Uurimistöö inimese papilloomiviiruse ja evolutsiooniliselt lähedase Macaca fascicularis papilloomiviiruse genoomi paljundamisest viirusnakkuse varajastes etappides

Väitekirja elektrooniline versioon ei sisalda publikatsiooneInimese papilloomiviirused (HPV-d) on kliiniliselt olulised viirused, kuna teatud viirustüüpide elutsükli käigus võib aset leida nakatunud raku muutumine kontrollimatu paljunemisvõimega kasvajarakuks. Selliste rakkude vohamine võib kaasa tuua HPV-seoseliste vähkkasvajate tekke, millest kõige levinum on emakakaelavähk. Kuna tänaseni ei ole õnnestunud välja arendada ravimeid väljakujunenud HPV-nakkuse kõrvaldamiseks, on HPV nakkusega seotud haiguste puhul selgelt määratletav täitmata ravivajadus. HPV-d nakatavad limaskestade või naha epiteelkoe keratinotsüüte ning viiruse elutsükkel on tihedalt seotud epiteelkoe uuenemisega, mille jäljendamine laboritingimustes on keeruline. See on oluliselt pärssinud HPV genoomse DNA paljundamise (replikatsiooni) uurimist ning selle molekulaarsete mehhanismide mõistmist, mis on ülioluline HPV-vastaste kõrgelt spetsiifiliste ravimite arendamisel. Meie uurimisrühm Tartu Ülikoolis on välja töötanud robustse, inimese U2OS rakuliinil põhineva mudelsüsteemi, mille abil antud uurimistöös kirjeldati HPV genoomse DNA esmast replikatsioonifaasi molekulaarsel tasemel. Replikatsiooni vaheproduktide täppisanalüüsi põhjal järeldati, et HPV esmane nakatumisjärgne DNA amplifitseerimine hõlmab kahte erinevat replikatsiooniviisi: kahesuunalist nn teeta-replikatsiooni ning ühesuunalist, arvatavasti rekombinatsioonist sõltuvat replikatsiooniviisi. Lisaks kirjeldati uurimistöös esmakordselt HPV-dele evolutsiooniliselt lähedaste viiruste, jaava makaagi (Macaca fascicularis) papilloomiviiruste (MfPV-de) DNA paljundamise ja geenide avaldumisega seotud molekulaarseid mehhanisme, kuna MfPV-d võivad olla sobivaimad viirused HPV-vastaste ravimikandidaatide testimiseks kasutatava loommudeli arendamiseks. Tulemuste põhjal järeldati, et MfPV ja kliiniliselt oluliste HPV tüüpide DNA paljundamine ja geenide avaldumine on molekulaarsel tasemel väga sarnane. Lisaks näidati, et vähkkasvajaid põhjustavate HPV-de replikatsiooni takistavad ravimikandidaadid pärsivad ka MfPV DNA paljundamist. Seega on antud uurimistöö tulemused aluseks edasisele arendustegevusele, mille eesmärgiks on välja töötada HPV-vastaste ravimite eelkliinilisteks testimisteks kasutatav uudne, HPV nakkust jäljendav loommudel.Human papillomaviruses (HPVs) are clinically important viruses as persistent infection with certain HPV types (high-risk types) might induce genetic instability and cancer development in host cells over the years. Although HPV-induced cancers are among the leading causes of cancer mortality worldwide (e.g., HPVs are almost always causative agents of cervical cancer), no effective therapeutics for ongoing HPV infection currently exist and the need for anti-HPV drugs to treat ongoing viral infections is strong. HPVs infect mucosal or cutaneous keratinocytes and have developed life-cycle strategies tightly linked to the renewal program of epithelial tissue. Mimicking this host differentiation-dependent viral life cycle under laboratory conditions is time consuming and complicated. Lack of a robust model system has hindered studying HPV DNA replication and elucidating molecular mechanisms behind it, which is a crucial prerequisite for developing effective therapeutic anti-HPV strategies. For this reason, our research group has developed a human U2OS cell line-based assay system that has proven to be an efficient tool for studying HPV genome replication. The present study made use of the U2OS cell line-based assay system. To shed more light on HPV DNA replication mechanisms, we analyzed episomal HPV replication structures generated during the initial phase of viral DNA replication. Two distinct DNA replication mechanisms were revealed to be involved in HPV genome replication - bidirectional theta-mode replication, and a unidirectional DNA replication mode which is likely associated with recombination-dependent replication. Additionally, this research characterizes cynomolgus macaque papillomaviruses (MfPVs) as potentially valuable for the development of an in vivo animal model for preclinical testing of anti-HPV therapeutics. The findings revealed that MfPVs and HPVs share similar characteristics in gene expression and DNA replication, including the finding that MfPV replication is sensitive to HPV-specific inhibitors. These findings justify the use of cynomolgus macaques as a relevant model system for developing therapeutic antiviral strategies.  https://www.ester.ee/record=b5250920~S

The molecular biology and HPV drug responsiveness of cynomolgus macaque papillomaviruses support their use in the development of a relevant in vivo model for antiviral drug testing.

Due to the extreme tissue and species restriction of the papillomaviruses (PVs), there is a great need for animal models that accurately mimic PV infection in humans for testing therapeutic strategies against human papillomaviruses (HPVs). In this study, we present data that demonstrate that in terms of gene expression during initial viral DNA amplification, Macaca fascicularis PV (MfPV) types 5 and 8 appear to be similar to mucosal oncogenic HPVs, while MfPV1 (isolated from skin) resembles most high-risk cutaneous beta HPVs (HPV5). Similarities were also observed in replication properties during the initial amplification phase of the MfPV genomes. We demonstrate that high-risk mucosal HPV-specific inhibitors target the transient replication of the MfPV8 genomes, which indicates that similar pathways are used by the high-risk HPVs and MfPVs during their genome replication. Taking all into account, we propose that Macaca fascicularis may serve as a highly relevant model for preclinical tests designed to evaluate therapeutic strategies against HPV-associated lesions

Identification and Tracking of Antiviral Drug Combinations

Combination therapies have become a standard for the treatment for HIV and hepatitis C virus (HCV) infections. They are advantageous over monotherapies due to better efficacy, reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify new synergistic combinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), echovirus 1 (EV1), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1) in vitro. We observed synergistic activity of nelfinavir with convalescent serum and with purified neutralizing antibody 23G7 against SARS-CoV-2 in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of nelfinavir with EIDD-2801 or remdesivir in Calu-3 cells. In addition, we showed synergistic activity of vemurafenib with emetine, homoharringtonine, anisomycin, or cycloheximide against EV1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar or niclosamide are synergistic against HCV infection in hepatocyte-derived Huh-7.5 cells, and that combinations of monensin with lamivudine or tenofovir are synergistic against HIV-1 infection in human cervical TZM-bl cells. These results indicate that synergy is achieved when a virus-directed antiviral is combined with another virus- or host-directed agent. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status.Peer reviewe