HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways.

R-loops are trimeric nucleic acid structures that form when an RNA molecule hybridizes with its complementary DNA strand, displacing the opposite strand. These structures regulate transcription as well as replication, but aberrant R-loops can form, leading to DNA breaks and genomic instability if unresolved. R-loop levels are elevated in many cancers as well as cells that maintain high-risk human papillomaviruses. We investigated how the distribution as well as function of R-loops changed between normal keratinocytes and HPV positive cells derived from a precancerous lesion of the cervix (CIN I). The levels of R-loops associated with cellular genes were found to be up to 10-fold higher in HPV positive cells than in normal keratinocytes while increases at ALU1 elements increased by up to 500-fold. The presence of enhanced R-loops resulted in altered levels of gene transcription, with equal numbers increased as decreased. While no uniform global effects on transcription due to the enhanced levels of R-loops were detected, genes in several pathways were coordinately increased or decreased in expression only in the HPV positive cells. This included the downregulation of genes in the innate immune pathway, such as DDX58, IL-6, STAT1, IFN-ß, and NLRP3. All differentially expressed innate immune genes dependent on R-loops were also associated with H3K36me3 modified histones. Genes that were upregulated by the presence of R-loops in HPV positive cells included those in the DNA damage repair such as ATM, ATRX, and members of the Fanconi Anemia pathway. These genes exhibited a linkage between R-loops and H3K36me3 as well as γH2AX histone marks only in HPV positive cells. These studies identify a potential link in HPV positive cells between DNA damage repair as well as innate immune regulatory pathways with R-loops and γH2AX/H3K36me3 histone marks that may contribute to regulating important functions for HPV pathogenesis.

p53-dependent R-loop formation and HPV pathogenesis.

R-loops are trimeric RNA: DNA hybrids that are important physiological regulators of transcription; however, their aberrant formation or turnover leads to genomic instability and DNA breaks. High-risk human papillomaviruses (HPV) are the causative agents of genital as well as oropharyngeal cancers and exhibit enhanced amounts of DNA breaks. The levels of R-loops were found to be increased up to 50-fold in cells that maintain high-risk HPV genomes and were readily detected in squamous cell cervical carcinomas in vivo but not in normal cells. The high levels of R-loops in HPV-positive cells were present on both viral and cellular sites together with RNase H1, an enzyme that controls their resolution. Depletion of RNase H1 in HPV-positive cells further increased R-loop levels, resulting in impaired viral transcription and replication along with reduced expression of the DNA repair genes such as FANCD2 and ATR, both of which are necessary for viral functions. Overexpression of RNase H1 decreased total R-loop levels, resulting in a reduction of DNA breaks by over 50%. Furthermore, increased RNase H1 expression blocked viral transcription and replication while enhancing the expression of factors in the innate immune regulatory pathway. This suggests that maintaining elevated R-loop levels is important for the HPV life cycle. The E6 viral oncoprotein was found to be responsible for inducing high levels of R-loops by inhibiting p53's transcriptional activity. Our studies indicate that high R-loop levels are critical for HPV pathogenesis and that this depends on suppressing the p53 pathway.

The Combination of TIM3-Based Checkpoint Blockade and Oncolytic Virotherapy Regresses Established Solid Tumors.

T-cell immunoglobulin and mucin domain 3 (TIM3) is emerging as a potential target for antibody-based checkpoint blockade. However, the efficacy of TIM3 blockade in combination with other treatment modalities, has not been extensively studied. In the current work we combined TIM3 blockade with myxoma virus-based oncolytic virotherapy (OV). Our results demonstrate that myxoma virus's ability to initiate an immense antitumor immune response complements the ability of TIM3 blockade to shift the tumor microenvironment to a more proinflammatory state. As a result, the combination of TIM3 blockade and OV is able to completely eradicate established disease, while neither monotherapy is effective. These data represent the first demonstration that OV can enhance the efficacy of TIM3 blockade and suggest that this treatment may need to be incorporated into more aggressive, combinatorial regimens in order to fulfill its potential as an immunotherapeutic.

UV Irradiation of Vaccinia Virus-Infected Cells Impairs Cellular Functions, Introduces Lesions into the Viral Genome, and Uncovers Repair Capabilities for the Viral Replication Machinery.

Vaccinia virus (VV), the prototypic poxvirus, encodes a repertoire of proteins responsible for the metabolism of its large dsDNA genome. Previous work has furthered our understanding of how poxviruses replicate and recombine their genomes, but little is known about whether the poxvirus genome undergoes DNA repair. Our studies here are aimed at understanding how VV responds to exogenous DNA damage introduced by UV irradiation. Irradiation of cells prior to infection decreased protein synthesis and led to an ∼12-fold reduction in viral yield. On top of these cell-specific insults, irradiation of VV infections at 4 h postinfection (hpi) introduced both cyclobutene pyrimidine dimer (CPD) and 6,4-photoproduct (6,4-PP) lesions into the viral genome led to a nearly complete halt to further DNA synthesis and to a further reduction in viral yield (∼35-fold). DNA lesions persisted throughout infection and were indeed present in the genomes encapsidated into nascent virions. Depletion of several cellular proteins that mediate nucleotide excision repair (XP-A, -F, and -G) did not render viral infections hypersensitive to UV. We next investigated whether viral proteins were involved in combatting DNA damage. Infections performed with a virus lacking the A50 DNA ligase were moderately hypersensitive to UV irradiation (∼3-fold). More strikingly, when the DNA polymerase inhibitor cytosine arabinoside (araC) was added to wild-type infections at the time of UV irradiation (4 hpi), an even greater hypersensitivity to UV irradiation was seen (∼11-fold). Virions produced under the latter condition contained elevated levels of CPD adducts, strongly suggesting that the viral polymerase contributes to the repair of UV lesions introduced into the viral genome. IMPORTANCE Poxviruses remain of significant interest because of their continuing clinical relevance, their utility for the development of vaccines and oncolytic therapies, and their illustration of fundamental principles of viral replication and virus/cell interactions. These viruses are unique in that they replicate exclusively in the cytoplasm of infected mammalian cells, providing novel challenges for DNA viruses. How poxviruses replicate, recombine, and possibly repair their genomes is still only partially understood. Using UV irradiation as a form of exogenous DNA damage, we have examined how vaccinia virus metabolizes its genome following insult. We show that even UV irradiation of cells prior to infection diminishes viral yield, while UV irradiation during infection damages the genome, causes a halt in DNA accumulation, and reduces the viral yield more severely. Furthermore, we show that viral proteins, but not the cellular machinery, contribute to a partial repair of the viral genome following UV irradiation.