Viral RNA Replication Research Articles

Activity, Template Preference, and Compatibility of Components of RNA Replicase of Eastern Equine Encephalitis Virus.

Eastern equine encephalitis virus (EEEV) usually cycles between Culiseta melanura mosquitoes and birds; however, it can also infect humans. EEEV has a positive-sense RNA genome that, in infected cells, serves as an mRNA for the P1234 polyprotein. P1234 undergoes a series of precise cleavage events producing four nonstructural proteins (nsP1-4) representing subunits of the RNA replicase. Here, we report the construction and properties of a trans-replicase for EEEV. The template RNA of EEEV was shown to be replicated by replicases of diverse alphaviruses. The EEEV replicase, on the other hand, demonstrated limited ability in replicating template RNAs originating from alphaviruses of the Semliki Forest virus complex. The replicase of EEEV was also successfully reconstructed from P123 and nsP4 components. The ability of EEEV P123 to form functional RNA replicases with heterologous nsP4s was more efficient using EEEV template RNA than heterologous alphavirus template RNA. This finding indicates that unlike with previously studied Semliki Forest complex alphaviruses, P123 and/or its processing products have a leading role in EEEV template RNA recognition. Infection of HEK293T cells harboring the EEEV template RNA with EEEV or Western equine encephalitis virus prominently activated expression of a reporter encoded in the template RNA; the effect was much smaller for infection with other alphaviruses and not detectable upon flavivirus infection. At the same time, EEEV infection resulted only in a limited activation of the template RNA of chikungunya virus. Thus, cells harboring reporter-carrying template RNAs can be used as sensitive and selective biosensors for different alphaviruses. IMPORTANCE Infection of EEEV in humans can cause serious neurologic disease with an approximately 30% fatality rate. Although human infections are rare, a record-breaking number was documented in 2019. The replication of EEEV has a unique requirement for host factors but is poorly studied, partly because the virus requires biosafety level 3 facilities which can limit the scope of experiments; at the same time, these studies are crucial for developing antiviral approaches. The EEEV trans-replicase developed here contributes significantly to research on EEEV, providing a safe and versatile tool for studying the virus RNA replication. Using this system, the compatibility of EEEV replicase components with counterparts from other alphaviruses was analyzed. The obtained data can be used to develop unique biosensors that provide alternative methods for detection, identification, quantitation, and neutralization of viable alphaviruses that are compatible with high throughput, semiautomated approaches.

SNAP23 is essential for germination of EV-D68 replication organelles

Picornaviruses rearrange host cell membranes to facilitate their own replication. Here we investigate the Qbc SNARE, SNAP23, which is found at the plasma membrane and plays roles in exocytosis. We found that knockdown of SNAP23 expression inhibits virus replication but not release from cells. Knocking down SNAP23 inhibits viral RNA replication and synthesis of structural proteins. Normal cellular levels of SNAP23 are required for an early step in virus production, prior to or at the stage of virus RNA replication. We report that SNAP23 knockdown generates large, electron-light structures, and that infection of cells with these structures does not alter them, and those cells fail to generate viral RNA replication sites. We suggest that SNAP23 may play a role in maintaining membranes and lipids needed for generating virus replication organelles. Further investigation is needed to determine the precise role of this crucial SNARE protein in EV-D68 replication.

Nonmuscle myosin IIA promotes the internalization of influenza A virus and regulates viral polymerase activity through interacting with nucleoprotein in human pulmonary cells

Influenza A virus (IAV), responsible for seasonal epidemics and recurring pandemics, represents a global threat to public health. Given the risk of a potential IAV pandemic, it is increasingly important to better understand virus-host interactions and develop new anti-viral strategies. Here, we reported nonmuscle myosin IIA (MYH9)-mediated regulation of IAV infection. MYH9 depletion caused a profound inhibition of IAV infection by reducing viral attachment and internalization in human lung epithelial cells. Surprisingly, overexpression of MYH9 also led to a significant reduction in viral productive infection. Interestingly, overexpression of MYH9 retained viral attachment, internalization, or uncoating, but suppressed the viral ribonucleoprotein (vRNP) activity in a minigenome system. Further analyses found that excess MYH9 might interrupt the formation of vRNP by interacting with the viral nucleoprotein (NP) and result in the reduction of the completed vRNP in the nucleus, thereby inhibiting subsequent viral RNA transcription and replication. Together, we discovered that MYH9 can interact with IAV NP protein and engage in the regulation of vRNP complexes, thereby involving viral replication. These findings enlighten new mechanistic insights into the complicated interface of host-IAV interactions, ultimately making it an attractive target for the generation of antiviral drugs.

Molecular architecture of the Chikungunya virus replication complex.

To better understand how positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) to synthesize, process, and transport viral RNA in virus-infected cells, we determined both the high-resolution structure of the core RNA replicase of chikungunya virus and the native RC architecture in its cellular context at subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase, the viral polymerase nsP4, which is in complex with nsP2 helicase-protease, sits in the central pore of the membrane-anchored nsP1 RNA-capping ring. The addition of a large cytoplasmic ring next to the C terminus of nsP1 forms the holo-RNA-RC as observed at the neck of spherules formed in virus-infected cells. These results represent a major conceptual advance in elucidating the molecular mechanisms of RNA virus replication and the principles underlying the molecular architecture of RCs, likely to be shared with many pathogenic (+) RNA viruses.

Surf4, cargo trafficking, lipid metabolism, and therapeutic implications.

Surfeit 4 is a polytopic transmembrane protein that primarily resides in the endoplasmic reticulum (ER) membrane. It is ubiquitously expressed and functions as a cargo receptor, mediating cargo transport from the ER to the Golgi apparatus via the canonical coat protein complex II (COPII)-coated vesicles or specific vesicles. It also participates in ER-Golgi protein trafficking through a tubular network. Meanwhile, it facilitates retrograde transportation of cargos from the Golgi apparatus to the ER through COPI-coated vesicles. Surf4 can selectively mediate export of diverse cargos, such as PCSK9 very low-density lipoprotein (VLDL), progranulin, α1-antitrypsin, STING, proinsulin, and erythropoietin. It has been implicated in facilitating VLDL secretion, promoting cell proliferation and migration, and increasing replication of positive-strand RNA viruses. Therefore, Surf4 plays a crucial role in various physiological and pathophysiological processes and emerges as a promising therapeutic target. However, the molecular mechanisms by which Surf4 selectively sorts diverse cargos for ER-Golgi protein trafficking remain elusive. Here, we summarize the most recent advances in Surf4, focusing on its role in lipid metabolism.

Paxlovid accelerates cartilage degeneration and senescence through activating endoplasmic reticulum stress and interfering redox homeostasis

BackgroundThe COVID-19 pandemic has become a huge threat to human health, infecting millions of people worldwide and causing enormous economic losses. Many novel small molecule drugs have been developed to treat patients with COVID-19, including Paxlovid, which block the synthesis of virus-related proteins and replication of viral RNA, respectively. Despite satisfactory clinical trial results, attention is now being paid to the long-term side effects of these antiviral drugs on the musculoskeletal system. To date, no study has reported the possible side effects, such as osteoarthritis, of Paxlovid. This study explored the effects of antiviral drug, Paxlovid, on chondrocyte proliferation and differentiation.MethodsIn this study, both in vitro and in vivo studies were performed to determine the effect of Paxlovid on chondrocyte degeneration and senescence. Furthermore, we explored the possible mechanism behind Paxlovid-induced acceleration of cartilage degeneration using transcriptome sequencing and related inhibitors were adopted to verify the downstream pathways behind such phenomenon.ResultsPaxlovid significantly inhibited chondrocyte extracellular matrix protein secretion. Additionally, Paxlovid significantly induced endoplasmic reticulum stress, oxidative stress, and downstream ferroptosis, thus accelerating the senescence and degeneration of chondrocytes. In vivo experiments showed that intraperitoneal injection of Paxlovid for 1 week exacerbated cartilage abrasion and accelerated the development of osteoarthritis in a mouse model.ConclusionsPaxlovid accelerated cartilage degeneration and osteoarthritis development, potentially by inducing endoplasmic reticulum stress and oxidative stress. Long-term follow-up is needed with special attention to the occurrence and development of osteoarthritis in patients treated with Paxlovid.

Identification of FDA-approved drugs against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) through computational virtual screening

The SARS-CoV-2 coronavirus is responsible for the COVID-19 outbreak, which overwhelmed millions of people worldwide; hence, there is an urgency to identify appropriate antiviral drugs. This study focuses on screening compounds that inhibit RNA-dependent RNA-polymerase (RdRp) essential for RNA synthesis required for replication of positive-strand RNA viruses. Computational screening against RdRp using Food and Drug Administration (FDA)-approved drugs identified ten prominent compounds with binding energies of more than − 10.00 kcal/mol, each a potential inhibitor of RdRp. These compounds’ binding energy is comparable to known RdRp inhibitors remdesivir (IC50 = 10.09 μM, SI = 4.96) and molnupiravir (EC50 = 0.67 − 2.66 µM) and 0.32–2.03 µM). Remdesivir and molnupiravir have been tested in clinical trial and remain authorized for emergency use in the treatment of COVID-19. In docking simulations, selected compounds are bound to the substrate-binding pocket of RdRp and showed hydrophobic and hydrogen bond interaction. For molecular dynamics simulation, capmatinib, pralsetinib, ponatinib, and tedizolid phosphate were selected from the initial ten candidate compounds. MD simulation indicated that these compounds are stable at 50-ns MD simulation when bound to RdRp protein. The screen hit compounds, remdesivir, molnupiravir, and GS-441524, are bound in the substrate binding pocket with good binding-free energy. As a consequence, capmatinib, pralsetinib, ponatinib, and tedizolid phosphate are potential new inhibitors of RdRp protein with potential of limiting COVID-19 infection by blocking RNA synthesis.

Substrate Specificity and Kinetics of RNA Hydrolysis by SARS-CoV-2 NSP10/14 Exonuclease.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes COVID-19, continues to evolve resistance to vaccines and existing antiviral therapies at an alarming rate, increasing the need for new direct-acting antiviral drugs. Despite significant advances in our fundamental understanding of the kinetics and mechanism of viral RNA replication, there are still open questions regarding how the proofreading exonuclease (NSP10/NSP14 complex) contributes to replication fidelity and resistance to nucleoside analogs. Through single turnover kinetic analysis, we show that the preferred substrate for the exonuclease is double-stranded RNA without any mismatches. Double-stranded RNA containing a 3'-terminal remdesivir was hydrolyzed at a rate similar to a correctly base-paired cognate nucleotide. Surprisingly, single-stranded RNA or duplex RNA containing a 3'-terminal mismatch was hydrolyzed at rates 125- and 45-fold slower, respectively, compared to the correctly base-paired double-stranded RNA. These results define the substrate specificity and rate of removal of remdesivir for the exonuclease and outline rigorous kinetic assays that could help in finding next-generation exonuclease inhibitors or nucleoside analogs that are able to evade excision. These results also raise important questions about the role of the polymerase/exonuclease complex in proofreading during viral replication. Addressing these questions through rigorous kinetic analysis will facilitate the search for desperately needed antiviral drugs to combat COVID-19.

Insight into the Hantaan virus RNA-dependent RNA polymerase inhibition using in-silico approaches.

The Hantaan virus (HTN) is a member of the hantaviridae family. It is a segmented type, negative-strand virus (sNSVs). It causes hemorrhagic fever with renal syndrome, which includes fever, vascular hemorrhage, and renal failure. This illness is one of the most serious hemorrhagic diseases in the world, and it is a major public health concern due to its high mortality rate. The Hantaan virus RNA-dependent RNA polymerase complex (RdRp) is involved in viral RNA transcription and replication for the survival and transmission of this virus. Therefore, it is a primary target for antiviral drug development. Interference with the endonucleolytic "cap-snatching" reaction by the HTN virus RdRp endonuclease domain is a particularly appealing approach for drug discovery against this virus. This RdRp endonuclease domain of the HTN virus has a metal-dependent catalytic activity. We targeted this metal-dependent enzymatic activity to identify inhibitors that can bind and disrupt this endonuclease enzyme activity using in-silico approaches i.e., molecular docking, molecular dynamics simulation, predicted absorption, distribution, metabolism, excretion, toxicity (ADMET) and drug-likeness studies. The docking studies showed that peramivir, and ingavirin compounds can effectively bind with the manganese ions and engage with other active site residues of this protein. Molecular simulations also showed stable binding of these ligands with the active site of HTN RdRp. Simulation analysis showed that they were in constant contact with the active site manganese ions and amino acid residues of the HTN virus endonuclease domain. This study will help in better understanding the HTN and related viruses.

Analysis of Expression Pattern of snoRNAs in Human Cells A549 Infected by Influenza A Virus.

Small nucleolar RNAs (snoRNAs) are a highly expressed class of non-coding RNAs known for their role in guiding post-transcriptional modifications of ribosomal RNAs and small nuclear RNAs. Emerging studies suggest that snoRNAs are also implicated in regulating other vital cellular processes, such as pre-mRNA splicing and 3'-processing of mRNAs, and in the development of cancer and viral infections. There is an emerging body of evidence for specific snoRNA's involvement in the optimal replication of RNA viruses. In order to investigate the expression pattern of snoRNAs during influenza A viral infection, we performed RNA sequencing analysis of the A549 human cell line infected by influenza virus A/Puerto Rico/8/1934 (H1N1). We identified 66 that were upregulated and 55 that were downregulated in response to influenza A virus infection. The increased expression of most C/D-box snoRNAs was associated with elevated levels of 5'- and 3'-short RNAs derived from this snoRNA. Analysis of the poly(A)+ RNA sequencing data indicated that most of the differentially expressed snoRNAs synthesis was not correlated with the corresponding host genes expression. Furthermore, influenza A viral infection led to an imbalance in the expression of genes responsible for C/D small nucleolar ribonucleoprotein particles' biogenesis. In summary, our results indicate that the expression pattern of snoRNAs in A549 cells is significantly altered during influenza A viral infection.

Synthetic Platforms for Characterizing and Targeting of SARS-CoV-2 Genome Capping Enzymes.

Essential viral enzymes have been successfully targeted to combat the diseases caused by emerging pathogenic RNA viruses (e.g., viral RNA-dependent RNA polymerase). Because of the conserved nature of such viral enzymes, therapeutics targeting these enzymes have the potential to be repurposed to combat emerging diseases, e.g., remdesivir, which was initially developed as a potential Ebola treatment, then was repurposed for COVID-19. Our efforts described in this study target another essential and highly conserved, but relatively less explored, step in RNA virus translation and replication, i.e., capping of the viral RNA genome. The viral genome cap structure disguises the genome of most RNA viruses to resemble the mRNA cap structure of their host and is essential for viral translation, propagation, and immune evasion. Here, we developed a synthetic, phenotypic yeast-based complementation platform (YeRC0M) for molecular characterization and targeting of SARS-CoV-2 genome-encoded RNA cap-0 (guanine-N7)-methyltransferase (N7-MTase) enzyme (nsp14). In YeRC0M, the lack of yeast mRNA capping N7-MTase in yeast, which is an essential gene in yeast, is complemented by the expression of functional viral N7-MTase or its variants. Using YeRC0M, we first identified important protein domains and amino acid residues that are essential for SARS-CoV-2 nsp14 N7-MTase activity. We also expanded YeRC0M to include key nsp14 variants observed in emerging variants of SARS-CoV-2 (e.g., delta variant of SARS-CoV-2 encodes nsp14 A394V and nsp14 P46L). We also combined YeRC0M with directed evolution to identify attenuation mutations in SARS-CoV-2 nsp14. Because of the high sequence similarity of nsp14 in emerging coronaviruses, these observations could have implications on live attenuated vaccine development strategies. These data taken together reveal key domains in SARS-CoV-2 nsp14 that can be targeted for therapeutic strategies. We also anticipate that these readily tractable phenotypic platforms can also be used for the identification of inhibitors of viral RNA capping enzymes as antivirals.

Topoisomerase 3b is dispensable for replication of a positive-sense RNA virus--murine coronavirus

A recent study demonstrated that a DNA-RNA dual-activity topoisomerase complex, TOP3B-TDRD3, is required for normal replication of positive-sense RNA viruses, including several human flaviviruses and coronaviruses; and the authors proposed that TOP3B is a target of antiviral drugs. Here we examined this hypothesis by investigating whether inactivation of Top3b can inhibit the replication of a mouse coronavirus, MHV, using cell lines and mice that are inactivated of Top3b or Tdrd3. We found that Top3b-KO or Tdrd3-KO cell lines generated by different CRISPR-CAS9 guide RNAs have variable effects on MHV replication. In addition, we did not find significant changes of MHV replication in brains or lungs in Top3B–KO mice. Moreover, immunostaining showed that Top3b proteins are not co-localized with MHV replication complexes but rather, localized in stress granules in the MHV-infected cells. Our results suggest that Top3b does not have a universal role in promoting replication of positive-sense RNA virus, and cautions should be taken when targeting it to develop anti-viral drugs.

Effects of Glutamine Starvation on SHVV Replication by Quantitative Proteomics Analysis

Snakehead vesiculovirus (SHVV), a strain of negative-stranded RNA viruses extracted from sick snakehead fish (Ophicephalus striatus), may pose a threat to the health of snakehead fish. Previous research has proved that the replication of SHVV can be significantly inhibited by glutamine starvation. To study how glutamine starvation inhibits SHVV replication, channel catfish ovary (CCO) cells with SHVV cultivated in the glutamine-free medium or the complete medium were used to investigate the differentially expressed proteins (DEPs). The results showed that 124 up-regulated and 246 down-regulated proteins were involved in many viral replication physiological processes, such as autophagy, post-translational modifications machinery, and functional pathways, including the PI3K-Akt signaling pathway and mTOR signaling pathway. Furthermore, a few proteins, such as Akt and Hsp90, which have been confirmed to be involved in the replication of RNA viruses, were also significantly differentially expressed. Taken together, our study demonstrated that glutamine starvation affects various functional pathways and the expression of some key proteins related to RNA viral replication, which will benefit future studies on the replication mechanisms of SHVV and the prevention of SHVV infection.

Honokiol and Alpha-Mangostin Inhibit Mayaro Virus Replication through Different Mechanisms.

Mayaro virus (MAYV) is an emerging arbovirus with an increasing circulation across the Americas. In the present study, we evaluated the potential antiviral activity of the following natural compounds against MAYV and other arboviruses: Sanguinarine, (R)-Shikonin, Fisetin, Honokiol, Tanshinone IIA, and α-Mangostin. Sanguinarine and Shikonin showed significant cytotoxicity, whereas Fisetin, Honokiol, Tanshinone IIA, and α-Mangostin were well tolerated in all the cell lines tested. Honokiol and α-Mangostin treatment protected Vero-E6 cells against MAYV-induced damage and resulted in a dose-dependent reduction in viral progeny yields for each of the MAYV strains and human cell lines assessed. These compounds also reduced MAYV viral RNA replication in HeLa cells. In addition, Honokiol and α-Mangostin disrupted MAYV infection at different stages of the virus life cycle. Moreover, Honokiol and α-Mangostin decreased Una, Chikungunya, and Zika viral titers and downmodulated the expression of E1 and nsP1 viral proteins from MAYV, Una, and Chikungunya. Finally, in Honokiol- and α-Mangostin-treated HeLa cells, we observed an upregulation in the expression of type I interferon and specific interferon-stimulated genes, including IFNα, IFNβ, MxA, ISG15, OAS2, MDA-5, TNFα, and IL-1β, which may promote an antiviral cellular state. Our results indicate that Honokiol and α-Mangostin present potential broad-spectrum activity against different arboviruses through different mechanisms.

Generation of viperin-knockout zebrafish by CRISPR/Cas9-mediated genome engineering and the effect of this mutation under VHSV infection

Viperin is an important virus-induced protein in animals that negatively participates in RNA viral replication and transcription. The reactive machinery of viperin suggests that it produces a regulatory molecule ddhCTP, which may affect immune regulation. In this study, we investigated the expression pattern of viperin in larval and adult stages of zebrafish by whole-mount in situ hybridization and reverse transcription-quantitative PCR (RT-qPCR). To elucidate the function of viperin, we generated a zebrafish knockout model using the CRISPR/Cas9 method and evaluated the mutation's effects under viral hemorrhagic septicemia virus (VHSV) infections. In zebrafish larvae, viperin was expressed in the brain region, eye, and pharynx, which was confirmed by cryosectioning. In adult zebrafish, blood cells showed the highest levels of viperin expression. In 5 dpf fish challenged with VHSV, the expression of the viral NP protein was significantly enhanced in viperin−/− compared to wild-type fish. In vitro VHSV propagation analysis indicated comparatively higher levels of virus propagation in viperin−/− fish. Mortality analysis confirmed higher mortality rates, and interferon gene expression analysis showed a strong upregulation of interferon (ifn)φ1 and 3 gene in viperin−/− fish infected with VHSV. This study describes the successful generation of a viperin-knockout model and the role of viperin during VHSV infections.

The Hierarchical Sequence Requirements of the H1 Subtype-Specific Noncoding Regions of Influenza A Virus.

The genome of influenza A virus consists of eight single-stranded viral RNA (vRNA) segments. The nonconserved noncoding regions (NCRs) at the 3' and 5' termini of each segment show extremely low divergence and mutation rate. They appear as segment specific among the eight segments and also subtype specific among different subtype-determinant hemagglutinin (HA) and neuraminidase (NA) segments. In order to acquire in-depth knowledge on the sequence requirements of the segment-specific or subtype-specific NCRs (ssNCRs), we, in the context of WSN (H1N1) reverse genetics, designed a virus random nucleotide selection assay (vRNSA) in which we generated pHW2000-HA plasmid libraries with random nucleotides in each grouped nucleotide positions in the 3' and 5' H1-ssNCRs, followed by virus rescue, serial passage, and deep sequencing. The resulting sequence logos present a visualized dynamic overview of the hierarchical sequence requirements of the 3' and 5' H1-ssNCRs. It showed that, in the process of continuous passage, the 3' H1-ssNCR, in general, stabilized more quickly than the 5' H1-ssNCR. The nucleotides close to the highly conserved 3' and 5' promoter regions showed higher sequence stringency than nucleotides away from the promoter regions. All stabilized sequences displayed a common feature of high A/U ratios. Especially with our mutational function analyses, we demonstrate that the 3' promoter-proximal nucleotides could cooperatively exert a direct effect on the transcription and replication of the HA segment. Together, these results provide in-depth knowledge for understanding the NCRs of influenza A virus. IMPORTANCE The segment-specific and subtype-specific nonconserved noncoding regions (ssNCRs) at both 3' and 5' ends of viral RNA segments of influenza A virus are largely conserved among the same segments of different viruses. However, the function-related sequence requirements of these ssNCRs remain unclear. In this study, through a novel self-designed vRNSA approach, we present a visualized dynamic overview diagram directly reflecting the hierarchical sequence requirements within and between the 3' and 5' H1-ssNCRs. The in-depth functional mutagenesis analyses further revealed that specific nucleotides in the 3' promoter-proximal region could cooperatively exert a direct effect on viral RNA transcription and replication. This work further advanced our knowledge in understanding the nonconserved noncoding regions of influenza A viruses.

Examining the role of EGR1 during viral infections.

Early growth response 1 (EGR1) is a multifunctional mammalian transcription factor capable of both enhancing and/or inhibiting gene expression. EGR1 can be activated by a wide array of stimuli such as exposure to growth factors, cytokines, apoptosis, and various cellular stress states including viral infections by both DNA and RNA viruses. Following induction, EGR1 functions as a convergence point for numerous specialized signaling cascades and couples short-term extracellular signals to influence transcriptional regulation of genes required to initiate the appropriate biological response. The role of EGR1 has been extensively studied in both physiological and pathological conditions of the adult nervous system where it is readily expressed in various regions of the brain and is critical for neuronal plasticity and the formation of memories. In addition to its involvement in neuropsychiatric disorders, EGR1 has also been widely examined in the field of cancer where it plays paradoxical roles as a tumor suppressor gene or oncogene. EGR1 is also associated with multiple viral infections such as Venezuelan equine encephalitis virus (VEEV), Kaposi's sarcoma-associated herpesvirus (KSHV), herpes simplex virus 1 (HSV-1), human polyomavirus JC virus (JCV), human immunodeficiency virus (HIV), and Epstein-Barr virus (EBV). In this review, we examine EGR1 and its role(s) during viral infections. First, we provide an overview of EGR1 in terms of its structure, other family members, and a brief overview of its roles in non-viral disease states. We also review upstream regulators of EGR1 and downstream factors impacted by EGR1. Then, we extensively examine EGR1 and its roles, both direct and indirect, in regulating replication of DNA and RNA viruses.

Application of a maximal-clique based community detection algorithm to gut microbiome data reveals driver microbes during influenza A virus infection.

Influenza A Virus (IAV) infection followed by bacterial pneumonia often leads to hospitalization and death in individuals from high risk groups. Following infection, IAV triggers the process of viral RNA replication which in turn disrupts healthy gut microbial community, while the gut microbiota plays an instrumental role in protecting the host by evolving colonization resistance. Although the underlying mechanisms of IAV infection have been unraveled, the underlying complex mechanisms evolved by gut microbiota in order to induce host immune response following IAV infection remain evasive. In this work, we developed a novel Maximal-Clique based Community Detection algorithm for Weighted undirected Networks (MCCD-WN) and compared its performance with other existing algorithms using three sets of benchmark networks. Moreover, we applied our algorithm to gut microbiome data derived from fecal samples of both healthy and IAV-infected pigs over a sequence of time-points. The results we obtained from the real-life IAV dataset unveil the role of the microbial families Ruminococcaceae, Lachnospiraceae, Spirochaetaceae and Prevotellaceae in the gut microbiome of the IAV-infected cohort. Furthermore, the additional integration of metaproteomic data enabled not only the identification of microbial biomarkers, but also the elucidation of their functional roles in protecting the host following IAV infection. Our network analysis reveals a fast recovery of the infected cohort after the second IAV infection and provides insights into crucial roles of Desulfovibrionaceae and Lactobacillaceae families in combating Influenza A Virus infection. Source code of the community detection algorithm can be downloaded from https://github.com/AniBhar84/MCCD-WN.

Architecture of the chikungunya virus replication organelle.

Alphaviruses are mosquito-borne viruses that cause serious disease in humans and other mammals. Along with its mosquito vector, the Alphavirus chikungunya virus (CHIKV) has spread explosively in the last 20 years, and there is no approved treatment for chikungunya fever. On the plasma membrane of the infected cell, CHIKV generates dedicated organelles for viral RNA replication, so-called spherules. Whereas structures exist for several viral proteins that make up the spherule, the architecture of the full organelle is unknown. Here, we use cryo-electron tomography to image CHIKV spherules in their cellular context. This reveals that the viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud. Biochemical assays show that the viral helicase-protease nsP2, while having no membrane affinity on its own, is recruited to membranes by nsP1. The tomograms further reveal that full-sized spherules contain a single copy of the viral genome in double-stranded form. Finally, we present a mathematical model that explains the membrane remodeling of the spherule in terms of the pressure exerted on the membrane by the polymerizing RNA, which provides a good agreement with the experimental data. The energy released by RNA polymerization is found to be sufficient to remodel the membrane to the characteristic spherule shape.

Therapeutic strategy targeting host lipolysis limits infection by SARS-CoV-2 and influenza A virus

The biosynthesis of host lipids and/or lipid droplets (LDs) has been studied extensively as a putative therapeutic target in diverse viral infections. However, directly targeting the LD lipolytic catabolism in virus-infected cells has not been widely investigated. Here, we show the linkage of the LD-associated lipase activation to the breakdown of LDs for the generation of free fatty acids (FFAs) at the late stage of diverse RNA viral infections, which represents a broad-spectrum antiviral target. Dysfunction of membrane transporter systems due to virus-induced cell injury results in intracellular malnutrition at the late stage of infection, thereby making the virus more dependent on the FFAs generated from LD storage for viral morphogenesis and as a source of energy. The replication of SARS-CoV-2 and influenza A virus (IAV), which is suppressed by the treatment with LD-associated lipases inhibitors, is rescued by supplementation with FFAs. The administration of lipase inhibitors, either individually or in a combination with virus-targeting drugs, protects mice from lethal IAV infection and mitigates severe lung lesions in SARS-CoV-2-infected hamsters. Moreover, the lipase inhibitors significantly reduce proinflammatory cytokine levels in the lungs of SARS-CoV-2- and IAV-challenged animals, a cause of a cytokine storm important for the critical infection or mortality of COVID-19 and IAV patients. In conclusion, the results reveal that lipase-mediated intracellular LD lipolysis is commonly exploited to facilitate RNA virus replication and furthermore suggest that pharmacological inhibitors of LD-associated lipases could be used to curb current COVID-19- and future pandemic outbreaks of potentially troublesome RNA virus infection in humans.