Role of miRNAs in regulating virus replication

Poliovirus interactions with host cells were investigated by studying the formation of ribonucleoprotein complexes at the 3' end of poliovirus negative-strand RNA which are presumed to be involved in viral RNA synthesis. It was previously shown that two host cell proteins with molecular masses of 36 and 38 kDa bind to the 3' end of viral negative-strand RNA at approximately 3 to 4 h after infection. We tested the hypothesis that preexisting cellular proteins are modified during the course of infection and are subsequently recruited to play a role in viral replication. It was demonstrated that the 38-kDa protein, either directly or indirectly, is the product of processing by poliovirus 3CD/3C proteinase. Only the modified 38-kDa protein, not its precursor protein, has a high affinity for binding to the 3' end of viral negative-strand RNA. This modification depends on proteolytically active proteinase, and a direct correlation between the levels of 3CD proteinase and the 38-kDa protein was demonstrated in infected tissue culture cells. The nucleotide (nt) 5-10 region (positive-strand numbers) of poliovirus negative-strand RNA is important for binding of the 38-kDa protein. Deletion of the nt 5-10 region in full-length, positive-strand RNA renders the RNA noninfectious in transfection experiments. These results suggest that poliovirus 3CD/3C proteinase processes a cellular protein which then plays an essential role during the viral life cycle.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression found in more than 200 diverse organisms. Although it is still not fully established if RNA viruses could generate miRNAs, there are examples of miRNA like sequences from RNA viruses with regulatory functions. In the case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several mechanisms that would make miRNAs impact the virus, like interfering with viral replication, translation and even modulating the host expression. In this study, we performed a machine learning based miRNA prediction analysis for the SARS-CoV-2 genome to identify miRNA-like hairpins and searched for potential miRNA-based interactions between the viral miRNAs and human genes and human miRNAs and viral genes. Overall, 950 hairpin structured sequences were extracted from the virus genome and based on the prediction results, 29 of them could be precursor miRNAs. Targeting analysis showed that 30 viral mature miRNA-like sequences could target 1,367 different human genes. PANTHER gene function analysis results indicated that viral derived miRNA candidates could target various human genes involved in crucial cellular processes including transcription, metabolism, defense system and several signaling pathways such as Wnt and EGFR signalings. Protein class-based grouping of targeted human genes showed that host transcription might be one of the main targets of the virus since 96 genes involved in transcriptional processes were potential targets of predicted viral miRNAs. For instance, basal transcription machinery elements including several components of human mediator complex (MED1, MED9, MED12L, MED19), basal transcription factors such as TAF4, TAF5, TAF7L and site-specific transcription factors such as STAT1 were found to be targeted. In addition, many known human miRNAs appeared to be able to target viral genes involved in viral life cycle such as S, M, N, E proteins and ORF1ab, ORF3a, ORF8, ORF7a and ORF10. Considering the fact that miRNA-based therapies have been paid attention, based on the findings of this study, comprehending mode of actions of miRNAs and their possible roles during SARS-CoV-2 infections could create new opportunities for the development and improvement of new therapeutics.

Vitamin D, often referred to as the “sunshine vitamin,” plays a crucial role in numerous physiological processes beyond its traditional association with bone health. It has a multifaceted role in the prevention of various diseases. Emerging research has highlighted its significance in immune modulation, cellular differentiation, and gene expression regulation, thereby influencing diverse health outcomes. A growing body of evidence suggests that vitamin D is intricately linked to immune system function, exerting both anti-inflammatory and immunomodulatory effects. Deficiency in this vitamin has been associated with an increased susceptibility to infections, autoimmune disorders, and chronic inflammatory diseases. Moreover, it has the potential to mitigate the risk of cardiovascular diseases. Vitamin D's impact on blood pressure regulation, endothelial function, and cholesterol metabolism underscores its role in maintaining cardiovascular health. The link between vitamin D deficiency and several types of cancer has garnered significant attention. Research suggests that vitamin D's anti-proliferative, pro-apoptotic, and anti-angiogenic properties may contribute to its protective effects against various cancers, including breast, prostate, and colorectal cancers. Furthermore, the role of vitamin D extends to mental health, with studies demonstrating its involvement in neurological functions and mood regulation. Deficiency has been associated with an increased risk of mood disorders such as depression and cognitive decline, emphasizing the importance of maintaining optimal vitamin D levels. This review also delves into the relationship between vitamin D and metabolic health. Vitamin D deficiency has been linked to insulin resistance, obesity, and type 2 diabetes. Adequate vitamin D levels positively influence insulin sensitivity, glucose homeostasis, and adipokine secretion. Thus, vitamin D's role in disease prevention extends far beyond its classical association with bone health. Its involvement in immune function, cardiovascular health, cancer prevention, mental well-being, and metabolic regulation highlights its multifaceted impact on various physiological systems. Public health strategies aimed at maintaining optimal vitamin D levels could have far-reaching implications for reducing the burden of chronic diseases and enhancing overall health outcomes. However, further research is warranted to elucidate the precise mechanisms underlying vitamin D's effects and to establish definitive guidelines for supplementation in disease prevention and management.

Identification of Viral MicroRNAs

Macrophages, as central components of innate immunity, feature significant heterogeneity. Numerus studies have revealed the pivotal roles of macrophages in the pathogenesis of liver fibrosis induced by various factors. Hepatic macrophages function to trigger inflammation in response to injury. They induce liver fibrosis by activating hepatic stellate cells (HSCs), and then inflammation and fibrosis are alleviated by the degradation of the extracellular matrix and release of anti-inflammatory cytokines. MicroRNAs (miRNAs), a class of small non-coding endogenous RNA molecules that regulate gene expression through translation repression or mRNA degradation, have distinct roles in modulating macrophage activation, polarization, tissue infiltration, and inflammation regression. Considering the complex etiology and pathogenesis of liver diseases, the role and mechanism of miRNAs and macrophages in liver fibrosis need to be further clarified. We first summarized the origin, phenotypes and functions of hepatic macrophages, then clarified the role of miRNAs in the polarization of macrophages. Finally, we comprehensively discussed the role of miRNAs and macrophages in the pathogenesis of liver fibrotic disease. Understanding the mechanism of hepatic macrophage heterogeneity in various types of liver fibrosis and the role of miRNAs on macrophage polarization provides a useful reference for further research on miRNA-mediated macrophage polarization in liver fibrosis, and also contributes to the development of new therapies targeting miRNA and macrophage subsets for liver fibrosis.

Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked to a bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). Together with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.

Chapter 22 - Ion channels in viruses

Introduction: Anal squamous cell carcinoma (ASCC) is a rare but increasingly common gastrointestinal malignancy, mainly associated with oncogenic human papillomaviruses (HPVs). The role of non-coding RNAs (ncRNAs) in tumorigenesis is recognized, but the impact of viral ncRNAs on host gene expression remains unclear. Methods: We re-analyzed total RNA-Seq data from 70 anal biopsies: 31 low-grade squamous intraepithelial lesions (LGSIL), 16 high-grade SIL (HGSIL), and 23 ASCC cases. Microbial composition was assessed taxonomically. Novel viral miRNAs were predicted using vsRNAfinder and linked to host targets using TargetScan and expression correlation analyses. Results: Microbial profiling revealed significant differences in abundance, with Alphapapillomaviruses types 9, 10, and 14 enriched across lesion grades. We identified 90 novel viral miRNAs and 177 significant anti-correlated miRNA–mRNA interactions. Target genes were enriched in pathways related to cell cycle, epithelial–mesenchymal transition, lipid metabolism, immune modulation, and viral replication. Discussion: Our findings suggest that HPV-derived miRNAs, including those from low-risk types, may contribute to neoplastic transformation by modulating host regulatory networks. Conclusion: This study highlights viral miRNAs as potential drivers of HPV-related anal cancer and supports their utility as early biomarkers and therapeutic targets in ASCC.

The family Flaviviridae comprises a wide variety of viruses that are distributed worldwide, some of which are associated with high rates of morbidity and mortality. There are neither vaccines nor antivirals for most flavivirus infections, reinforcing the importance of research on different aspects of the viral life cycle. During infection, cytoplasmic accumulation of RNA fragments mainly originating from the 3′ UTRs, which have been designated subgenomic flavivirus RNAs (sfRNAs), has been detected. It has been shown that eukaryotic exoribonucleases are involved in viral sfRNA production. Additionally, viral and human small RNAs (sRNAs) have also been found in flavivirus-infected cells, especially microRNAs (miRNAs). miRNAs were first described in eukaryotic cells and in a mature and functional state present as single-stranded 18–24 nt RNA fragments. Their main function is the repression of translation through base pairing with cellular mRNAs, besides other functions, such as mRNA degradation. Canonical miRNA biogenesis involves Drosha and Dicer, however miRNA can also be generated by alternative pathways. In the case of flaviviruses, alternative pathways have been suggested. Both sfRNAs and miRNAs are involved in viral infection and host cell response modulation, representing interesting targets of antiviral strategies. In this review, we focus on the generation and function of viral sfRNAs, sRNAs and miRNAs in West Nile, dengue, Japanese encephalitis, Murray Valley encephalitis and yellow fever infections, as well as their roles in viral replication, translation and cell immune response evasion. We also give an overview regarding other flaviviruses and the generation of cellular miRNAs during infection.

Epstein-Barr virus (EBV) is a human specific gammaherpesvirus that establishes lifelong infection in >95% of all adults and is associated with multiple cancers, including B cell lymphomas. Murine gammaherpesvirus 68 (MHV68) is a natural rodent pathogen that is genetically and biologically related to EBV and importantly provides a tractable animal model to study viral and host factors that impact chronic infection. Both of these viruses have a natural tropism for B cells and usurp B cell differentiation to drive a robust polyclonal germinal center response, which is needed to establish the latent viral reservoir in memory B cells. The factors which these viruses use to usurp the host germinal center response to establish chronic infection are not clearly defined. Using MHV68 we discovered that global IL-17RA signaling as well as T-cell intrinsic IL-17RA signaling is proviral and supports the gammaherpesvirus-driven germinal center response needed to establish chronic infection. Loss of global and T-cell intrinsic IL-17RA signaling resulted in a significant attenuation in the germinal center response as well as viral latency and reactivation. Given the B cell tropism of MHV68 we generated a B cell specific model of IL-17RA deficiency to understand the significance of IL-17RA signaling in virally infected cells. Loss of IL-17RA signaling in B cells during MHV68 infection resulted in an attenuated germinal center response as well as reduced viral reactivation and latency, similar to what was observed in the loss of global and T-cell intrinsic IL-17RA signaling. This indicates that IL-17RA signaling in both virally infected and uninfected cells play an important proviral role in the establishment of chronic gammaherpesvirus infection. WMed Start up funds and an American Cancer Society Postdoctoral Award (134165-PF-19-176-01-MPC) which was used to generate the B cell specific IL-17RA deficient mice.

Serous ovarian cancer (SEOC) is the deadliest gynecologic malignancy. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate gene expression and protein translation. MiRNAs are also encoded by viruses with the intent of regulating their own genes and those of the infected cells. This is the first study assessing viral miRNAs in SEOC. MiRNAs sequencing data from 487 SEOC patients were downloaded from the TCGA website and analyzed through in-house sequencing pipeline. To cross-validate TCGA analysis, we measured the expression of miR-H25 by quantitative immunofluorescence in an additional cohort of 161 SEOC patients. Gene, miRNA expression, and cytotoxicity assay were performed on multiple ovarian cancer cell lines transfected with miR-H25 and miR-BART7. Outcome analysis was performed using multivariate Cox and Kaplan-Meier method. Viral miRNAs are more expressed in SEOC than in normal tissues. Moreover, Herpetic viral miRNAs (miR-BART7 from EBV and miR-H25 from HSV-2) are significant and predictive biomarkers of outcome in multivariate Cox analysis. MiR-BART7 correlates with resistance to first line chemotherapy and early death, whereas miR-H25 appears to impart a protective effect and long term survival. Integrated analysis of gene and viral miRNAs expression suggests that miR-BART7 induces directly cisplatin-resistance, while miR-H25 alters RNA processing and affects the expression of noxious human miRNAs such as miR-143. This is the first investigation linking viral miRNA expression to ovarian cancer outcome. Viral miRNAs can be useful to develop biomarkers for early diagnosis and as a potential therapeutic tool to reduce SEOC lethality. Citation Format: Deep Pandya, Marisa Mariani, Mark McHugh, Mirko Andreoli, Steven Sieber, Shiquan He, Candice Dowell-Martino, Paul Fiedler, Giovanni Scambia, Cristiano Ferlini. Herpes virus microRNA expression and significance in serous ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3994. doi:10.1158/1538-7445.AM2015-3994

Reactivation of Latent HIV-1 by Inhibition of BRD4

Deletion of miRNAs in bone marrow prevents streptozotocin-induced murine autoimmune diabetes but deletion of miR-155 does not

Polyomaviruses are small, circular dsDNA viruses that can cause cancer. Alternative splicing of polyomavirus early transcripts generates large and small tumor antigens (LT, ST) that play essential roles in viral replication and tumorigenesis. Some polyomaviruses also express middle tumor antigens (MTs) or alternate LT open reading frames (ALTOs), which are evolutionarily related but have distinct gene structures. MTs are a splice variant of the early transcript whereas ALTOs are overprinted on the second exon of the LT transcript in an alternate reading frame and are translated via an alternative start codon. Merkel cell polyomavirus (MCPyV), the only human polyomavirus that causes cancer, encodes an ALTO but its role in the viral lifecycle and tumorigenesis has remained elusive. Here, we show MCPyV ALTO acts as a tumor suppressor and is silenced in Merkel cell carcinoma (MCC). Rescuing ALTO in MCC cells induces growth arrest and activates NF-κB signaling. ALTO activates NF-κB by binding SQSTM1 and TRAF2&3 via two N-Terminal Activating Regions (NTAR1+2), resembling Epstein-Barr virus (EBV) Latent Membrane Protein 1 (LMP1). Following activation, NF-κB dimers bind the MCPyV noncoding control region (NCCR) and downregulate early transcription. Beyond MCPyV, NTAR motifs are conserved in other polyomavirus ALTOs, which activate NF-κB signaling, but are lacking in MTs that do not. Furthermore, polyomavirus ALTOs downregulate their respective viral early transcription in an NF-κB- and NTAR-dependent manner. Our findings suggest that ALTOs evolved to suppress viral replication and promote viral latency and that MCPyV ALTO must be silenced for MCC to develop.

Exosomes are membrane-enclosed vesicles actively released into the extracellular space, whose content reflect the physiological/pathological state of the cells they originate from. These vesicles participate in cell-to-cell communication and transfer of biologically active proteins, lipids, and RNAs. Their role in viral infections is just beginning to be appreciated. RNA viruses are an important class of pathogens and affect millions of people worldwide. Recent studies on Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), human T-cell lymphotropic virus (HTLV), and Dengue Virus (DENV) have demonstrated that exosomes released from infected cells harbor and deliver many regulatory factors including viral RNA and proteins, viral and cellular miRNA, and other host functional genetic elements to neighboring cells, helping to establish productive infections and modulating cellular responses. Exosomes can either spread or limit an infection depending on the type of pathogen and target cells, and can be exploited as candidates for development of antiviral or vaccine treatments. This review summarizes recent progress made in understanding the role of exosomes in RNA virus infections with an emphasis on their potential contribution to pathogenesis.