Cutaneous and mucosal human papillomaviruses differ in net surface charge, potential impact on tropism

Papillomaviruses can roughly be divided into two tropism groups, those infecting the skin, including the genus beta PVs, and those infecting the mucosa, predominantly genus alpha PVs. The L1 capsid protein determines the phylogenetic separation between beta types and alpha types and the L1 protein is most probably responsible for the first interaction with the cell surface. Virus entry is a known determinant for tissue tropism and to study if interactions of the viral capsid with the cell surface could affect HPV tropism, the net surface charge of the HPV L1 capsid proteins was analyzed and HPV-16 (alpha) and HPV-5 (beta) with a mucosal and cutaneous tropism respectively were used to study heparin inhibition of uptake. The negatively charged L1 proteins were all found among HPVs with cutaneous tropism from the beta- and gamma-PV genus, while all alpha HPVs were positively charged at pH 7.4. The linear sequence of the HPV-5 L1 capsid protein had a predicted isoelectric point (pI) of 6.59 and a charge of -2.74 at pH 7.4, while HPV-16 had a pI of 7.95 with a charge of +2.98, suggesting no interaction between HPV-5 and the highly negative charged heparin. Furthermore, 3D-modelling indicated that HPV-5 L1 exposed more negatively charged amino acids than HPV-16. Uptake of HPV-5 (beta) and HPV-16 (alpha) was studied in vitro by using a pseudovirus (PsV) assay. Uptake of HPV-5 PsV was not inhibited by heparin in C33A cells and only minor inhibition was detected in HaCaT cells. HPV-16 PsV uptake was significantly more inhibited by heparin in both cells and completely blocked in C33A cells

Transcriptional activation of the human papillomavirus type 5 and 16 long control region in cells from cutaneous and mucosal origin

Human papillomavirus type-16 (HPV-16) infects mucosal epithelium and is the most common type found in cervical cancer. HPV-5 infects cornified epithelium and is the most common type found on normal skin and belongs to the types frequently associated with skin cancers of Epidermodysplasia verruciformis patients. One factor by which this anatomical tropism could be determined is the regulation of HPV gene expression in the host cell. The HPV long control region (LCR) contains cis-responsive elements that regulate HPV transcription and the epithelial tropism of HPV is determined by epithelial specific constitutive enhancers in the LCR. Since HPV-16 and other types infecting the mucosa differ in host cell from HPV types infecting skin, it has been hypothesized that it is the combination of ubiquitous transcription factors working in concert in the host cell that determines the cell-type-specific expression. To study if HPV tropism could be determined by differences in transcriptional regulation we have cloned the transcriptional regulating region, LCR, from HPV-16 and HPV-5 and studied the activation of a reporter gene in cell lines with different origin. To analyse promoter activity we transfected the plasmids into four different cell lines; HaCaT, C33A, NIKS and W12E and the efficiency of HPV-5 and HPV-16 LCR in the different cell lines was compared. In HaCaT cells, with a skin origin, the HPV-5 LCR was two-fold more efficient in transcriptional activation compared to the HPV-16 LCR. In cervical W12E cells the HPV-16 LCR was almost 2-fold more effective in activating transcription compared to the HPV-5 LCR. The ability to initiate transcription in the other cell lines was independent on cell origin and HPV-type

Pentavalent Sialic Acid Conjugates Block Coxsackievirus A24 Variant and Human Adenovirus Type 37–Viruses That Cause Highly Contagious Eye Infections

Coxsackievirus A24 variant (CVA24v) and human adenovirus 37 (HAdV-37) are leading causative agents of the severe and highly contagious ocular infections acute hemorrhagic conjunctivitis and epidemic keratoconjunctivitis, respectively. Currently, neither vaccines nor antiviral agents are available for treating these diseases, which affect millions of individuals worldwide. CVA24v and HAdV-37 utilize sialic acid as attachment receptors facilitating entry into host cells. Previously, we and others have shown that derivatives based on sialic acid are effective in preventing HAdV-37 binding and infection of cells. Here, we designed and synthesized novel pentavalent sialic acid conjugates and studied their inhibitory effect against CVA24v and HAdV-37 binding and infection of human corneal epithelial cells. The pentavalent conjugates are the first reported inhibitors of CVA24v infection and proved efficient in blocking HAdV-37 binding. Taken together, the pentavalent conjugates presented here form a basis for the development of general inhibitors of these highly contagious ocular pathogens

Human papillomavirus tropism : determinants of viral tissue specificity

Cervical cancer is the second most common cancer among women worldwide and human papillomavirus (HPV) is a prerequisit for the development of this cancer. HPV belongs to the Papillomaviridae family and infects the basal layer of epithelial cells where it generally progresses into warts or condylomas. HPV can only reproduce in differentiating epithelia and it is therefore difficult to study the natural infection of HPV. More than 100 HPV types exist and they are divided into different genera based on their L1 open reading frame sequence. Most of the HPV types in the alpha-papillomavirus genus infect the mucosal epithelium while HPVs from the beta-papillomavirus genus usually infect cutaneous epithelial cells. Presently, it is not known what decides the anatomical tropism and our aim was to study determinants of this tropism. By using HPV virus like particles (VLP) and pseudovirus we found that VLPs from the two alpha-papillomaviruses HPV-6 and HPV-16 interacted with cell-surface heparan sulfate (HS) for initial attachment. When we labelled HPV VLPs with a fluorescent dye to study internalization HPV-6 was more strongly inhibited than HPV-16. Furthermore, a pseudovirus infection assay demonstrated that the beta-papillomavirus HPV-5 was less dependent on HS for infection than HPV-16. By analyzing the isoelectric point (p1) of the HPV L1 capsid protein we found that alpha HPV types were more positively charged than beta HPV types. Also, HPV-6 had a higher positive charge than HPV-16. Thus, the inhibition of the negatively charged heparin against HPV infection was clearly related to the charge of the HPV L1 capsid. This suggested that the initial interaction could be one of the determinants of tropism although not the sole factor. Lactoferrin is a protein found in milk, saliva, semen, tear fluid and endocervical secretions that has antiviral activities. Both human and bovine lactoferrin inhibited HPV infection but we found no significant differences in inhibition of alpha- and beta-papillomavirus infection. We could however demonstrate that different lactoferricins, small peptide derivates from the N-terminal part of lactoferrin, were able to inhibit HPV infection. This antiviral activity depended on lactoferricin peptide, HPV type and cell origin. The regulation of HPV gene expression in the host cell could also determine HPV tropism. The HPV long control region (LCR) contains cis-responsive elements that regulate HPV transcription and the epithelial tropism of HPV is determined by epithelial specific constitutive enhancers in the LCR. It has been hypothesized that the combination of transcription factors in the host cell determines the cell-type-specific expression. In cells with a skin origin the HPV-5 LCR was twice as efficient in transcriptional activation compared to HPV-16 LCR, while in cervical cells the HPV-16 LCR was almost twice as effective in activating transcription compared to HPV-5 LCR. To conclude, alpha- and beta-papillomaviruses differed regarding their ability to infect cells and regulate viral gene expression. These abilities corresponded with their natural host cells and suggested that HPV anatomical tropism could be determined at several steps in the HPV life cycle

Attenuation of extubation responses: Comparison of prior treatment with verapamil and dexmedetomidine

Background: Tracheal extubation is almost always associated with stress response, airway response, and arrhythmias. There are many ways to attenuate this stress response. We have compared verapamil and dexmedetomidine on attenuation of these responses. Materials and Methods: Thirty patients (American Society of Anaesthesiologists Grade I, II) scheduled for spinal surgeries under general anaesthesia were randomly divided into two groups. At the end of surgery, after return of spontaneous efforts (bispectral index >80), in “Group V” verapamil 0.1 mg/kg and in “Group D” dexmedetomidine 0.3 mcg/kg were administered as bolus intravenously over one minute. Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were recorded just before (T0) and 2 min after intravenous administration of study medications (TM), just after oral suction (TS), immediately after extubation (TE) and 1, 3, 5 and 10 min postextubation (E1, E3, E5, E10). Duration of emergence and extubation, quality of extubation, Richmond Agitation Sedation Scale (RASS) score and time to reach modified Aldrete score ≥9 were compared. Results: HR, SBP, DBP, MAP were higher in Group V than Group D, but statistically insignificant (P > 0.05). Extubation quality scores was 1 for 20%, 2 for 60% and 3 for 20% patients in Group V, whereas 1 in 80%, 2 in 20% in Group D. There was occurrence of bradycardia within 2 min of administration of drug in one patient in Group D. RASS score was in the range of -1 to +1 in >90% patient in Group V, whereas -3 to -1 in 80% cases in Group D. Conclusion: Single dose of dexmedetomidine (0.3 mcg/kg) given before extubation produced significant attenuation of circulatory and airway responses during extubation as compared to verapamil (0.1 mg/kg)

Exploring divalent conjugates of 5-N-acetyl-neuraminic acid as inhibitors of coxsackievirus A24 variant (CVA24v) transduction

Coxsackievirus A24 variant (CVA24v) is responsible for several outbreaks and two pandemics of the highly contagious eye infection acute hemorrhagic conjunctivitis (AHC). Currently, neither prevention (vaccines) nor treatments (antivirals) are available for combating this disease. CVA24v attaches to cells by binding Neu5Ac-containing glycans on the surface of cells which facilitates entry. Previously, we have demonstrated that pentavalent Neu5Ac conjugates attenuate CVA24v infection of human corneal epithelial (HCE) cells. In this study, we report on the structure-based design of three classes of divalent Neu5Ac conjugates, with varying spacer lengths, and their effect on CVA24v transduction in HCE cells. In relative terms, the most efficient class of divalent Neu5Ac conjugates are more efficient than the pentavalent Neu5Ac conjugates previously reported