Cleavage of the HPV16 Minor Capsid Protein L2 during Virion Morphogenesis Ablates the Requirement for Cellular Furin during De Novo Infection

Infections by high-risk human papillomaviruses (HPV) are the causative agents for the development of cervical cancer. As with other non-enveloped viruses, HPVs are taken up by the cell through endocytosis following primary attachment to the host cell. Through studies using recombinant pseudovirus particles (PsV), many host cellular proteins have been implicated in the process. The proprotein convertase furin has been demonstrated to cleave the minor capsid protein, L2, post-attachment to host cells and is required for infectious entry by HPV16 PsV. In contrast, using biochemical inhibition by a furin inhibitor and furin-negative cells, we show that tissue-derived HPV16 native virus (NV) initiates infection independent of cellular furin. We show that HPV16 L2 is cleaved during virion morphogenesis in differentiated tissue. In addition, HPV45 is also not dependent on cellular furin, but two other alpha papillomaviruses, HPV18 and HPV31, are dependent on the activity of cellular furin for infection.     

Inactivation of Polyomavirus SV40 as Surrogate for Human Papillomaviruses by Chemical Disinfectants

Human papillomaviruses (HPV) are non-enveloped DNA viruses infecting cutaneous and mucosal squamous epithelia. Sexually transmitted HPV-types that are carcinogenic to humans such as HPV16 can induce cervical and other anogenital cancers. Virus transmission through fomites such as inadequately disinfected gynecological equipment is a further potential transmission route. Since HPV cannot be easily grown in cell culture, polyomavirus SV40 has been used as a surrogate virus when testing the virucidal activity of chemical disinfectants. So far, studies that have compared the virucidal activity of different disinfectants against HPV and SV40 are lacking. Here, we evaluated the susceptibility of HPV16 pseudovirus and SV40 to seven active biocidal substances using quantitative suspension tests. Ethanol, glutaraldehyde (GTA), dodecyldipropylentriamin (DPTA), and ortho-phthalaldehydes (OPA) were able to reduce the infectivity of HPV16 pseudovirus >99.99% after 5 min. In contrast, isopropanol, peracetic acid (PAA), and quaternary ammonium compounds with alkylamines (QAC) only led to a slight or no reduction in infectivity. Concerning SV40, only GTA (60 min contact time), PAA, and OPA had virus-inactivating effects. In conclusion, the virucidal activity of three out of seven disinfectants tested was different for HPV16 pseudovirus and SV40. In this study, SV40 was shown to be a reliable surrogate virus for HPV when testing isopropanol-, GTA-, QAC-, and OPA-based disinfectants.     

The Tetraspanin CD151 in Papillomavirus Infection

Human papillomaviruses (HPV) are non-enveloped DNA tumor viruses that infect skin and mucosa. The most oncogenic subtype, HPV16, causes various types of cancer, including cervical, anal, and head and neck cancers. During the multistep process of infection, numerous host proteins are required for the delivery of virus genetic information into the nucleus of target cells. Over the last two decades, many host-cell proteins such as heparan sulfate proteoglycans, integrins, growth factor receptors, actin and the tetraspanin CD151 have been described to be involved in the process of infectious entry of HPV16. Tetraspanins have the ability to organize membrane microdomains and to directly influence the function of associated molecules, including binding of receptors to their ligands, receptor oligomerization and signal transduction. Here, we summarize the current knowledge on CD151, and CD151-associated partners during HPV infection and discuss the underlying mechanisms.     

Persistent Human Papillomavirus Infection

Simple SummaryThe success of HPV as an infectious agent lies not within its ability to cause disease, but rather in the adeptness of the virus to establish long-term persistent infection. The ability of HPV to replicate and maintain its genome in a stratified epithelium is contingent on the manipulation of many host pathways. HPVs must abrogate host anti-viral defense programs, perturb the balance of cellular proliferation and differentiation, and hijack DNA damage signaling and repair pathways to replicate viral DNA in a stratified epithelium. Together, these characteristics contribute to the ability of HPV to achieve long-term and persistent infection and to its evolutionary success as an infectious agent.AbstractPersistent infection with oncogenic human papillomavirus (HPV) types is responsible for ~5% of human cancers. The HPV infectious cycle can sustain long-term infection in stratified epithelia because viral DNA is maintained as low copy number extrachromosomal plasmids in the dividing basal cells of a lesion, while progeny viral genomes are amplified to large numbers in differentiated superficial cells. The viral E1 and E2 proteins initiate viral DNA replication and maintain and partition viral genomes, in concert with the cellular replication machinery. Additionally, the E5, E6, and E7 proteins are required to evade host immune responses and to produce a cellular environment that supports viral DNA replication. An unfortunate consequence of the manipulation of cellular proliferation and differentiation is that cells become at high risk for carcinogenesis.     

The Process of Filopodia Induction during HPV Infection

Human Papillomavirus 16 (HPV16) infects mucosal and epithelial cells and has been identified as a high-risk HPV type that is an etiologic agent of human cancers. The initial infectious process, i.e., the binding of the virus particle and its entry into the host cell, has been studied extensively, although it is not fully understood. There is still a gap in understanding the steps by which the virus is able to cross the plasma membrane after receptor binding. In this study, we demonstrate that after HPV16 comes into contact with a plasma membrane receptor, there are cytoskeletal changes resulting in an increase of filopodia numbers. This increase in filopodia numbers was transient and was maintained during the first two hours after virus addition. Our data show that there is a statistically significant increase in infection when filopodia numbers are increased by the addition of drug and virus simultaneously, and a decrease in virus infection when filopodia formation is inhibited. We describe that HPV16 binding results in the activation of Cdc42 GTPase that in turn results in an increase in filopodia. siRNA directed at Cdc42 GTPase resulted in a statistically significant reduction of infection and a corresponding lack of filopodia induction.     

E4 expression of human papillomavirus in cervical samples with different cytology categories

In some circumstances, infection with human papillomavirus (HPV), especially HPV type 16 (HPV16), progresses to cervical cancer. Viral E4 expression reflects viral replication and translation and its presence may rule out a latent infectious stage. Twenty cervical cytology samples with known HPV 16 infection of each cytological category namely, negative for intraepithelial lesion (NIL), low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesion (HSIL) and squamous carcinoma (SCC) were investigated by single-step quantitative RT PCR for HPV 16 E4 mRNA, which was not found in any NIL sample but in all LSIL and HSIL samples. Thus, E4 expression assay should be useful for determining precancerous states and may be suitable as an adjunct in cervical HPV testing.     

The Role of Human Papillomavirus in Human Immunodeficiency Virus Acquisition in Men who have Sex with Men: A Review of the Literature

Human Papillomavirus (HPV) infection is the most common sexually transmitted infection (STI) worldwide. Incidence rates of HPV infection among human immunodeficiency virus (HIV)-infected individuals are well documented and are several-fold higher than among HIV-uninfected individuals. Few studies have demonstrated an increased risk for acquiring HIV infection in those with HPV infection, and this risk seems to be higher when HPV strains are of high-risk oncogenic potential. The estimated prevalence of high-risk oncogenic HPV infection is highest in men who have sex with men (MSM), a particularly vulnerable group with high prevalence rates of HIV infection and other STIs. In this paper, we provide a comprehensive review of the available literature on the role of HPV infection in HIV acquisition. Our review includes data from cross-sectional and longitudinal studies.     

Potential role of human papilloma virus in the pathogenesis of gastric cancer

AIM:To demonstrate the presence and biological activity of human papilloma virus(HPV)in gastric cancer(GAC)tissues.METHODS:The study involved 84 surgically treated patients with gastric adenocarcinoma,regardless of the clinical stage of the disease.The presence of HPV DNA of high oncogenic risk types in formalin-fixed,paraffinembedded tumor samples was determined using quantitative polymerase chain reaction analysis.A stringentprotocol of prevention of cross-and environmental contamination was applied during DNA isolation,and amplification,as well as confirmation of the biological activity of the virus in tumor cells,was implemented.The study utilized the Real-time High Risk HPV test,which detects the DNA of 14 HPV subtypes that are considered to have high oncogenic potential.The overexpression of the p16INK4a protein assessed immunohistochemically was considered confirmation of the HPV infection.RESULTS:Among the 89 patients initially included in the study group,diagnostic results were obtained for84 individuals.In five cases,either the histopathological material was too scant to isolate the necessary amount of DNA,or the isolated DNA was significantly degraded,resulting in the failure of internal control amplification within the predefined number of 35 cycles.Those patients were excluded from further analysis.The amplification of HPV DNA was demonstrated in none of the84 tissue samples;thus,all cases were considered to have a negative DNA status of highly oncogenic HPV subtypes.Immunohistochemical staining provided diagnostic results for all of the examined tissue samples,and excluded the accumulation of the p16INK4a protein in tumor cells,thus confirming the lack of active HPV infection in all of the individuals.CONCLUSION:The study does not confirm the presence or biological activity of HPV in tumor tissues.Thus,the relationship between GAC and HPV infection,in the Central European population seems doubtful.     

人乳头瘤病毒16型L1的表达、病毒样颗粒组装及其免疫原性研究

目的 在原核表达系统中表达HPV16 L1蛋白,纯化后在体外自组装成VLPs并鉴定。方法 优化GenBank中HPV16 L1基因序列并截短C末端25个氨基酸,构建至原核表达载体pET-28a上,获得重组表达载体pET28a-16L1△C25。采用镍亲和层析法纯化超声上清,于体外解组装-重组装HPV16 VLPs,采用动态光散射和透射电镜进行形貌分析,纯化后于第0、2和4周免疫小鼠,假病毒中和试验检测HPV16 VLPs免疫后血清中和抗体。结果 双酶切和测序结果表明成功构建pET28a-16L1△C25重组质粒,诱导表达后,经SDS-PAGE和Western blotting分析显示表达的L1蛋白大部分以可溶性形式存在,纯化后的蛋白样品于体外重新组装,动态光散射和透射电镜能够观察到形态与天然病毒颗粒相似的VLPs,第6周小鼠血清中和抗体滴度Log10平均值达到4.43。结论 利用原核表达系统成功表达了截短型HPV16 L1蛋白,并于体外组装成结构完整的VLPs,且具有较好的免疫原性,为低成本HPV预防性疫苗的研发奠定基础。     

Human papillomavirus prevalence, cervical abnormalities and risk factors among female sex workers in Lima, Peru

Female sex workers (FSWs) are at high risk of human papillomavirus (HPV) infection. Questionnaires were administered to 200 FSWs aged 18-26 years in Lima, Peru, to gather risk behaviours, and cervical swab samples were collected for Pap smears and HPV DNA testing as part of a longitudinal study. Participants reported a median of 120 clients in the past month, and 99.2% reported using condoms with clients. The prevalence of any HPV in cervical samples was 66.8%; 34 (17.1%) participants had prevalent HPV 16 or 18, and 92 (46.2%) had one or more oncogenic types. Fifteen women had abnormal Pap smears, 13 of which were HPV DNA positive. Fewer years since first sex was associated with oncogenic HPV prevalence in a model adjusted for previous sexually transmitted infection (STI) status and condom use with partners (prevalence ratio = 0.77, 95% confidence interval [CI] = 0.60-0.97). Our data confirm the high rates of HPV transmission among FSWs in Peru, highlighting the need for early and effective strategies to prevent cervical cancer.     

Human papillomavirus subtype 16 is common in Pakistani women with cervical carcinoma.

INTRODUCTION: Human papillomavirus (HPV) is recognized as a major causative agent for cervical carcinomas. Based on their oncogenic potential, HPV subtypes have been divided into high- and low-risk. In Pakistan, screening for HPV in female patients is not commonly practiced, and as a consequence, the degree of HPV prevalence and its correlation with cervical cancer is unknown. OBJECTIVE: In this study, we have attempted to estimate the prevalence of HPV infection, and also the HPV subtype profile, among Pakistani women with cervical cancer from varied geographical, racial, and social backgrounds within Pakistan. METHODOLOGY: Women visiting two tertiary care hospitals in Karachi, diagnosed with carcinoma of the cervix within the past 15 years, were analyzed for HPV subtypes in their cancer specimens. Retrospectively, 60 paraffin-embedded cervical cancer biopsies were examined for the presence of HPV DNA. After DNA extraction from these samples, polymerase chain reaction (PCR) was used to amplify the HPV L1 gene using the consensus (general) primers, and primers specific for subtypes 16 and 18. RESULTS: Of the 60 samples analyzed, only one sample was HPV negative; the rest of the samples were positive for the presence of HPV. Of the 59 HPV positive samples, 56 showed the presence of HPV16 and one sample was positive for HPV18; HPV subtype could not be determined in two samples. CONCLUSION: Our results show a strong relationship between HPV infection and cervical cancer among Pakistani women. These results underscore the need to implement regular HPV screening for Pakistani women. An early diagnosis of HPV infection will allow better health management to reduce the risk of developing cervical cancer.     

Incidence,prevalence, and clearance of type-specific human papillomavirus infections: The Young Women's Health Study

The natural history of type-specific human papillomavirus (HPV) infections was examined in a cohort of 331 women aged 18-35 years who self-referred for routine gynecological care. Participants underwent a gynecological examination at baseline and at approximately 4 and approximately 10 months after baseline. Cervical samples were collected for HPV testing and genotyping at each visit, as was information on reproductive, sexual, and medical histories. The rate of new HPV infections was 2.9% per month; the highest rates were observed for HPV types 16, 39, 84, and 51. Among women who tested negative for HPV at baseline, the cumulative probability of acquiring an oncogenic HPV strain during a 12-month follow-up period was 0.32, compared with 0.18 for nononcogenic strains. Women who had had >/=1 new male sex partner in the recent past were significantly more likely to acquire a new HPV infection (relative hazard, 2.39; 95% confidence interval, 1.20-4.76). The median time to clearance of infection was significantly longer for oncogenic strains (9.8 months) than for nononcogenic strains (4.3 months).     

人乳头状瘤病毒16型伪病毒载体对肝癌细胞系HepG2细胞的杀伤研究

目的 构建基于人乳头状瘤病毒（HPV）16型的伪病毒载体,并检测其对肝癌细胞系HepG2细胞的杀伤活性。方法利用昆虫什状病毒表达系统表达病毒蛋白,在体外将病毒蛋白包装白喉毒（DT）A链表达型质粒,形成伪病毒。透射电镜观察病毒样颗粒（VLP）的结构,并转染肝癌细胞系HepG2。乳酸脱氢酶释放法检测对肝癌细胞系HepG2的杀伤能力。结果透射电镜观察显示病毒蛋白可自我组装成VLP,在转染肝癌细胞系HepG2后,乳酸脱氢酶释放法成功检测到伪病毒埘肝癌细胞系HepG2的杀伤作用。结论基于HPV16的新型伪病毒载体能成功转染肝癌细胞系HepG2,并产生杀伤活性,为肝癌的基因治疗提供了一种可选择的方法。     

Viperin,an IFN-Stimulated Protein,Delays Rotavirus Release by Inhibiting Non-Structural Protein 4 (NSP4)-Induced Intrinsic Apoptosis

Viral infections lead to expeditious activation of the host’s innate immune responses, most importantly the interferon (IFN) response, which manifests a network of interferon-stimulated genes (ISGs) that constrain escalating virus replication by fashioning an ill-disposed environment. Interestingly, most viruses, including rotavirus, have evolved numerous strategies to evade or subvert host immune responses to establish successful infection. Several studies have documented the induction of ISGs during rotavirus infection. In this study, we evaluated the induction and antiviral potential of viperin, an ISG, during rotavirus infection. We observed that rotavirus infection, in a stain independent manner, resulted in progressive upregulation of viperin at increasing time points post-infection. Knockdown of viperin had no significant consequence on the production of total infectious virus particles. Interestingly, substantial escalation in progeny virus release was observed upon viperin knockdown, suggesting the antagonistic role of viperin in rotavirus release. Subsequent studies unveiled that RV-NSP4 triggered relocalization of viperin from the ER, the normal residence of viperin, to mitochondria during infection. Furthermore, mitochondrial translocation of NSP4 was found to be impeded by viperin, leading to abridged cytosolic release of Cyt c and subsequent inhibition of intrinsic apoptosis. Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain. Collectively, the present study demonstrated the role of viperin in restricting rotavirus egress from infected host cells by modulating NSP4 mediated apoptosis, highlighting a novel mechanism behind viperin’s antiviral action in addition to the intricacy of viperin–virus interaction.     

Age-specific prevalence, incidence, and duration of human papillomavirus infections in a cohort of 290 US men

BACKGROUND: Human papillomavirus (HPV) infections cause disease in men and women, and male-to-female HPV transmission influences the risk of cancer in females. The purpose of the present study was to describe the overall and age-specific incidence and clearance of HPV infections in men. METHODS: In a prospective cohort study of 290 men aged 18-44 years, participants were examined at baseline and every 6 months, with a mean duration of follow-up of 15.5 months. RESULTS: The period prevalence was 52.8% for any, 31.7% for oncogenic, and 30.0% for nononcogenic HPV infection. The 12-month cumulative risk of acquiring a new HPV infection was 29.2%. Incidences of HPV types 6, 11, 16, and 18 were 2.8, 0.5, 4.8, and 0.8 per 1000 person-months, respectively. The median time to clearance of any HPV infection was 5.9 months (95% confidence interval, 5.7-6.1 months), with comparable times to clearance for oncogenic and nononcogenic infections. Approximately 75% of men tested negative for any HPV 12 months after initial HPV detection. Age was not significantly associated with HPV incidence or duration of infection in men. CONCLUSION: HPV infection in men was common, with relatively rapid rates of acquisition and clearance.     

Circumcision and human papillomavirus infection in men: a site-specific comparison

BACKGROUND: Lack of circumcision has been identified as a risk factor for male genital human papillomavirus (HPV) infection, although this association has not been consistently supported. METHODS: Specimens for HPV testing were collected from a cohort of 379 (primarily heterosexual) adult males. HPV prevalence in the glans penis and coronal sulcus, penile shaft, scrotum, semen, and urine was compared by circumcision status. RESULTS: Overall, HPV DNA prevalence ranged from 6% in semen to 52% in the penile shaft. The prevalence of any HPV infection in the glans/corona was significantly higher in uncircumcised men (46%) than in circumcised men (29%) (odds ratio [OR], 1.96 [95% confidence interval (CI), 1.02-3.75], adjusted for demographic characteristics and sexual history). Uncircumcised men also had an increased risk of oncogenic HPV infection (adjusted OR, 2.51 [95% CI, 1.11-5.69]) and infection with multiple HPV types (adjusted OR, 3.56 [95% CI, 1.50-8.50]). Among uncircumcised men, HPV prevalence in the foreskin (44%) was comparable to that in the glans/corona, and type-specific positivity was observed between the 2 sites (kappa=0.52). CONCLUSIONS: Uncircumcised men have an increased risk of HPV infection, including with oncogenic HPV, specifically localized to the glans/corona, possibly because of its proximity to the foreskin, which may be particularly vulnerable to infection.     

Laryngeal squamous cell carcinoma in HIV-positive patients: lack of association with human papillomavirus infection

Objectives Neoplasms associated with human papillomavirus (HPV) infection occur at increased frequency in patients with HIV infection/AIDS. Although laryngeal squamous cell carcinomas (LSCCs) in HIV-positive patients are uncommon, a higher incidence of this malignancy in HIV-positive patients than in the general population has been reported. As a proportion of LSCCs are associated with HPV in the general population, the clinicopathological features of a series of LSCCs developing in HIV-positive patients were evaluated to investigate the possible relationship with HPV infection, and infection with other oncogenic viruses.
Methods All HIV-positive patients with LSCC diagnosed at a single institution from 1998 to 2007 were retrospectively evaluated. The clinicopathological features were analysed and tissues were tested by polymerase chain reaction (PCR), using the short PCR fragment 10 (SPF10) primer, a highly sensitive method for HPV DNA detection. Immunohistochemical studies for HIV p24, p16^INK4a and p53 were performed. Epstein–Barr virus (EBV) and human herpes virus 8 (HHV-8) were also investigated.
Results Six out of 4987 HIV-infected patients seen in this period in the Infectious Diseases Department developed LSCC (median age 41.5 years; male to female ratio 1:1). All patients were heavy smokers and the tumours presented at an advanced clinical stage. HPV was not detected in any tumour, not even in two patients with coexisting HPV-associated gynaecological neoplasm. Staining for HIV p24 and p16^INK4a was negative, whereas p53 was positive in half the cases. EBV and HHV-8 were also negative.
Conclusion LSCC developing in HIV-positive patients is an infrequent neoplasm, not usually associated with HPV infection. It develops in young, heavy smokers and presents at an advanced clinical stage.     

What is the significance of the HPV epidemic?

Human papillomavirus (HPV) is the most common sexually transmitted infection. The incidence of this infection has been on the rise in recent times. It is estimated that approximately 6 million new HPV infections are acquired each year in the United States alone, and prevalence data suggest that as many as 24 million American adults--that is, 1 in 5--may be infected with HPV. Unfortunately, there is little public awareness and knowledge about the infection and its sequelae. It is well known that more than 90% of cases of anogenital warts are caused by HPV. HPV has been implicated in cancers of the cervix, vulva, vagina, penis, anus, and oropharynx. The virus is a necessary cause of cervical cancer. HPV DNA is detected in almost 100% of cases of cervical cancer. There have been major strides in recent years in the prevention of this infection and consequently, of diseases related to it. Vaccines are available and licensed in some countries. Two HPV vaccines are available: a quadrivalent (HPV types 6, 11, 16, and 18) vaccine and a bivalent (HPV types 16 and 18) vaccine. Both vaccines show a more than 90% protection against persistent HPV infection for up to 5 years after vaccination. The role of the vaccine in males is still controversial. The vaccination cost, however, is beyond the reach of many individuals in developing countries where 80% of cervical cancer cases of are found. Many countries in Africa are battling with HIV, malaria, tuberculosis, maternal mortality, and childhood illness. Nevertheless, with increased awareness, political will, and engagement by pharmaceutical countries, HPV vaccines may become affordable in these countries.     

Propagation of infectious human papillomavirus type 16 by using an adenovirus and Cre/LoxP mechanism

Human papillomavirus type 16 (HPV16) infection is a major risk factor for the development of squamous cell cancers of the cervix and of the head and neck. A major barrier to understanding the progression from initial infection to cancer has been the lack of in vitro models that allow infection, replication, and persistence of the viral genome as an episome in differentiated epithelial cells. To overcome this barrier, we designed an adenoviral delivery vector that contained a full HPV16 genome flanked by LoxP homologous recombination sites and a fluorescent reporter that was expressed only after the HPV genome was excised by Cre recombinase. This system delivered circular HPV16 genomes to cervical epithelial cells and well differentiated human airway epithelia. After delivery, the HPV16 genome replicated and persisted as an episome in cervical keratinocytes. These cells developed an immortalized phenotype and a dysplastic epithelial appearance. Moreover, induction of differentiation led to the expression of late genes and production of infectious HPV16 virions. This work provides a means of introducing biologically active HPV genomes into epithelial cells, which are normally difficult to transfect. These methods allow the study of HPV genome replication and gene expression in the earliest stages of HPV genome establishment, and they may provide a means to study nononcogenic HPV viral types.