Biology subjects

Palinski, R.

Publications and source records attributed to Palinski, R..

4 recordsLinked to original sources

bioRxiv2022

Beta human papillomavirus 8E6 promotes alternative end-joining

Double strand breaks (DSBs) are one of the most lethal DNA lesions in cells. Previous studies show that the E6 protein of beta-human papillomavirus (HPV8 E6) impairs two major DSB repair pathways homologous recombination (HR) and non-homologous end-joining (NHEJ). However, HPV8 E6 delays but does not eliminate DSB repair capability of cells. How DSBs are repaired in cells with HPV8 E6 remains to be studied. We hypothesis that HPV8 E6 promotes a backup DSB repair pathway, alternative end-joining (Alt-EJ). Using CAS9 based Alt-EJ reporters, we show that HPV8 E6 promotes Alt-EJ. Further, using small molecule inhibitors, CRISPR/CAS9 gene knockout, and HPV8 E6 mutant, we find that HPV8 E6 promotes Alt-EJ by binding p300, an acetyltransferase that facilitates DSB repair by HR and NHEJ. Finally, we analyzed whole genome sequencing data from genomes of human foreskin keratinocytes expressing HPV8 E6 and found they displayed an increased frequency of deletions bearing the microhomology signatures of Alt-EJ. This study fills the knowledge gap how DSB is repaired in cells with HPV8 E6 and the mutagenic consequences of HPV8 E6 mediated p300 destabilization. Broadly, this study supports the hypothesis that beta-HPV promotes cancer formation by increasing genomic instability.

cancer biology↗

bioRxiv2022

Beta HPV8 E6 Induces Micronuclei Formation and Promotes Chromothripsis

Cutaneous beta genus human papillomaviruses ({beta}-HPV) are suspected to promote the development of non-melanoma skin cancer (NMSC) by destabilizing the host genome. Multiple studies have established the genome destabilizing capacities of {beta}-HPV proteins E6 and E7 as a co-factor with UV. However, the E6 protein from {beta}-HPV8 (HPV8 E6) induces tumors in mice without UV exposure. Here, we examined a UV-independent mechanism of HPV8 E6-induced genome destabilization. We showed that HPV8 E6 reduced the abundance of anaphase bridge resolving helicase, Bloom syndrome protein (BLM). The diminished BLM was associated with increased segregation errors and micronuclei. These HPV8 E6-induced micronuclei had disordered micronuclear envelopes yet retained replication and transcription competence. HPV8 E6 decreased antiproliferative responses to micronuclei and time-lapse imaging revealed HPV8 E6 promoted cells with micronuclei to complete mitosis. Finally, whole genome sequencing revealed that HPV8 E6 induced chromothripsis in 9 chromosomes. These data provide insight into mechanisms by which HPV8 E6-induces genome instability independent of UV exposure. ImportanceSome beta genus human papillomaviruses ({beta}-HPVs) may promote skin carcinogenesis by inducing mutations in the host genome. Supporting this, the E6 protein from {beta}-HPV8 (8E6) promotes skin cancer in mice with or without UV exposure. Many mechanisms by which 8E6 increases mutations caused by UV have been elucidated, but less is known about how 8E6 induces mutations without UV. We address that knowledge gap by showing 8E6 causes mutations stemming from mitotic errors. Specifically, 8E6 reduces the abundance of BLM, a helicase that resolves and prevents anaphase bridges. This hinders anaphase bridge resolution and increases their frequency. 8E6 makes the micronuclei that can result from anaphase bridges more common. These micronuclei often have disrupted envelopes yet retain localization of nuclear-trafficked proteins. 8E6 promotes the growth of cells with micronuclei and causes chromothripsis, a mutagenic process where hundreds to thousands of mutations occur in a chromosome.

cancer biology↗

bioRxiv2021

Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors.

Beta human papillomavirus ({beta}-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks in DNA (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. They are not thought to occur in the same cell at the same time. By destabilizing p300, {beta}-HPV type 8 protein E6 ({beta}-HPV8 E6) attenuates both repair pathways. However, {beta}-HPV8 E6 delays rather than abrogates DSB repair. Thus, {beta}-HPV8 E6 may cause DSBs to be repaired through a more mutagenic process. To evaluate this, immunofluorescence microscopy was used to detect colocalization, formation, and resolution of DSB repair complexes following damage. Flow cytometry and immunofluorescence microscopy were used to determine the cell cycle distribution of repair complexes. The resulting data show that {beta}-HPV8 E6 causes HR factors (RPA70 and RAD51) to colocalize with a persistent NHEJ factor (pDNA-PKcs). RPA70 complexes gave way to RAD51 complexes as in canonical HR, but RAD51 and pDNA-PKcs colocalization did not resolve within 32 hours of damage. The persistent RAD51 foci occur in G1 phase, consistent with recruitment after NHEJ fails. Chemical inhibition of p300, p300 knockout cells, and an {beta}-HPV8 E6 mutant demonstrate that these phenotypes are the result of {beta}-HPV8 E6-meidated p300 destabilization. Mutations associated with DSB repair were identified using next generation sequencing after a CAS9-induced DSB. {beta}-HPV8 E6 increases the frequency of mutations (>15 fold) and deletions (>20 fold) associated with DSB repair. These data suggest that {beta}-HPV8 E6 causes abnormal DSB repair where both NHEJ and HR occur at the same lesion and that his leads to deletions as the single stranded DNA produced during HR is removed by NHEJ. Author SummaryOur previous work shows that a master transcription regulator, p300, is required for two major DNA double strand break (DSB) repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). By degrading p300, beta genus Human Papillomavirus 8 protein E6 ({beta}-HPV8 E6) hinders DNA-PKcs activity, which is a key factor of NHEJ. {beta}-HPV8 E6 also stalls HR via p300 degradation resulting in the persistence of a core factor, RAD51. NHEJ and HR are known competitive to each other and only one pathway can be initiated to repair a DSB. Particularly, NHEJ tends to be used in G1 phase and HR occurs in S/G2 phase. Here, we show that {beta}-HPV8 E6 allows NHEJ and HR to occur at the same break site. This is expected to be mutagenic because HR generates overhangs while NHEJ removes them. Further, we show that {beta}-HPV8 E6 allows HR to occur in G1, which cannot be finished due to the lack of homologous templates. Finally, our sequencing results show that {beta}-HPV8 E6 significantly increases genomic variations including deletions and insertions following CAS9 induced DSB. This study supports the hypothesis that {beta}-HPV8 infections increases genomic instability.

cancer biology↗

bioRxiv2019

Into the deep (sequence) of the foot-and-mouth disease virus gene pool: bottlenecks and adaptation during infection in naïve and vaccinated cattle

Foot-and-mouth disease virus (FMDV), like many RNA viruses, infects hosts as a population of closely related viruses referred to as a quasispecies. The behavior of this quasispecies has not been described in detail over the full course of infection in a natural host species. In this study, virus samples taken from vaccinated and non-vaccinated cattle up to 35 days post experimental infection with FMDV A24-Cruzeiro were analyzed by deep-sequencing. Vaccination induced significant differences compared to viruses from non-vaccinated cattle. in virus substitution rates, entropy, and evidence for adaptation. Genomic variation detected during early infection was found to reflect the diversity inherited from the source virus (inoculum), whereas by 12 days post infection (dpi) dominant viruses were defined by newly acquired mutations. In most serially sampled cattle, mutations conferring recognized fitness gain occurred within numerous genetic backgrounds, often associated with selective sweeps. Persistent infections always included multiple FMDV subpopulations, suggesting independently maintained foci of infection within the nasopharyngeal mucosa. Although vaccination prevented disease, subclinical infection in this group was associated with very early bottlenecks which subsequently reduced the diversity within the virus population. This implies an added consequence of vaccination in the control of foot-and-mouth disease. Viruses sampled from both animal cohorts contained putative antigenic escape mutations. However, these mutations occurred during later stages of infection, at which time transmission between animals is less likely to occur. ImportancePreparedness and control of foot-and-mouth disease virus have substantial, yet distinct implications in endemic and free regions. Viral evolution and emergence of novel strains are of critical concern in both settings. The factors that contribute to the asymptomatic carrier state, a common form of long-term FMDV infection in cattle and other species, are important but not well-understood. This experimental study of foot-and-mouth disease virus in cattle explored the evolution of the pathogen through detailed sampling and analytical methods in both vaccinated and non-vaccinated hosts. Significant differences were identified between the viruses subclinically infecting vaccinated animals and those causing clinical disease in the non-vaccinated cohort. These results can benefit vaccination programs and contribute to the understanding of persistent infection of cattle.

microbiology↗