Furin Cleavage of L2 during Papillomavirus Infection: Minimal Dependence on Cyclophilins.

UNLABELLED: Despite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during human papillomavirus 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, the Propionibacterium shermanii transcarboxylase domain (PSTCD), to the N terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin, and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurred on both the HaCaT cell surface and extracellular matrix (ECM). Contrary to current models, experiments on the involvement of cyclophilins revealed little, if any, role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 (2RHKR5) and Arg12 (9RTKR12). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12, as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues revealed that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage. IMPORTANCE: Furin cleavage of minor capsid protein L2 during papillomavirus infection has been difficult to directly visualize and quantify, confounding efforts to study this important step of HPV infection. Fusion of a small protein domain to the N terminus greatly facilitates direct visualization of the cleavage product, revealing important characteristics of this critical process. Contrary to the current model, we found that cleavage is largely independent of cyclophilins, suggesting that cyclophilins act either in parallel to or downstream of furin to trigger exposure of a conserved N-terminal L2 epitope (RG-1) during infection. Based on this finding, we strongly caution against using L2 RG-1 epitope exposure as a convenient but indirect proxy of furin cleavage.

Maternal influenza immunization and birth outcomes of stillbirth and spontaneous abortion: a systematic review and meta-analysis.

Despite strong evidence that maternal influenza vaccination during pregnancy is safe, uptake of influenza vaccination during pregnancy remains low. We identified studies that assessed outcomes of stillbirth or spontaneous abortion after administration of influenza vaccine during pregnancy. We conducted a literature search in November 2013 that yielded 447 total citations. After removal of duplicates and studies deemed not relevant based on the title and abstract, 36 records underwent a full text review and 7 studies were included in the final review. Where possible, adjusted results were included in the meta-analysis. Women in the influenza vaccine group had a lower likelihood of stillbirth (relative risk [RR], 0.73; 95% confidence interval [CI], .55-.96); this association was similar when restricted to the H1N1pdm09 vaccine (RR, 0.69; 95% CI, .53-.90). The pooled estimate for spontaneous abortion was not significant (RR, 0.91; 95% CI, .68-1.22). These analyses add to the evidence base for the safety of influenza vaccination in pregnancy.

Perceptions of personal belief vaccine exemption policy: a survey of Arizona vaccine providers.

BACKGROUND: As exemptions to school-entry requirements rise, vaccination rates in Arizona school children are approaching levels that may threaten public health. Understanding the interactions physicians have with vaccine-hesitant parents, as well as the opinions physicians hold regarding vaccination, exemption, and exemption policies, are critical to our understanding of, and ability to affect, vaccination exemption rates among children. METHODS: Survey responses were elicited from practitioners listed in The Arizona Partnership for Immunization and the Arizona Medical Association databases using a multi-pronged recruitment approach. Respondents provided data regarding their practice, comfort with parental refusal of individual vaccines, opinions about the beliefs held by parents that seek exemptions, parent education strategies, issues regarding providing care to unvaccinated children, and potential changes to Arizona policy. RESULTS: A total of 152 practitioners providing care to a wide geographic and economic population of Arizona responded to the survey. Respondents were generally strong advocates of all immunizations but were more accepting of parents' desires to refuse hepatitis B and rotavirus vaccines. Almost all providers indicated that they see patients whose parents request to refuse or delay from vaccinations at least occasionally (88% and 97%, respectively). Only 37% of respondents indicated that they would be supportive of a policy requiring them to sign off on a parent's decision to refuse vaccination. CONCLUSIONS: Vaccination providers in Arizona are generally very supportive of childhood immunizations but have varying comfort with exemption from individual vaccines. Responding providers tended to not support a requirement for a physician's signature for vaccine exemptions due to varying concerns regarding the implementation of such a practice.