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•DNA and several RNA viruses demand access to the cell nucleus for replication.•The nuclear envelope (NE) is a critical barrier for viral entry and egress.•Viral entry occurs via ...nuclear pores, by awaiting NE breakdown or NE remodelling.•Nuclear pores, parapore transport and NE destabilization are used for viral egress.•Insights into nuclear bypass pathways can reveal new antiviral therapeutic strategies.
Viruses are obligatory intracellular parasites. For their efficient replication, many require access to the nuclear interior. Yet, only few viral particles are small enough to passively diffuse through the nuclear pore complexes, calling for alternative strategies to bypass the nuclear envelope barrier. Some viruses will await mitotic nuclear envelope breakdown to gain access, whereas others will exploit more active means, for instance by hijacking nuclear pore transport or by directly targeting constituents of the nuclear envelope so as to remodel and temporarily perturb its integrity. After replication, newly produced viral DNA complexes need to cross the same barrier to exit the nucleus and enter the cytoplasm, where the final stages of virion maturation take place. There are also different flavours to the feat of nuclear egress that vary in delicacy and intensity. In this review, we define the major entry and egress strategies that are exploited by different viruses and describe the molecular details thereof. Ultimately, a deeper understanding of these pathways may help identifying molecular targets for blocking viral reproduction or spreading.
Cisplatin-induced hypomagnesemia is described in humans and rats, but the underlying mechanisms are still unclear. Recent studies have shown that epidermal growth factor (EGF) stimulates Mg(2+) ...re-absorption in the distal convoluted tubule via the Mg(2+) channel TRPM6. This study investigates the role of TRPM Mg(2+) channels, claudines, and EGF in the Mg(2+) homeostasis in a rat model of cisplatin-induced nephrotoxicity. Wistar rats were given 2.5 mg/kg cisplatin per week for 3 weeks and were euthanized 4 or 9 weeks after the first administration. The cisplatin treatment significantly increased the fractional excretion of Mg(2+). Real-time RT-PCR and/or Western blots were performed to assess the renal expression TRPM6, TRPM7, claudin-16, claudin-19, EGF, EGF receptor (EGFR) and EGFR-pathway components. The renal mRNA expression of TRPM6 and EGF showed a significant decrease after cisplatin treatment, while the TRPM7, claudin-16 and EGFR expressions remained stable. The claudin-19 mRNA expression was significantly upregulated after cisplatin treatment. Western blotting confirmed the mRNA expression data for the claudins, but an showed upregulation of EGFR only at week 9. The role of the EGFR pathway, involving Pi3-AKT-Rac1, in cisplatin-induced nephropathy, could not be substantiated in further detail. This study shows that cisplatin treatment results in EGF and TRPM6 downregulation in the rat kidney, causing renal Mg(2+) loss. Our results are in line with the hypothesis that EGF influences the renal expression or activation of TRPM6 and plays a significant role in Mg(2+) loss in medication-induced nephropathy.
Cutaneous warts are infectious disorders caused by human papillomavirus (HPV). A recent study revealed that the HPV genotype influences the natural course and response to treatment for plantar warts, ...suggesting that HPV genotyping could potentially be used to optimize wart treatment schemes. For this purpose, a wart‐associated HPV genotyping assay was developed. The assay was subjected to an intensive validation process including, i.a., empiric determination of the annealing temperature, primer‐probe optimization, evaluation of the analytical specificity and sensitivity, viral load quantification, and qualitative as well as quantitative analysis of intra‐run repeatability and inter‐run reproducibility. The newly developed assay was employed in a small‐scale HPV genotyping study of wart biopsies (n = 50). The assay exhibited an analytical type‐specific sensitivity and specificity of 100% (95% confidence interval CI: 83.9%–100%). The limit of quantification of the tested sequences corresponded to less than 17 viral copies/µl, while the limit of detection was less than 5 copies/µl. Very good to excellent agreements were gained between intra‐ and inter‐run measurements (κ = 0.85–1.00) and coefficients of variation of the quantitative agreements were less then 3%. 22.5% (95% CI: 11%–39%) of the analyzed biopsies were negative for the tested HPV types, while 35% (95% CI: 21%–52%) contained multiple infections. The wart‐associated HPV quantitative polymerase chain reaction assay was proven to be highly sensitive and specific. Multiple HPV infections were detected in 35% of lesions, contradicting the current literature claiming that in immunocompetent patients only 4%–16% of warts exhibit multiple HPV infections. This assay is qualified to be implemented in development of future genotype specific wart treatment strategies.
Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that ...lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in
LMNB1
knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that
LMNB1
knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.
Graphical abstract
Persistent high‐risk human papillomavirus (HPV) infection is strongly associated with development of high‐grade cervical intraepithelial neoplasia or cancer (CIN3+). In single type infections, serial ...type‐specific viral‐load measurements predict the natural history of the infection. In infections with multiple HPV‐types, the individual type‐specific viral‐load profile could distinguish progressing HPV‐infections from regressing infections. A case‐cohort natural history study was established using samples from untreated women with multiple HPV‐infections who developed CIN3+ (n = 57) or cleared infections (n = 88). Enriched cell pellet from liquid based cytology samples were subjected to a clinically validated real‐time qPCR‐assay (18 HPV‐types). Using serial type‐specific viral‐load measurements (≥3) we calculated HPV‐specific slopes and coefficient of determination (R2) by linear regression. For each woman slopes and R2 were used to calculate which HPV‐induced processes were ongoing (progression, regression, serial transient, transient). In transient infections with multiple HPV‐types, each single HPV‐type generated similar increasing (0.27copies/cell/day) and decreasing (−0.27copies/cell/day) viral‐load slopes. In CIN3+, at least one of the HPV‐types had a clonal progressive course (R2 ≥ 0.85; 0.0025copies/cell/day). In selected CIN3+ cases (n = 6), immunostaining detecting type‐specific HPV 16, 31, 33, 58 and 67 RNA showed an even staining in clonal populations (CIN3+), whereas in transient virion‐producing infections the RNA‐staining was less in the basal layer compared to the upper layer where cells were ready to desquamate and release newly‐formed virions. RNA‐hybridization patterns matched the calculated ongoing processes measured by R2 and slope in serial type‐specific viral‐load measurements preceding the biopsy. In women with multiple HPV‐types, serial type‐specific viral‐load measurements predict the natural history of the different HPV‐types and elucidates HPV‐genotype attribution.
What's new?
It is well known that individual high‐risk human papilloma virus (HPV) genotypes can cause cervical cancer, but the risk posed by infections with multiple viral genotypes is less clear. Here the authors examined genotype‐specific viral loads over time in women infected with multiple HPV genotypes. While in women with no cervical intraepithelial neoplasia (CIN), infection dynamics of multiple or single genotypes were similar, in women with advanced lesions (CIN3+) progressive clonal populations could be clearly identified. The authors propose three serial genotype‐specific viral load measurements to identify progressive clonal viral populations as a new screening tool for cervical cancer in women infected with multiple HPV genotypes.
Background Persistent high-risk (hr) human papillomavirus (HPV) infection is a necessary cause of cervical cancer. Cervical cancer is a major public health problem in Sub-Saharan Africa including ...South Africa. This study investigated the prevalence of and factors associated with hr-HPV infection among women attending a tertiary hospital in Gauteng Province, South Africa. Methods Cervical samples were collected from 526 participants aged greater than or equal to 18 years using a Cervex BrushR Combi and tested for hr-HPV types on the Abbott m2000 analyzer using the Abbott RealTime HR HPV assay. Samples that tested hr-HPV deoxyribonucleic acid (DNA)-positive were further tested for hr-HPV E6/E7 messenger ribonucleic acid (mRNA) using the APTIMAR HPV assay on the Panther system (Hologic, Inc.). Sociodemographic data were collected using a self-administered questionnaire. Binomial regression analysis was used to assess factors associated with hr-HPV infection. Results Overall hr-HPV DNA prevalence was 48.1% (95%CI: 43.8-52.4%). Of the hr-HPV DNA-positives, 24.5% (95%CI: 19.3-30.1) had HPV-16; 12.3% (95%CI: 8.5-16.9) had HPV-18 and 87.4% (95%CI: 82.6-91.2) had other 12 h-HPVs. Of the samples positive for hr-HPV DNA, 84.2% (95%CI: 79.1-88.5) (213/253) were positive for hr-HPV E6/E7 mRNA. Advanced age was an important factor linked to hr-HPV E6/E7 mRNA positivity. Based on multivariate binomial regression analysis, unemployment (PR: 1.50; 95%CI: 1.23-1.83) and being married (PR: 0.61; 95%CI: 0.47-0.81) were identified as statistically significant (p < 0.0001) predictive and protective factors, respectively, for hr-HPV infection. Conclusions The prevalence of hr-HPV infection was high. Furthermore, hr-HPV DNA-positive samples had a high hr-HPV E6/E7 mRNA prevalence. The presence of hr-HPV E6/E7mRNA indicates active infection and thus a greater risk of developing the cervical disease. Therefore, HPV mRNA testing could be a better test to monitor women who are positive with Pap smear before colposcopy is performed to reduce the burden of referrals. Keywords: Human papillomavirus, E6/E7 mRNA, Prevalence, Risk factors, HPV 16, HPV 18
Human papillomaviruses (HPV) are small, non‐enveloped DNA viruses, which upon chronic infection can provoke cervical and head‐and‐neck cancers. Although the infectious life cycle of HPV has been ...studied and a vaccine is available for the most prevalent cancer‐causing HPV types, there are no antiviral agents to treat infected patients. Hence, there is a need for novel therapeutic entry points and a means to identify them. In this work, we have used high‐content microscopy to quantitatively investigate the early phase of HPV infection. Human cervical cancer cells and immortalized keratinocytes were exposed to pseudoviruses (PsV) of the widespread HPV type 16, in which the viral genome was replaced by a pseudogenome encoding a fluorescent reporter protein. Using the fluorescent signal as readout, we measured differences in infection between cell lines, which directly correlated with host cell proliferation rate. Parallel multiparametric analysis of nuclear organization revealed that HPV PsV infection alters nuclear organization and inflates promyelocytic leukemia protein body content, positioning these events at the early stage of HPV infection, upstream of viral replication. Time‐resolved analysis revealed a marked heterogeneity in infection kinetics even between two daughter cells, which we attribute to differences in viral load. Consistent with the requirement for mitotic nuclear envelope breakdown, pharmacological inhibition of the cell cycle dramatically blunted infection efficiency. Thus, by systematic image‐based single cell analysis, we revealed phenotypic alterations that accompany HPV PsV infection in individual cells, and which may be relevant for therapeutic drug screens.
Abstract
Cutaneous warts are benign skin lesions caused by the human papillomavirus (HPV). Even though they are considered benign, they can have a considerable impact on the quality of life and cause ...serious illness in certain immunocompromised populations. Studies have shown that the efficacy of wart treatment is dependent on the causative HPV type. Therefore, in this article, we aim to determine the HPV genotype-specific prevalence in cutaneous warts of a Flemish population as part of the Omnivirol-Salycilic acid randomized controlled trial. Swab samples of cutaneous warts (n = 269) were collected during enrollment. The DNA extraction was performed on the automated NucliSENS® easyMAG® system (bioMérieux). The samples were analyzed with two separate in-house PCR assays capable of detecting the most prevalent cutaneous HPV types (i.e. wart-associated HPV qPCR) as well as the most relevant mucosal types (i.e. RIATOL qPCR assay). In total, the type-specific prevalence of 30 distinct HPV genotypes was determined. The beta-globin gene was used as a cellularity control and for viral load quantification. Data concerning wart persistence, previous treatment, wart type, and other relevant wart and patient characteristics was collected through a baseline questionnaire. The study population consisted mostly of persistent warts considering that 98% (n = 263) of the sampled skin lesions were older than six months and 92% (n = 247) had undergone previous treatment. The most prominent wart type was the mosaic verruca plantaris (42%, n = 113). The most prevalent HPV types were cutaneous HPV types 27 (73%, n = 195), 57 (63%, n = 169), and 2 (42%, n = 113). Only 2% (n = 6) of the lesions was HPV negative. The highest median viral loads were observed with HPV27 and 57 (i.e. 6.29E+04 and 7.47E+01 viral copies per cell respectively). The multivariate analysis found significant associations between wart persistence and certain wart types, the number of warts, and HPV genotypes. Based on these findings, persistent warts are more likely to: (1) be verruca vulgaris, verruca plantaris simple or mosaic, (2) to manifest as multiple warts, (3) and to be negative for HPV type 2 or 4. These characteristics can be useful in the clinical setting for future risk stratification when considering treatment triage and management.
Trial registration
: NCT05862441, 17/05/2023 (retrospectively registered).
Human papillomaviruses are the most important causative agents for invasive cervical cancer development. HPV type-specific vaccination and HPV cervical cancer screening methods are being widely ...recommended to control the disease but the epidemiology of the circulating HPV types may vary locally. The circulating HPV-strains have never been assessed in Burundi. This study determined the prevalence and genotype-specific distribution of HPV in four different strata in Burundi: HIV-infected or non-infected and women living in rural or urban areas. Implications for HPV diagnosis and vaccine implementation was discussed.
Four cross-sectional surveys were conducted in Burundi (2013 in a rural area and 2016 in urban area) among HIV-infected and uninfected women living in rural and urban areas. Liquid-Based Cytology (LBC) and HPV genotyping were performed and risk factors for HPV infection and cervical pre-cancer lesions were determined using logistic regression model.
HPV prevalence was very high in urban area with significant differences between HIV-positive and negative women (p<0.0001). In fact, 45.7% of HIV-positive participants were infected with any HPV type and all were infected with at least one HR/pHR-HPV type. Among the HIV-negative participants, 13.4% were HPV-infected, of whom, only four women (2.7%) were infected with HR/pHR-HPV types. In rural area, HPV infection did not significantly differ between HIV-positive and negative women (30.0% and 31.3% respectively; p = 0.80). In urban area, multiple infections with HR/pHR-HPV types were detected in 13.9% and 2.7% among HIV-positive and negative women respectively (p<0.0001), whereas in rural area, multiple infections with HR/pHR-HPV types were detected in 4.7% and 3.3% of HIV-positive and negative women respectively (p = 0.56). The most prevalent HR/pHR-HPV types in HIV-positive women living in urban area were HPV 52, 51, 56, 18 and 16 types. In HIV-negative women living in urban area, the most prevalent HR/pHR-HPV types were HPV 66, 67, 18, 45 and 39 types. In HIV-positive women living in rural area, the most prevalent HR/pHR-HPV types were HPV 66, 16, 18 and 33 types. In HIV-negative women living in rural area, the most prevalent HR/pHR-HPV types were HPV 16, 66, 18, 35 and 45 types. Independent risk factors associated with cervical lesions were HPV and HIV infections.
There is a high burden of HR and pHR-HPV infections, in particular among HIV-infected women living in urban area. The study points out the need to introduce a comprehensive cervical cancer control programme adapted to the context. This study shows that the nonavalent vaccine covers most of the HR/pHR-HPV infections in rural and urban areas among HIV-infected and uninfected women.
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