Human papillomaviruses sensitize cells to DNA damage induced apoptosis by targeting the innate immune sensor cGAS.

The cyclic GMP-AMP synthase (cGAS) is a critical regulator of the innate immune response acting as a sensor of double-strand DNAs from pathogens or damaged host DNA. Upon activation, cGAS signals through the STING/TBK1/IRF3 pathway to induce interferon expression. Double stranded DNA viruses target the cGAS pathway to facilitate infection. In HPV positive cells that stably maintain viral episomes, the levels of cGAS were found to be significantly increased over those seen in normal human keratinocytes. Furthermore the downstream effectors of the cGAS pathway, STING and IRF3, were fully active in response to signaling from the secondary messenger cGAMP or poly (dA:dT). In HPV positive cells cGAS was detected in both cytoplasmic puncta as well as in DNA damage induced micronuclei. E6 was responsible for increased levels of cGAS that was dependent on inhibition of p53. CRISPR-Cas9 mediated knockout of cGAS prevented activation of STING and IRF3 but had a minimal effect on viral replication. A primary function of cGAS in HPV positive cells was in response to treatment with etoposide or cisplatin which lead to increased levels of H2AX phosphorylation and activation of caspase 3/7 cleavage while having only a minimal effect on activation of homologous recombination repair factors ATM, ATR or CHK2. In HPV positive cells cGAS was found to regulate the levels of the phosphorylated non-homologous end-joining kinase, DNA-PK, which may contribute to H2AX phosphorylation along with other factors. Importantly cGAS was also responsible for increased levels of DNA breaks along with enhanced apoptosis in HPV positive cells but not in HFKs. This study identifies an important and novel role for cGAS in mediating the response of HPV positive cells to chemotherapeutic drugs.

Human Papillomaviruses Target the DNA Damage Repair and Innate Immune Response Pathways to Allow for Persistent Infection.

Persistent infection with high-risk human papillomaviruses (HPVs) is the major risk factor associated with development of anogenital and oropharyngeal cancers. Initial infection by HPVs occurs into basal epithelial cells where viral genomes are established as nuclear episomes and persist until cleared by the immune response. Productive replication or amplification occurs upon differentiation and is dependent upon activation of the ataxia-telangiectasia mutated (ATM), ataxia telangiectasia and RAD3-related (ATR) DNA damage repair (DDR) pathways. In addition to activating DDR pathways, HPVs must escape innate immune surveillance mechanisms by antagonizing sensors, adaptors, interferons and antiviral gene expression. Both DDR and innate immune pathways are key host mechanisms that crosstalk with each other to maintain homeostasis of cells persistently infected with HPVs. Interestingly, it is still not fully understood why some HPV infections get cleared while others do not. Targeting of these two processes with antiviral therapies may provide opportunities for treatment of cancers caused by high-risk HPVs.

New advances in our understanding of the "unique" RNase L in host pathogen interaction and immune signaling.

Ever since the discovery of the existence of an interferon (IFN)-regulated ribonuclease, significant advances have been made in understanding the mechanism and associated regulatory effects of its action. What had been studied initially as a "unique" endoribonuclease is currently known as ribonuclease L (RNase L where "L" stands for latent). Some of the key developments include discovery of the RNase L signaling pathway, its structural characterization, and its molecular cloning. RNase L has been implicated in antiviral and antibacterial defense, as well as in hereditary prostate cancer. RNase L is activated by 2'-5' linked oligoadenylates (2-5A), which are synthesized by the oligoadenylate synthetases (OASs), a family of IFN-regulated pathogen recognition receptors that sense double-stranded RNAs. Activated RNase L cleaves single stranded RNAs, including viral RNAs and cellular RNAs. The catalytic activity of RNase L has been found to lead into the activation of several cellular signaling pathways, including those involved in autophagy, apoptosis, IFN-ß production, NLRP3 inflammasome activation leading to IL-1ß secretion, inhibition of cell migration, and cell adhesion. In this review, we will highlight the newest advances in our understanding of the catalytic role of RNase L in the context of different cellular pathways and extend the scope of these findings to discussion of potential therapeutic targets for antimicrobial drug development.

Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus.

Single-stranded circular RNAs (circRNAs), generated through 'backsplicing', occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gamma-herpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate circRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N6-methyladenosine (m6A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.

OAS-RNase L innate immune pathway mediates the cytotoxicity of a DNA-demethylating drug.

Drugs that reverse epigenetic silencing, such as the DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (AZA), have profound effects on transcription and tumor cell survival. AZA is an approved drug for myelodysplastic syndromes and acute myeloid leukemia, and is under investigation for different solid malignant tumors. AZA treatment generates self, double-stranded RNA (dsRNA), transcribed from hypomethylated repetitive elements. Self dsRNA accumulation in DNMTi-treated cells leads to type I IFN production and IFN-stimulated gene expression. Here we report that cell death in response to AZA treatment occurs through the 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway. OASs are IFN-induced enzymes that synthesize the RNase L activator 2-5A in response to dsRNA. Cells deficient in RNase L or OAS1 to 3 are highly resistant to AZA, as are wild-type cells treated with a small-molecule inhibitor of RNase L. A small-molecule inhibitor of c-Jun NH2-terminal kinases (JNKs) also antagonizes RNase L-dependent cell death in response to AZA, consistent with a role for JNK in RNase L-induced apoptosis. In contrast, the rates of AZA-induced and RNase L-dependent cell death were increased by transfection of 2-5A, by deficiencies in ADAR1 (which edits and destabilizes dsRNA), PDE12 or AKAP7 (which degrade 2-5A), or by ionizing radiation (which induces IFN-dependent signaling). Finally, OAS1 expression correlates with AZA sensitivity in the NCI-60 set of tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. These studies may eventually lead to pharmacologic strategies for regulating the antitumor activity and toxicity of AZA and related drugs.

Maximum walking speed in multiple sclerosis assessed with visual perceptive computing.

BACKGROUND: Gait is often impaired in people with multiple sclerosis (PwMS), but detailed assessment of gait impairment in research and care remains challenging. In a previous pilot study we reported the feasibility of visual perceptive computing (VPC) for gait assessment in PwMS using the Short Maximum Speed Walk (SMSW), which assesses gait on recording distances confined to less than 4 meters. OBJECTIVE: To investigate the equivalence of SMSW to rater-based timed 25ft. walk (T25FW) in a large cohort of PwMS, and to investigate the association of SMSW-derived gait parameters with clinical disability, as well as subjective and objective gait impairment, in order to validate the SMSW as a quick and objective measure of clinical relevance possibly superior to T25FW. METHODS: 95 PwMS and 60 healthy controls (HC) performed the SMSW using a VPC system with Microsoft Kinect. All participants received two immediate retests to establish test-retest-reliability. Both PwMS and HC performed the T25FW. PwMS were rated according to the Expanded Disability Status Scale (EDSS) and answered the 12-item Multiple Sclerosis Walking Scale (MSWS-12) as a measure of self-perceived walking impairment. RESULTS: PwMS showed reduced average speed (p<0.001) and higher mediolateral deviation (p = 0.002) during SMSW than HC. Average speed was the most reliable SMSW parameter in PwMS and HC (intra-class correlation coefficient (ICC) in PwMS = 0.985, and in HC = 0.977). Average speed declined with age in PwMS and HC (r in PwMS = -0.648, and in HC = -0.452, both p<0.001). Correlation of SMSW average speed and T25FW speed was high in both groups (r in PwMS = 0.783, and in HC = 0.747, both p<0.001) and mean difference (0.0013 m/s) between methods was below smallest detectable change. Average speed correlated well with both clinical disability based on EDSS (r = -0.586, p<0.001) and self-perceived walking impairment based on MSWS-12 (r = -0.546, p<0.001). CONCLUSION: VPC-assessed walking parameters during SMSW can reliably detect gait disturbance in PwMS over very short distance. Specifically, maximum gait speed can be obtained with high accuracy in this simple test set-up. Cross-sectional associations with disability and self-perceived walking impairment support clinical relevance. Given its objectivity in a simple test set-up, SMSW is superior to T25FW.

Validity of visual perceptive computing for static posturography in patients with multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) patients frequently have postural control impairment but quantitative posturography is difficult to perform in clinical care. Recent technology facilitates new posturography approaches. OBJECTIVE: To evaluate construct validity of visual perceptive computing (VPC) for static posturography to study postural control in MS patients. METHODS: A total of 90 MS patients and 59 healthy controls (HCs) performed three stance tests: open, closed and tandem stance. Static posturography was performed using a VPC system with Microsoft Kinect. Clinical assessments included Expanded Disability Status Scale (EDSS), Timed-25-Foot-Walk, Short-Maximum-Speed-Walk and 12-item MS Walking Scale (MSWS-12) questionnaire. Reliability was assessed with intra-class correlation coefficients at retest. RESULTS: As a group, MS patients performed worse than HCs in all tests. The closed stance test showed best applicability and reliability. With closed eyes, in 36.7% of patients, the three-dimensional mean angular sway velocity (MSV-3D) was above HCs' 95th percentile. Higher MSV-3D was associated with decreased walking speed (p < 0.001); worse clinical scores, mainly attributable to the cerebellar functional system score (p < 0.001); and reflected in self-reported walking disability (MSWS-12, p < 0.001). CONCLUSION: Postural control can be reliably assessed by VPC-based static posturography in patients with MS. Abnormal postural control seems to predominantly reflect involvement of cerebellar circuits with impact on gait and walking disability.

Ranking of Dystonia Severity by Pairwise Video Comparison.

BACKGROUND: Reviewers of dystonia rating scales agree on the need to assess symptoms more comprehensively. During the development of a quantitative dystonia assessment by video-perceptive computing, we devised a video-based severity ranking as a procedure to create a validation standard without the use of clinical scales. METHODS: Thirty-four patients with dystonia (17 with dystonic tremor) and 2 controls were assessed with clinical scales and video-recordings of 24 short movement tasks. Two to 4 raters compared multiple permutations of videos from 22 subjects, including 2 healthy controls, until a complete rank order was achieved. Inter-rater agreement was expressed as normalized Kendall tau distance. Spearman correlations of video rank order with clinical scales and self-rating were repeated for tremor/nontremor subgroups. RESULTS: Normalized Kendall tau distances were <0.3 for 15 items. The video rank order for sitting and head movements correlated with clinical scales for the whole group (rho 0.52-0.87) and in the subgroup without tremor. In the tremor subgroup such correlation was perceived in the 2 items involving sitting. Video rank order correlated with quality of life self-rating only in 1 item (arms held in front, palm down). CONCLUSIONS: The agreement of video rankings between raters is remarkable. The lack of correlation in the tremor subgroup in several items may be interpreted as tremor being considered in video comparisons but not in clinical scales. This supports video-based ranking as a more comprehensive rating of dystonia and as a possible validation instrument applicable in situations in which no reference standard is available.

Murine AKAP7 has a 2',5'-phosphodiesterase domain that can complement an inactive murine coronavirus ns2 gene.

Viral 2',5'-phosphodiesterases (2',5'-PDEs) help disparate RNA viruses evade the antiviral activity of interferon (IFN) by degrading 2',5'-oligoadenylate (2-5A) activators of RNase L. A kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) to localize and organize cyclic AMP (cAMP) signaling during diverse physiological processes. Among more than 43 AKAP isoforms, AKAP7 appears to be unique in its homology to viral 2',5'-PDEs. Here we show that mouse AKAP7 rapidly degrades 2-5A with kinetics similar to that of murine coronavirus (mouse hepatitis virus [MHV]) strain A59 ns2 and human rotavirus strain WA VP3 proteins. To determine whether AKAP7 could substitute for a viral 2',5'-PDE, we inserted AKAP7 cDNA into an MHV genome with an inactivated ns2 gene. The AKAP7 PDE domain or N-terminally truncated AKAP7 (both lacking a nuclear localization motif), but not full-length AKAP7 or a mutant, AKAP7(H185R), PDE domain restored the infectivity of ns2 mutant MHV in bone marrow macrophages and in livers of infected mice. Interestingly, the AKAP7 PDE domain and N-terminally deleted AKAP7 were present in the cytoplasm (the site of MHV replication), whereas full-length AKAP7 was observed only in nuclei. We suggest the possibility that viral acquisition of the host AKAP7 PDE domain might have occurred during evolution, allowing diverse RNA viruses to antagonize the RNase L pathway. Importance: Early virus-host interactions determine whether an infection is established, highlighting the need to understand fundamental mechanisms regulating viral pathogenesis. Recently, our laboratories reported a novel mode of regulation of the IFN antiviral response. We showed that the coronavirus MHV accessory protein ns2 antagonizes the type I IFN response, promoting viral replication and hepatitis. ns2 confers virulence by cleaving 2',5'-oligoadenylate (2-5A) activators of RNase L in macrophages. We also reported that the rotavirus VP3 C-terminal domain (VP3-CTD) cleaves 2-5A and that it may rescue ns2 mutant MHV. Here we report that a cellular protein, AKAP7, has an analogous 2',5'-phosphodiesterase (2',5'-PDE) domain that is able to restore the growth of chimeric MHV expressing inactive ns2. The proviral effect requires cytoplasmic localization of the AKAP7 PDE domain. We speculate that AKAP7 is the ancestral precursor of viral proteins, such as ns2 and VP3, that degrade 2-5A to evade the antiviral activity of RNase L.