treatment

Human papillomavirus G-rich regions as potential antiviral drug 1 targets

International audienceHerein we report for the first time the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding which may have an impact on transcription, replication and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G-quadruplex conformation upon interaction with a well-characterized highly selective G4-ligand, PhenDC3, which may have animpact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G-quadruplex structures, were found to form single stable complexes in the presence of two other G4-ligands, C8and Pyridostatin, respectively. Additionally, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G-quadruplexes may be an alternative route for the development of novel antiviral therapies

Human Papillomavirus G-Rich Regions as Potential Antiviral Drug Targets

International audienceAbstract G‐quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G‐quartets, the length and positions of loops, flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4‐prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G‐quadruplex or not. In this article, we adapted the well‐known FRET‐melting assay to characterize G4 in batch, where the sequence to be tested is added, as an unlabeled competitor, to a system composed of a dual‐labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G‐quadruplexes. In this so‐called FRET‐MC (melting competition) assay, G4‐forming competitors lead to a marked decrease of the ligand‐induced stabilization effect (∆ T m ), while non‐specific competitors (e.g., single‐ or double‐stranded sequences) have little effect. Sixty‐five known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized