Isogenic sets of T. vaginalis harboring different combinations of trichomonasviruses reveal insights into host–virus interactions

Abstract

Trichomonasviruses are a genus of double stranded RNA (dsRNA) viruses that persist in the obligate human protozoan parasite T. vaginalis (Tvag). Tvag is the causative agent of trichomoniasis, the most common nonviral sexually transmitted infection (STI) worldwide. These viruses, commonly known as TVV1, TVV2, TVV3, TVV4, and TVV5, have been implicated in increasing the severity of trichomoniasis by eliciting greater pelvic inflammation. Unlike many familiar eukaryotic viruses, trichomonasviruses are not thought to have an extracellular lifecycle and instead are believed to be transmitted vertically as the parasite divides. The presence of trichomonasviruses is not thought to be detrimental to the health of the parasite, but rigorous investigation into the effects of trichomonasviruses on their host has to date been challenging. Because uninfected parasites are not thought to be able to be infected with trichomonasviruses, previous studies into the effects of trichomonasviruses on their host have largely relied on comparing virus-positive isolates to virus-negative isolates. However, gene expression differences between these isolates have confounded studies. In 2022, Narayanasamy et al. reported that a cytidine nucleoside analog, 2’-C-methylcytidine (2CMC) was effective in clearing Tvag isolates of infection with TVV1, TVV2, and TVV3. In this study, we extended the results of Narayanasamy et al. to demonstrate that 2CMC is effective against in reducing viral RNA abundance of representative strains of all five TVV species. In doing so, we identified differences in species-specific differences in 2CMC susceptibility and harness those susceptibility differences to generate isogenic sets of Tvag clones derived from the same parent isolate that harbored different combinations of trichomonasviruses. Using these newly generated isogenic sets, we investigated effects of viral infection on Tvag growth, survival, and adherence to human cells. While we saw no apparent difference in the growth rate of virus-positive and virus-negative Tvag clones in ideal conditions, our results suggested that virus-positive Tvag clones have an increased ability to survive in harsh conditions and a decreased ability to adhere to human cells. To understand the mechanism underlying these results, we performed differential gene expression analysis using three isogenic Tvag sets and revealed a number of differentially expressed genes between cured and uncured Tvag clones, including the putative transcription factor TvMyb4, which was consistently upregulated across Tvag clones harboring trichomonasviruses. We provide evidence suggesting involvement of TvMyb4 in the persistence of trichomonasviruses and in the survival differences between virus-positive and virus-negative clones. Together, the results presented in this work reveal new insights about differences between trichomonasvirus species and the relationship between trichomonasviruses and their host, providing valuable information about both an important human pathogen and a valuable model system for viral persistence.