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1.  Flaviviruses Are Sensitive to Inhibition of Thymidine Synthesis Pathways 
Journal of Virology  2013;87(17):9411-9419.
Dengue virus has emerged as a global health threat to over one-third of humankind. As a positive-strand RNA virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. Therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. In a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and floxuridine inhibited dengue virus infections at low micromolar concentrations. Here, we demonstrate that all serotypes of dengue virus, as well as West Nile virus, are highly sensitive to both methotrexate and floxuridine, whereas other RNA viruses (Sindbis virus and vesicular stomatitis virus) are not. Interestingly, flavivirus replication was restored by folinic acid, a thymidine precursor, in the presence of methotrexate and by thymidine in the presence of floxuridine, suggesting an unexpected role for thymidine in flavivirus replication. Since thymidine is not incorporated into RNA genomes, it is likely that increased thymidine production is indirectly involved in flavivirus replication. A possible mechanism is suggested by the finding that p53 inhibition restored dengue virus replication in the presence of floxuridine, consistent with thymidine-less stress triggering p53-mediated antiflavivirus effects in infected cells. Our data reveal thymidine synthesis pathways as new and unexpected therapeutic targets for antiflaviviral drug development.
doi:10.1128/JVI.00101-13
PMCID: PMC3754125  PMID: 23824813
2.  Influence of Clinical Experience and Productivity on Emergency Medicine Faculty Teaching Scores 
Background
Commonly cited barriers to effective teaching in emergency medicine include lack of time, competing demands for patient care, and a lack of formal teaching experience. Teaching may be negatively affected by demands for increased clinical productivity, or positively influenced by clinical experience.
Objective
To examine the association between faculty teaching scores and clinical productivity, years of clinical experience, and amount of clinical contact with resident physicians.
Methods
We conducted a retrospective, observational study with existing data on full-time faculty at a high-volume, urban emergency medicine residency training program for academic year 2008–2009. Residents rated faculty on 9 domains of teaching, including willingness to teach, enthusiasm for teaching, medical knowledge, preparation, and communication. Clinical productivity data for relative value units per hour and number of patients per hour, years of clinical experience, and annual clinical hours were obtained from existing databases.
Results
For the 25 core faculty members included in the study, there was no relationship between faculty teaching scores and clinical productivity measures (relative value units per hour: r2  =  0.01, P  =  .96, patients per hour: r2  =  0.00, P  =  .76), or between teaching scores and total clinical hours with residents (r2  =  0.07, P  =  .19). There was a significant negative relationship between years of experience and teaching scores (r2  =  0.27, P < .01).
Conclusions
Our study demonstrated that teaching scores for core emergency medicine faculty did not correlate with clinical productivity or amount of clinical contact with residents. Teaching scores were inversely related to number of years of clinical experience, with more experienced faculty earning the lowest teaching scores. Further study is necessary to determine if there are clinical measures that identify good educators.
doi:10.4300/JGME-D-11-00193.1
PMCID: PMC3546571  PMID: 24294418
3.  APOBEC3G Restricts HIV-1 to a Greater Extent than APOBEC3F and APOBEC3DE in Human Primary CD4+ T Cells and Macrophages 
Journal of Virology  2013;87(1):444-453.
APOBEC3 proteins inhibit HIV-1 replication in experimental systems and induce hypermutation in infected patients; however, the relative contributions of several APOBEC3 proteins to restriction of HIV-1 replication in the absence of the viral Vif protein in human primary CD4+ T cells and macrophages are unknown. We observed significant inhibition of HIV-1Δvif produced in 293T cells in the presence of APOBEC3DE (A3DE), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H haplotype II (A3H HapII) but not APOBEC3B (A3B), APOBEC3C (A3C), or APOBEC3H haplotype I (A3H HapI). Our previous studies showed that Vif amino acids Y40RHHY44 are important for inducing proteasomal degradation of A3G, whereas amino acids 14DRMR17 are important for degradation of A3F and A3DE. Here, we introduced substitution mutations of 40YRHHY44 and 14DRMR17 in replication-competent HIV-1 to generate vif mutants NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 to compare the antiviral activity of A3G to the combined antiviral activity of A3F and A3DE in activated CD4+ T cells and macrophages. During the first 15 days (round 1), in which multiple cycles of viral replication occurred, both the NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutants replicated in activated CD4+ T cells and macrophages, and only the NL4-3 YRHHY>A5 mutant showed a 2- to 4-day delay in replication compared to the wild type. During the subsequent 27 days (round 2) of cultures initiated with peak virus obtained from round 1, the NL4-3 YRHHY>A5 mutant exhibited a longer, 8- to 10-day delay and the NL4-3 DRMR>A4 mutant exhibited a 2- to 6-day delay in replication compared to the wild type. The NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutant proviruses displayed G-to-A hypermutations primarily in GG and GA dinucleotides as expected of A3G- and A3F- or A3DE-mediated deamination, respectively. We conclude that A3G exerts a greater restriction effect on HIV-1 than A3F and A3DE.
doi:10.1128/JVI.00676-12
PMCID: PMC3536366  PMID: 23097438
4.  Induction of the Cellular MicroRNA, Hs_154, by West Nile Virus Contributes to Virus-Mediated Apoptosis through Repression of Antiapoptotic Factors 
Journal of Virology  2012;86(9):5278-5287.
MicroRNAs (miRNAs) are a class of noncoding small RNAs that regulate multiple cellular processes, as well as the replication and pathogenesis of many DNA viruses and some RNA viruses. Examination of cellular miRNA profiles in West Nile virus (WNV)-infected HEK293 and SK-N-MC cells revealed increased expression of multiple miRNA species. One of these miRNAs, Hs_154, was significantly induced not only in WNV-infected neuronal cells in culture but also in the central nervous system tissues of infected mice and, upon transfection, caused a significant reduction in viral replication. Analysis of mRNA transcripts enriched through immunoprecipitation of the RNA-induced silencing complex identified several transcripts that contain seed sequence matches to Hs_154 in their 3′ untranslated regions (UTRs). Two of these targets, the CCCTC-binding factor (CTCF) and the epidermal growth factor receptor (EGFR)-coamplified and overexpressed protein (ECOP/VOPP1) proteins display reduced expression in WNV-infected cells, and the 3′ UTRs of these transcripts were sufficient to cause downregulation of expression in infected cells or in cells transfected with Hs_154, findings consistent with miRNA targeting of these transcripts. CTCF and ECOP have been shown to be associated with cell survival, implicating miRNA-directed repression of these targets in WNV-induced cell death. Consistent with this hypothesis, expression of these genes in WNV-infected cells results in a reduction in the number of cells undergoing apoptosis. These observations suggest that induction of Hs_154 expression after WNV infection modulates the apoptotic response to WNV and that cellular miRNA expression can be quickly altered during WNV infection to control aspects of the host response.
doi:10.1128/JVI.06883-11
PMCID: PMC3347395  PMID: 22345437
5.  Inhibition of Dengue Virus Infections in Cell Cultures and in AG129 Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence ▿  
Journal of Virology  2011;85(19):10154-10166.
The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5′ cyclization sequence (5′CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an “in vivo-ready” version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.
doi:10.1128/JVI.05298-11
PMCID: PMC3196423  PMID: 21795337
6.  Virus activated filopodia promote human papillomavirus type 31 uptake from the extracellular matrix 
Virology  2008;381(1):16-21.
Human papillomaviruses (HPVs), etiological agents of epithelial tumors and cancers, initiate infection of basal human keratinocytes (HKs) facilitated by wounding. Virions bind to HKs and their secreted extracellular matrix (ECM), but molecular roles for wounding or ECM binding during infection are unclear. Herein we demonstrate HPV31 activates signals promoting cytoskeletal rearrangements and virion transport required for internalization and infection. Activation of tyrosine and PI3 kinases precedes induction of filopodia whereon virions are transported toward the cell body. Coupled with loss of ECM bound virions this supports a model whereby virus activated filopodial transport contributes to increased and protracted virion uptake into susceptible cells.
doi:10.1016/j.virol.2008.08.040
PMCID: PMC3253369  PMID: 18834609
papillomavirus; filopodia; signaling; keratinocyte; cytoskeleton; extracellular matrix
7.  High-Content Assay to Identify Inhibitors of Dengue Virus Infection 
Abstract
Dengue virus (DENV) infections are vectored by mosquitoes and constitute one of the most prevalent infectious diseases in many parts of the world, affecting millions of people annually. Current treatments for DENV infections are nonspecific and largely ineffective. In this study, we describe the adaptation of a high-content cell-based assay for screening against DENV-infected cells to identify inhibitors and modulators of DENV infection. Using this high-content approach, we monitored the inhibition of test compounds on DENV protein production by means of immunofluorescence staining of DENV glycoprotein envelope, simultaneously evaluating cytotoxicity in HEK293 cells. The adapted 384-well microtiter-based assay was validated using a small panel of compounds previously reported as having inhibitory activity against DENV infections of cell cultures, including compounds with antiviral activity (ribavirin), inhibitors of cellular signaling pathways (U0126), and polysaccharides that are presumed to interfere with virus attachment (carrageenan). A screen was performed against a collection of 5,632 well-characterized bioactives, including U.S. Food and Drug Administration–approved drugs. Assay control statistics show an average Z' of 0.63, indicative of a robust assay in this cell-based format. Using a threshold of >80% DENV inhibition with <20% cellular cytotoxicity, 79 compounds were initially scored as positive hits. A follow-up screen confirmed 73 compounds with IC50 potencies ranging from 60 nM to 9 μM and yielding a hit rate of 1.3%. Over half of the confirmed hits are known to target transporters, receptors, and protein kinases, providing potential opportunity for drug repurposing to treat DENV infections. In summary, this assay offers the opportunity to screen libraries of chemical compounds, in an effort to identify and develop novel drug candidates against DENV infections.
doi:10.1089/adt.2010.0321
PMCID: PMC2962577  PMID: 20973722
8.  Identification of Specific Determinants of Human APOBEC3F, APOBEC3C, and APOBEC3DE and African Green Monkey APOBEC3F That Interact with HIV-1 Vif ▿  
Journal of Virology  2010;84(24):12599-12608.
Human APOBEC3F (hA3F) and human APOBEC3G (hA3G) are potent anti-human immunodeficiency virus (anti-HIV) host factors that suppress viral replication by hypermutating the viral genome, inhibiting reverse transcription, and hindering integration. To overcome hA3F and hA3G, HIV-1 encodes Vif, which binds and targets these host proteins for proteasomal degradation. Previously, we reported that the hA3F-Vif interactions that lead to hA3F degradation are located in the region comprising amino acids 283 to 300. We have now performed mutational analysis of this region and found that the 289EFLARH294 amino acids contribute to hA3F-Vif binding and are critical for A3F's sensitivity to Vif. Mutants in which E289 is mutated significantly increase hA3F's ability to inhibit viral infectivity in the presence of Vif, and coimmunoprecipitation assays show that binding of Vif to the E289K mutant is decreased. We examined the role of the EFLARH sequence in other A3 proteins, including human A3C (hA3C), human A3DE (hA3DE), African green monkey A3F (agmA3F), and rhesus macaque A3F (rhA3F). hA3C, hA3DE, and agmA3F were all susceptible to degradation induced by HIV-1 Vif, while rhA3F was not. Mutagenesis of the glutamate in the EFLARH sites of hA3C, hA3DE, and agmA3F decreases the susceptibilities of these proteins to Vif-induced degradation. Together, these results indicate that the EFLARH region in hA3F, hA3C, hA3DE, and agmA3F interacts with HIV-1 Vif and that this interaction plays a role in the Vif-mediated proteasomal degradation of these A3 proteins. These studies identify a conserved region in 3 of 7 human A3 proteins that is critical for degradation mediated by HIV-1 Vif and provide structural insights into the hA3F-Vif interactions that could facilitate the development of a novel class of anti-HIV agents.
doi:10.1128/JVI.01437-10
PMCID: PMC3004357  PMID: 20943965
9.  Reporting of complex interventions in clinical trials: development of a taxonomy to classify and describe fall-prevention interventions 
Trials  2011;12:125.
Background
Interventions for preventing falls in older people often involve several components, multidisciplinary teams, and implementation in a variety of settings. We have developed a classification system (taxonomy) to describe interventions used to prevent falls in older people, with the aim of improving the design and reporting of clinical trials of fall-prevention interventions, and synthesis of evidence from these trials.
Methods
Thirty three international experts in falls prevention and health services research participated in a series of meetings to develop consensus. Robust techniques were used including literature reviews, expert presentations, and structured consensus workshops moderated by experienced facilitators. The taxonomy was refined using an international test panel of five health care practitioners. We assessed the chance corrected agreement of the final version by comparing taxonomy completion for 10 randomly selected published papers describing a variety of fall-prevention interventions.
Results
The taxonomy consists of four domains, summarized as the "Approach", "Base", "Components" and "Descriptors" of an intervention. Sub-domains include; where participants are identified; the theoretical approach of the intervention; clinical targeting criteria; details on assessments; descriptions of the nature and intensity of interventions. Chance corrected agreement of the final version of the taxonomy was good to excellent for all items. Further independent evaluation of the taxonomy is required.
Conclusions
The taxonomy is a useful instrument for characterizing a broad range of interventions used in falls prevention. Investigators are encouraged to use the taxonomy to report their interventions.
doi:10.1186/1745-6215-12-125
PMCID: PMC3127768  PMID: 21586143
10.  P Body-Associated Protein Mov10 Inhibits HIV-1 Replication at Multiple Stages ▿ †  
Journal of Virology  2010;84(19):10241-10253.
Recent studies have shown that APOBEC3G (A3G), a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is localized to cytoplasmic mRNA-processing bodies (P bodies). However, the functional relevance of A3G colocalization with P body marker proteins has not been established. To explore the relationship between HIV-1, A3G, and P bodies, we analyzed the effects of overexpression of P body marker proteins Mov10, DCP1a, and DCP2 on HIV-1 replication. Our results show that overexpression of Mov10, a putative RNA helicase that was previously reported to belong to the DExD superfamily and was recently reported to belong to the Upf1-like group of helicases, but not the decapping enzymes DCP1a and DCP2, leads to potent inhibition of HIV-1 replication at multiple stages. Mov10 overexpression in the virus producer cells resulted in reductions in the steady-state levels of the HIV-1 Gag protein and virus production; Mov10 was efficiently incorporated into virions and reduced virus infectivity, in part by inhibiting reverse transcription. In addition, A3G and Mov10 overexpression reduced proteolytic processing of HIV-1 Gag. The inhibitory effects of A3G and Mov10 were additive, implying a lack of functional interaction between the two inhibitors. Small interfering RNA (siRNA)-mediated knockdown of endogenous Mov10 by 80% resulted in a 2-fold reduction in virus production but no discernible impact on the infectivity of the viruses after normalization for the p24 input, suggesting that endogenous Mov10 was not required for viral infectivity. Overall, these results show that Mov10 can potently inhibit HIV-1 replication at multiple stages.
doi:10.1128/JVI.00585-10
PMCID: PMC2937795  PMID: 20668078
11.  MULTIPLE WAYS OF TARGETING APOBEC3/VIF INTERACTIONS FOR ANTI-HIV-1 DRUG DEVELOPMENT 
Trends in pharmacological sciences  2009;30(12):638-646.
Human immunodeficiency virus type 1 (HIV-1) infections and the resulting acquired immunodeficiency syndrome (AIDS) pandemic remain a global challenge in the absence of a protective vaccine and because of rapid selection of drug-resistant viral variants in response to all currently available antiviral therapies. Development of new and highly active antiviral agents would greatly facilitate effective clinical management of HIV-1 infections and delay the onset of AIDS. Recent advances in our understanding of intracellular immunity conferred by host cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F), and the mechanism by which the virally encoded Virion Infectivity Factor (Vif) protein induces their proteasomal degradation, provide fresh opportunities for the development of novel antiviral treatments. Interestingly, the interactions between Vif-A3G and Vif-A3F that overcome this host defense mechanism are structurally distinct, and provide two potential targets for antiviral drug development. This review provides an overview of the current knowledge of APOBEC3/Vif interactions and recent efforts to target these interactions for antiviral drug development.
doi:10.1016/j.tips.2009.09.006
PMCID: PMC2787722  PMID: 19837465
12.  Antagonism of human adiponectin receptors and their membrane progesterone receptor paralogs by TNFα and a ceramidase inhibitor. † 
Biochemistry  2009;48(24):5504-5506.
The Progestin and AdipoQ Receptor (PAQR) family of proteins comprises three distinct structural classes, each with seemingly different agonist specificities. For example, Class I receptors, like the human adiponectin receptors (AdipoR1 and AdipoR2), sense proteins with a particular three-dimensional fold, while Class II receptors are non-classical membrane receptors for the steroid hormone progesterone. Using a previously developed heterologous expression system to study PAQR receptor activity, we demonstrate that human PAQRs from all three classes are antagonized by both 1S,2R-D-erythro-2-N-myristoylamino)-1-phenyl-1-propanol, a ceramidase inhibitor, and TNFα, a homolog of adiponectin that functions antagonistically to both adiponectin and progesterone in human cells.
doi:10.1021/bi9006258
PMCID: PMC2789275  PMID: 19453184
13.  Adiponectin identified as an agonist for PAQR3/RKTG using a yeast-based assay system 
The PAQR family of proteins comprises an intriguing group of newly discovered receptors. Although the agonist is known for 5 of the 11 human PAQRs, most are considered “orphan” receptors. We developed a yeast-based assay system for PAQR receptor activity that can be used to identify agonists for PAQRs of unknown function. Using this system, we found that the proteinaceous hormone adiponectin functions as an agonist of PAQR3, a previously uncharacterized member of this family. This is not surprising given that PAQR3 is most closely related to PAQR1 (AdipoR1) and PAQR2 (AdipoR2), which also sense adiponectin. The identification of adiponectin as an agonist for PAQR3 is of considerable clinical relevance because adiponectin suppresses the proliferation of tumor cells and it has been reported that PAQR3 suppresses tumorigenesis. Thus, the interaction between PAQR3 and adiponectin may help explain the antiproliferative properties of adiponectin.
doi:10.1080/10799890902729456
PMCID: PMC2792888  PMID: 19519172
Adiponectin receptor; PAQR3; RKTG; Yeast; AdipoR1
14.  Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors 
Steroids  2008;73(11):1160-1173.
The nuclear progesterone receptor (nPR) mediates many of the physiological effects of progesterone by regulating the expression of genes, however, progesterone also exerts non-transcriptional (non-genomic) effects that have been proposed to rely on a receptor that is distinct from nPR. Several members of the Progestin and AdipoQ Receptor (PAQR) family were recently identified as potential mediators of these non-genomic effects. Membranes from cells expressing these proteins, called mPRα, mPRβ and mPRγ, were shown to specifically bind progesterone and have G-protein coupled receptor (GPCR) characteristics, although other studies dispute these findings. To clarify the role of these mPRs in non-genomic progesterone signaling, we established an assay for PAQR functional evaluation using heterologous expression in Saccharomyces cerevisiae. Using this assay, we demonstrate unequivocally that mPRα, mPRβ and mPRγ can sense and respond to progesterone with EC50 values that are physiologically relevant. Agonist profiles also show that mPRα, mPRβ and mPRγ are activated by ligands, such as 17α-hydroxyprogesterone, that are known to activate non-genomic pathways but not nPR. These results strongly suggest that these receptors may indeed function as the long-sought-after membrane progesterone receptors. Additionally, we show that two uncharacterized PAQRs, PAQR6 and PAQR9, are also capable of responding to progesterone. These mPR-like PAQRs have been renamed mPRδ (PAQR6) and mPRε (PAQR9). Additional characterization of mPRγ and mPRα indicate that their progesterone-dependent signaling in yeast does not require heterotrimeric G-proteins, thus calling into question the characterization of the mPRs as a novel class of G-protein coupled receptor.
doi:10.1016/j.steroids.2008.05.003
PMCID: PMC2597464  PMID: 18603275
membrane progestin receptors; progesterone; yeast; adiponectin; osmotin
15.  Caveolin-1-Dependent Infectious Entry of Human Papillomavirus Type 31 in Human Keratinocytes Proceeds to the Endosomal Pathway for pH-Dependent Uncoating▿  
Journal of Virology  2008;82(19):9505-9512.
High-risk human papillomaviruses (HPVs) are small nonenveloped DNA viruses with a strict tropism for squamous epithelium. The viruses are causative agents of cervical cancer and some head and neck cancers, but their differentiation-dependent life cycles have made them difficult to study in simple cell culture. Thus, many aspects of early HPV infection remain mysterious. We recently showed the high-risk HPV type 31 (HPV31) enters its natural host cell type via caveola-dependent endocytosis, a distinct mechanism from that of the closely related HPV16 (Smith et al., J. Virol. 81:9922-9931, 2007). Here, we determined the downstream trafficking events after caveolar entry of HPV31 into human keratinocytes. After initial plasma membrane binding, HPV31 associates with caveolin-1 and transiently localizes to the caveosome before trafficking to the early endosome and proceeding through the endosomal pathway. Caveosome-to-endosome transport was found to be Rab5 GTPase dependent. Although HPV31 capsids were observed in the lysosome, Rab7 GTPase was dispensable for HPV31 infection, suggesting that viral genomes escape from the endosomal pathway prior to Rab7-mediated capsid transport. Consistent with this, the acidic pH encountered by HPV31 within the early endosomal pathway induces a conformational change in the capsid resulting in increased DNase susceptibility of the viral genome, which likely aids in uncoating and/or endosomal escape. The entry and trafficking route of HPV31 into human keratinocytes represents a unique viral pathway by which the virions use caveolar entry to eventually access a low-pH site that appears to facilitate endosomal escape of genomes.
doi:10.1128/JVI.01014-08
PMCID: PMC2546945  PMID: 18667513
16.  Human Papillomavirus Type 31 Uses a Caveolin 1- and Dynamin 2-Mediated Entry Pathway for Infection of Human Keratinocytes▿  
Journal of Virology  2007;81(18):9922-9931.
Papillomaviruses are species-specific and epitheliotropic DNA viruses that cause tumors in their natural hosts. Certain infections with genital human papillomavirus (HPV) types are causally related to cervical cancer development. Most papillomaviruses are thought to infect cells via a clathrin-dependent pathway, yet no studies have determined the entry route in permissive host epithelial cells. Employing fluorescently labeled and native virions, we tested the effects of dominant-negative and biochemical inhibitors of cellular endocytosis pathways. Infections of human keratinocytes, a natural host cell type for HPVs, were assessed visually and by infectious entry assays. We found that HPV type 31 (HPV31) entry and initiation of early infection events require both caveolin 1 and dynamin 2 and occur independently of clathrin-mediated endocytosis. Treatment with chlorpromazine and filipin had opposing effects on HPV31 and HPV16 infection. HPV31 entry was remarkably slow, with a half-time of ≈14 h, whereas the entry half-time of HPV16 was 4 h. Consistent with a caveola-mediated entry pathway for HPV31, the virions associated with detergent-resistant lipid rafts. During a 16-h microscopic tracking of HPV31 and HPV16 virions, no colocalization of the two viral types was observed. These data suggest that HPV31 and HPV16 virions use distinct routes for host epithelial cell entry.
doi:10.1128/JVI.00988-07
PMCID: PMC2045393  PMID: 17626097
17.  Human Papillomavirus Type 31b Infection of Human Keratinocytes Does Not Require Heparan Sulfate 
Journal of Virology  2005;79(11):6838-6847.
Oncogenic human papillomaviruses (HPVs) are difficult to study experimentally as they replicate at low levels in vivo. This has precluded the purification of high-risk HPV virions from in vivo lesions. Virus-like particles (VLPs) and pseudovirions from low- and high-risk HPV types can emulate various aspects of HPV virion attachment and infections. These studies suggest that HPV infection is mediated by α6-integrin and/or heparan-sulfonated receptors. However, whether VLPs and pseudovirions accurately reflect the infection process of HPV virions has not been verified. We generated infectious HPV31b virions from organotypic (raft) tissues and performed experimental infections in a variety of cells. Successful infection following viral attachment, internalization, and nuclear transport was assayed by detecting newly synthesized, spliced HPV transcripts using reverse transcription (RT)-PCR or RT-quantitative PCR. Most human epithelial cells were infected with HPV31b at a multiplicity of infection as low as 1 to 10 viral genome equivalents per cell. HPV31b infection was detected in other cell lines, including COS-7 monkey kidney cells, but higher viral multiplicities of infection were required. Heparin preparations of various molecular weights or heparinase I treatment of cells prevented HPV31b infection of COS-7 cells and C-33A human cervical cancer cells in reproducible and dose-dependent manners. However, these reagents were unable to block infection of human keratinocytes, including HaCaT and N/TERT-1 cells and low-passage human foreskin keratinocytes. These data suggest that HPV31b infection of human keratinocytes, the natural host cell for HPV infections in vivo, does not require a heparan-sulfonated receptor, whereas heparan sulfate is important for infection of some other cells.
doi:10.1128/JVI.79.11.6838-6847.2005
PMCID: PMC1112118  PMID: 15890923

Results 1-17 (17)