Translocation of the papillomavirus L2/vDNA complex across the limiting membrane requires the onset of mitosis.

The human papillomavirus type 16 (HPV16) L2 protein acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of this critical process remain poorly characterized. We have developed a system based on subcellular compartmentalization of the enzyme BirA and its cognate substrate to detect membrane translocation of L2-BirA from incoming virions. We find that L2 translocation requires transport to the TGN and is strictly dependent on entry into mitosis, coinciding with mitotic entry in synchronized cells. Cell cycle arrest causes retention of L2/vDNA at the TGN; only release and progression past G2/M enables translocation across the limiting membrane and subsequent infection. Microscopy of EdU-labeled vDNA reveals a rapid and dramatic shift in vDNA localization during early mitosis. At late G2/early prophase vDNA egresses from the TGN to a pericentriolar location, accumulating there through prometaphase where it begins to associate with condensed chromosomes. By metaphase and throughout anaphase the vDNA is seen bound to the mitotic chromosomes, ensuring distribution into both daughter nuclei. Mutations in a newly defined chromatin binding region of L2 potently blocked translocation, suggesting that translocation is dependent on chromatin binding during prometaphase. This represents the first time a virus has been shown to functionally couple the penetration of limiting membranes to cellular mitosis, explaining in part the tropism of HPV for mitotic basal keratinocytes.

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.

A transmembrane domain and GxxxG motifs within L2 are essential for papillomavirus infection.

During cellular invasion, human papillomavirus type 16 (HPV16) must transfer its viral genome (vDNA) across the endosomal membrane prior to its accumulation at nuclear PML bodies for the establishment of infection. After cellular uptake, the capsid likely undergoes pH-dependent disassembly within the endo-/lysosomal compartment, thereby exposing hidden domains in L2 that facilitate membrane penetration of L2/vDNA complexes. In an effort to identify regions of L2 that might physically interact with membranes, we have subjected the L2 sequence to multiple transmembrane (TM) domain prediction algorithms. Here, we describe a conserved TM domain within L2 (residues 45 to 67) and investigate its role in HPV16 infection. In vitro, the predicted TM domain adopts an alpha-helical structure in lipid environments and can function as a real TM domain, although not as efficiently as the bona fide TM domain of PDGFR. An L2 double point mutant renders the TM domain nonfunctional and blocks HPV16 infection by preventing endosomal translocation of vDNA. The TM domain contains three highly conserved GxxxG motifs. These motifs can facilitate homotypic and heterotypic interactions between TM helices, activities that may be important for vDNA translocation. Disruption of some of these GxxxG motifs resulted in noninfectious viruses, indicating a critical role in infection. Using a ToxR-based homo-oligomerization assay, we show a propensity for this TM domain to self-associate in a GxxxG-dependent manner. These data suggest an important role for the self-associating L2 TM domain and the conserved GxxxG motifs in the transfer of vDNA across the endo-/lysosomal membrane.

Human papillomavirus type 16 does not require cathepsin L or B for infection.

Cathepsin L (CatL) and cathepsin B (CatB) are lysosomal proteases that many viruses utilize for capsid disassembly. We tested whether CatL and CatB are required for infection by human papillomavirus type 16 (HPV16). CatL- and CatB-deficient mouse embryonic fibroblasts had higher levels of infection when compared with wild-type cells. Similar results were obtained in HaCaT keratinocytes treated with CatL- or CatB-specific small interfering RNA. Thus, CatL and CatB are not required for HPV16 infection but instead appear to restrict infection.

Opposing effects of bacitracin on human papillomavirus type 16 infection: enhancement of binding and entry and inhibition of endosomal penetration.

Cell invasion by human papillomavirus type 16 (HPV16) is a complex process relying on multiple host cell factors. Here we describe an investigation into the role of cellular protein disulfide isomerases (PDIs) by studying the effects of the commonly used PDI inhibitor bacitracin on HPV16 infection. Bacitracin caused an unusual time-dependent opposing effect on viral infection. Enhanced cellular binding and entry were observed at early times of infection, while inhibition was observed at later times postentry. Bacitracin was rapidly taken up by host cells and colocalized with HPV16 at late times of infection. Bacitracin had no deleterious effect on HPV16 entry, capsid disassembly, exposure of L1/L2 epitopes, or lysosomal trafficking but caused a stark inhibition of L2/viral DNA (vDNA) endosomal penetration and accumulation at nuclear PML bodies. γ-Secretase has recently been implicated in the endosomal penetration of L2/vDNA, but bacitracin had no effect on γ-secretase activity, indicating that blockage of this step occurs through a γ-secretase-independent mechanism. Transient treatment with the reductant ß-mercaptoethanol (ß-ME) was able to partially rescue the virus from bacitracin, suggesting the involvement of a cellular reductase activity in HPV16 infection. Small interfering RNA (siRNA) knockdown of cellular PDI and the related PDI family members ERp57 and ERp72 reveals a potential role for PDI and ERp72 in HPV infection.

The acoustic characteristics of professional opera singers performing in chorus versus solo mode.

In this study, members of a professional opera chorus were recorded using close microphones, while singing in both choral and solo modes. The analysis included computation of long-term average spectra (LTAS) for the two song sections performed and calculation of singing power ratio (SPR) and energy ratio (ER), which provide an indication of the relative energy in the singer's formant region. Vibrato rate and extent were determined from two matched vowels, and SPR and ER were calculated for these vowels. Subjects sung with equal or more power in the singer's formant region in choral versus solo mode in the context of the piece as a whole and in individual vowels. There was no difference in vibrato rate and extent between the two modes. Singing in choral mode, therefore, required the ability to use a similar vocal timbre to that required for solo opera singing.

Development of an auditory-perceptual rating instrument for the operatic singing voice.

Although considerable progress has been made in the development of acoustic and physiological measures of operatic singing voice, there is still no widely accepted objective tool for the evaluation of its multidimensional features. Auditory-perceptual evaluation, therefore, remains an important evaluation method for singing pedagogues, voice scientists, and clinicians who work with opera singers. Few investigators, however, have attempted to develop standard auditory-perceptual tools for evaluation of the operatic voice. This study aimed to pilot test a new auditory-perceptual rating instrument for operatic singing voice. Nine expert teachers of operatic singing used the instrument to rate the singing voices of 21 professional opera chorus artists from a national opera company. The findings showed that the instrument has good face validity, that it can be legitimately treated as a psychometrically sound scale, and that raters can use the scale consistently, both between and within judges. This new instrument, therefore, has the potential to allow opera singers, their teachers, voice care clinicians, and researchers to evaluate the important auditory-perceptual features of operatic voice quality.

PhiXing-it, displaying foreign peptides on bacteriophage ΦX174.

Although bacteriophage φX174 is easy to propagate and genetically tractable, it is use as a peptide display platform has not been explored. One region within the φX174 major spike protein G tolerated 13 of 16 assayed insertions, ranging from 10 to 75 amino acids. The recombinant proteins were functional and incorporated into infectious virions. In the folded protein, the peptides would be icosahedrally displayed within loops that extend from the protein׳s ß-barrel core. The well-honed genetics of φX174 allowed permissive insertions to be quickly identified by the cellular phenotypes associated with cloned gene expression. The cloned genes were easily transferred from plasmids to phage genomes via recombination rescue. Direct ELISA validated several recombinant virions for epitope display. Some insertions conferred a temperature-sensitive (ts) protein folding defect, which was suppressed by global suppressors in protein G, located too far away from the insertion to directly alter peptide display.

Comparison of the MTI Photoscreener and the Welch-Allyn SureSight autorefractor in a tertiary care center.

INTRODUCTION: The MTI photoscreener (MTI) and the Welch-Allyn SureSight autorefractor are commonly used for preschool vision screening. We compared both of these methods on 100 consecutive patients in a prospective, randomized, masked, clinical trial conducted at a tertiary care center. METHODS: One hundred patients between 1 and 6 years of age were included in the study. All participants underwent a comprehensive eye examination with cycloplegic refraction. Examination failure analysis was done on the SureSight data using the manufacturer's referral criteria, the Vision in Preschoolers study (VIP) 90% specificity criteria, the VIP 94% specificity criteria, and the referral criteria proposed by Rowatt and colleagues. RESULTS: Data were successfully obtained on 76% of children using the SureSight and 96% with the MTI. The sensitivity and specificity of the SureSight to detect clinically significant amblyogenic factors using the manufacturer's criteria was 96.6 and 38.1%, using the VIP 90% criteria was 79.3 and 64.3%, using the VIP 94% criteria was 67.2 and 69.0%, and using criteria proposed by Rowatt and colleagues was 62.1 and 73.8%. The sensitivity and specificity of the MTI photoscreener was 94.8 and 88.1%, respectively. CONCLUSIONS: Using the manufacturer's referral criteria, the SureSight had a sensitivity equal to the MTI photoscreener; however, the specificity was low and over-referrals were anticipated. As specificity levels were increased, a substantial number of children with amblyogenic risk factors were not appropriately identified within our study population..

Therelationship between professional operatic soprano voice and high range spectral energy.

Operatic sopranos need to be audible over an orchestra yet they are not considered to possess a singer's formant. As in other voice types, some singers are more successful than others at being heard and so this work investigated the frequency range of the singer's formant between 2000 and 4000 Hz to consider the question of extra energy in this range. Such energy would give an advantage over an orchestra, so the aims were to ascertain what levels of excess energy there might be and look at any relationship between extra energy levels and performance level. The voices of six operatic sopranos (national and international standard) were recorded performing vowel and song tasks and subsequently analyzed acoustically. Measures taken from vowel data were compared with song task data to assess the consistency of the approaches. Comparisons were also made with regard to two conditions of intended projection (maximal and comfortable), two song tasks (anthem and aria), two recording environments (studio and anechoic room), and between subjects. Ranking the singers from highest energy result to lowest showed the consistency of the results from both vowel and song methods and correlated reasonably well with the performance level of the subjects. The use of formant tuning is considered and examined.