ETHNIC-SPECIFIC HUMAN PAPILLOMAVIRUS TYPE-16 VARIANTS EXHIBIT DISTINCT MODES OF HOST ENTRY AND INFECTION
Open Access
- Author:
- Lengel, Kathleen
- Graduate Program:
- Biomedical Sciences
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- December 03, 2018
- Committee Members:
- Craig Matthew Meyers, Thesis Advisor/Co-Advisor
Neil David Christensen, Committee Member
Laura Carrel, Committee Member - Keywords:
- HPV
Virus entry - Abstract:
- Human papillomavirus type 16 (HPV16) is a high-risk type of mucosal HPV and persistent infection accounts for nearly 50% of all cervical cancers worldwide. HPVs are highly diverse in their sequences; there are over 200 types of HPV and within these types are sequence differences classified as variants containing less than 2% difference with respect to their major capsid protein, L1, sequence. Population studies have laid down vital groundwork for in-depth characterization of HPV16 variants such as determination of relative carcinogenicity and predominant histological phenotypes observed in diseased epithelia. All these studies have supported the hypothesis that ethnic background and geographical distribution are key determinants of HPV16 infectivity, persistence, and disease severity. Variant lineages of African (AFR) and Asian (As) decent (Non-European; non-EUR) have a greater likelihood to persist and cause cervical carcinogenesis compared to the prototype (EUR). In order to elucidate the cause of different disease outcomes, many groups have investigated HPV16 properties variant gene There are no whole-genome studies of the HPV16 variant life-cycle and infectivity and these are needed to determine how specific genetic variations could be related to ethnicity through in vitro functional and mechanistic analyses. Attachment and entry studies were done using HPV HR types derived from stratified and differentiated epithelium and the results suggest a difference in cellular requirements for binding and infection. Variant HPVs are at least 98% similar in their L1 sequence; could variant lineages harbor small nucleotide polymorphisms (SNPs) within L1 (or L2), that may affect capsid conformation or important interactions with cellular molecules, resulting in a novel preferred cell entry pathway? We created immortalized cervical cell lines containing EUR and non-EUR variants of HPV16 and generated native infectious virus using the organotypic raft culture system, mimicking differentiated epithelium, to achieve recapitulation of the native HPV life-cycle in vitro. We treated infections with furin inhibitor, heparin sulfate at low and high treatment concentrations, and tested infection of CHO cells deficient in glycosaminoglycans (GAGs). We tested the relative infectivity of variant NVs with and without various treatments to determine essential cell surface interactions for cell entry. Three EUR variant lineages, 114B, 114K, and W12, do not require cellular furin activity and are independent of binding to HSPGs or other GAGs. In contrast, when treated with furin inhibitor, non-EUR lineage NA 3724 shows a ~60 % decrease in infectivity (NA could be dependent on some basal level of furin activity for efficient infection) and AFR2 2716, like HPV18 (HR HPV responsible for ~20 % of cervical cancers), was completely inhibited indicating infection is furin-dependent. Heparin sulfate treatment abolished HPV18 infectivity at both the low and high heparin concentrations, suggesting HS binding is necessary for infection. In these studies, AFR2 2716 behaved similar to NA 3724 in the response to heparin treatment. Low amounts of HS are enough to knock infectivity down ~70 % with the high concentration HS treatment inhibiting ~90 % of viral entry. In contrast, non-EUR variants show a moderate decrease with HPV18 exhibiting nearly 80 % inhibition. Our data show that even small sequence variations within HPV16 are sufficient to elicit very different behavior compared to the prototype virus. This study highlights the importance of WGS and deep sequencing for identification of ethnic variants in addition to with-in host single nucleotide polymorphisms (SNPs) because a minor change can have big effects on this small DNA tumor virus.