Inhibition of cellular RNA methyltransferase abrogates influenza virus capping and replication.

Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

Structural basis for substrate selection by the SARS-CoV-2 replicase.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.

Proteolytic Processing of the Coronavirus Replicase Nonstructural Protein 14 Exonuclease Is Not Required for Virus Replication but Alters RNA Synthesis and Viral Fitness.

Coronaviruses (CoVs) initiate replication by translation of the positive-sense RNA genome into the replicase polyproteins connecting 16 nonstructural protein domains (nsp1-16), which are subsequently processed by viral proteases to yield mature nsp. For the betacoronavirus murine hepatitis virus (MHV), total inhibition of translation or proteolytic processing of replicase polyproteins results in rapid cessation of RNA synthesis. The nsp5-3CLpro (Mpro) processes nsps7-16, which assemble into functional replication-transcription complexes (RTCs), including the enzymatic nsp12-RdRp and nsp14-exoribonuclease (ExoN)/N7-methyltransferase. The nsp14-ExoN activity mediates RNA-dependent RNA proofreading, high-fidelity RNA synthesis, and replication. To date, the solved partial RTC structures, biochemistry, and models use or assume completely processed, mature nsp. Here, we demonstrate that in MHV, engineered deletion of the cleavage sites between nsp13-14 and nsp14-15 allowed recovery of replication-competent virus. Compared to wild-type (WT) MHV, the nsp13-14 and nsp14-15 cleavage deletion mutants demonstrated delayed replication kinetics, impaired genome production, altered abundance and patterns of recombination, and impaired competitive fitness. Further, the nsp13-14 and nsp14-15 mutant viruses demonstrated mutation frequencies that were significantly higher than with the WT. The results demonstrate that cleavage of nsp13-14 or nsp14-15 is not required for MHV viability and that functions of the RTC/nsp14-ExoN are impaired when assembled with noncleaved intermediates. These data will inform future genetic, structural, biochemical, and modeling studies of coronavirus RTCs and nsp 13, 14, and 15 and may reveal new approaches for inhibition or attenuation of CoV infection. IMPORTANCE Coronavirus replication requires proteolytic maturation of the nonstructural replicase proteins to form the replication-transcription complex. Coronavirus replication-transcription complex models assume mature subunits; however, mechanisms of coronavirus maturation and replicase complex formation have yet to be defined. Here, we show that for the coronavirus murine hepatitis virus, cleavage between the nonstructural replicase proteins nsp13-14 and nsp14-15 is not required for replication but does alter RNA synthesis and recombination. These results shed new light on the requirements for coronavirus maturation and replication-transcription complex assembly, and they may reveal novel therapeutic targets and strategies for attenuation.

Conserved Lysine Residues of Hepatitis B Virus Core Protein Are Not Required for Covalently Closed Circular DNA Formation.

Hepatitis B virus (HBV) core protein (HBc), the building block of the viral capsid, plays a critical role throughout the HBV life cycle. There are two highly conserved lysine residues, namely, K7 and K96, on HBc, which have been proposed to function at various stages of viral replication, potentially through lysine-specific posttranslational modifications (PTMs). Here, we substituted K7 and K96 with alanine or arginine, which would also block potential PTMs on these two lysine residues, and tested the effects of these substitutions on HBV replication and infection. We found that the two lysine residues were dispensable for all intracellular steps of HBV replication. In particular, all mutants were competent to form the covalently closed circular DNA (cccDNA) via the intracellular amplification pathway, indicating that K7 and K96, or any PTMs of these residues, were not essential for nucleocapsid uncoating, a prerequisite for cccDNA formation. Furthermore, we found that K7A and K7R mutations did not affect de novo cccDNA formation and RNA transcription during infection, indicating that K7 or any PTMs of this residue were dispensable for HBV infection. In addition, we demonstrated that the HBc K7 coding sequence (AAA), as part of the HBV polyadenylation signal UAUAAA, was indispensable for viral RNA production, implicating this cis requirement at the RNA level, instead of any function of HBc-K7, likely constrains the identity of the 7th residue of HBc. In conclusion, our results provided novel insights regarding the roles of lysine residues on HBc, and their coding sequences, in the HBV life cycle. IMPORTANCE Hepatitis B virus (HBV) infection remains a public health burden that affects 296 million individuals worldwide. HBV core protein (HBc) is involved in almost all steps in the HBV life cycle. There are two conserved lysine residues on HBc. Here, we found that neither of them is essential for HBV intracellular replication, including the formation of covalently closed circular DNA (cccDNA), the molecular basis for establishing and sustaining the HBV infection. However, K96 is critical for virion morphogenesis, while the K7 coding sequence, but not HBc-K7 itself, is indispensable, as part of the RNA polyadenylation signal, for HBV RNA production from cccDNA. Our results provide novel insights regarding the role of the conserved lysine residues on HBc, and their coding sequences, in viral replication, and should facilitate the development of antiviral drugs against the HBV capsid protein.

5-Iodotubercidin inhibits SARS-CoV-2 RNA synthesis.

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).

The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme.

SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5' capping of viral RNAs. The formation of the 5' 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5' triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5' end of viral RNA via a 5' to 5' triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.

Contribution of autophagy machinery factors to HCV and SARS-CoV-2 replication organelle formation.

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.

The predicted stem-loop structure in the 3'-end of the human norovirus antigenomic sequence is required for its genomic RNA synthesis by its RdRp.

The norovirus genome consists of a single positive-stranded RNA. The mechanism by which this single-stranded RNA genome is replicated is not well understood. To reveal the mechanism underlying the initiation of the norovirus genomic RNA synthesis by its RNA-dependent RNA polymerase (RdRp), we used an in vitro assay to detect the complementary RNA synthesis activity. Results showed that the purified recombinant RdRp was able to synthesize the complementary positive-sense RNA from a 100-nt template corresponding to the 3'-end of the viral antisense genome sequence, but that the RdRp could not synthesize the antisense genomic RNA from the template corresponding to the 5'-end of the positive-sense genome sequence. We also predicted that the 31 nt region at the 3'-end of the RNA antisense template forms a stem-loop structure. Deletion of this sequence resulted in the loss of complementary RNA synthesis by the RdRp, and connection of the 31 nt to the 3'-end of the inactive positive-sense RNA template resulted in the gain of complementary RNA synthesis by the RdRp. Similarly, an electrophoretic mobility shift assay further revealed that the RdRp bound to the antisense RNA specifically, but was dependent on the 31 nt at the 3'-end. Therefore, based on this observation and further deletion and mutation analyses, we concluded that the predicted stem-loop structure in the 31 nt end and the region close to the antisense viral genomic stem sequences are both important for initiating the positive-sense human norovirus genomic RNA synthesis by its RdRp.

Enterovirus 71 2A Protease Inhibits P-Body Formation To Promote Viral RNA Synthesis.

Several viruses have been proven to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation remains unclear, and the detailed molecular mechanisms and functions of picornavirus regulation of P-bodies are limited. Here, we show the crucial role of 2A protease in inhibiting P-bodies to promote viral replication during enterovirus 71 infection. Moreover, we found that the activity of 2A protease is essential to inhibit P-body formation, which was proven by the result that infection with EV71-2AC110S, a 2A protease activity-inactivated recombinant virus, failed to block the formation of P-bodies. Furthermore, we show that DDX6, a scaffolding protein of P-bodies, interacted with viral RNA to facilitate viral replication rather than viral translation, by using a Renilla luciferase mRNA reporter system and nascent RNA capture assay. Altogether, our data first demonstrate that the 2A protease of enterovirus inhibits P-body formation to facilitate viral RNA synthesis by recruiting the P-body components to viral RNA. IMPORTANCE Processing bodies (P-bodies) are constitutively present in eukaryotic cells and play an important role in the mRNA cycle, including regulation of gene expression and mRNA degradation. The P-body is the structure that viruses manipulate to facilitate their survival. Here, we show that the 2A protease alone was efficient to block P-body formation during enterovirus 71 infection, and its activity is essential. When the assembly of P-bodies was blocked by 2A protease, DDX6 and 4E-T, which were required for P-body formation, bound to viral RNA to facilitate viral RNA synthesis. We propose a model revealing that EV71 manipulates P-body formation to generate an environment that is conducive to viral replication by facilitating viral RNA synthesis: 2A protease blocked P-body assembly to make it possible for virus to take advantage of P-body components.

Remdesivir for the treatment of Covid-19: the value of biochemical studies.

The nucleotide analogue prodrug remdesivir remains the only FDA-approved antiviral small molecule for the treatment of infection with SARS-CoV-2. Biochemical studies revealed that the active form of the drug targets the viral RNA-dependent RNA polymerase and causes delayed chain-termination. Delayed chain-termination is incomplete, but the continuation of RNA synthesis enables a partial escape from viral proofreading. Remdesivir becomes embedded in the copy of the RNA genome that later serves as a template. Incorporation of an incoming nucleotide triphosphate is now inhibited by the modified template. Knowledge on the mechanism of action matters. Enzymatic inhibition links to antiviral effects in cell cultures, animal models and viral load reduction in patients, which provides the logical chain that is expected for a direct acting antiviral. Hence, remdesivir also serves as a benchmark in current drug development efforts that will hopefully lead to orally available treatments to the benefit of a broader population.

Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity.

Dengue virus (DENV) is spread from human to human through the bite of the female Aedes aegypti mosquito and leads to about 100 million clinical infections yearly. Treatment options and vaccine availability for DENV are limited. Defective interfering particles (DIPs) are considered a promising antiviral approach but infectious virus contamination has limited their development. Here, a DENV-derived DIP production cell line was developed that continuously produced DENV-free DIPs. The DIPs contained and could deliver to cells a DENV serotype 2 subgenomic defective-interfering RNA, which was originally discovered in DENV infected patients. The DIPs released into cell culture supernatant were purified and could potently inhibit replication of all DENV serotypes in cells. Antiviral therapeutics are limited for many viral infection. The DIP system described could be re-purposed to make antiviral DIPs for many other RNA viruses such as SARS-CoV-2, yellow fever, West Nile and Zika viruses.

Functionally active cyclin-dependent kinase 9 is essential for porcine reproductive and respiratory syndrome virus subgenomic RNA synthesis.

Cyclin-dependent kinase 9 (CDK9) is a key regulator of RNA-polymerase II and a candidate therapeutic target for various virus infections such as respiratory syncytial virus, herpes simplex virus, human adenovirus, human cytomegalovirus, hepatitis virus B, and human papillomavirus. We employed CDK9-IN-1, a selective CDK9 inhibitor, to investigate the role of CDK9 in porcine reproductive and respiratory syndrome virus (PRRSV) infection. CDK9-IN-1 dose-dependently reduced PRRSV replication without cytotoxicity in the infected cells. The antiviral activity of CDK9-IN-1 was further confirmed by evaluating the effects of lentivirus-mediated CDK9 knockdown or CDK9 overexpression on PRRSV infection. Briefly, the depletion of CDK9 significantly inhibited viral replication, while the overexpression of CDK9 promoted viral replication. PRRSV infection also enhanced the nuclear export of CDK9 without affecting CDK9 protein expression. Viral replication cycle analyses further revealed that functionally active CDK9 in the cytosol advanced viral subgenomic RNA synthesis. Collectively, our data illustrated that CDK9 was a new host factor that was involved in PRRSV subgenomic RNA synthesis, and CDK9 inhibitor, CDK9-IN-1 was a promising antiviral candidate for PRRSV infection.

Mapping of sequences in the 5' region and 3' UTR of tomato ringspot virus RNA2 that facilitate cap-independent translation of reporter transcripts in vitro.

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5'-viral genome-linked protein (VPg) and have a 3' polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3' untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5' regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5' region and 3' UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5' region and 3' UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro.

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40-80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.

Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.

Remdesivir: Critical Clinical Appraisal for COVID 19 Treatment.

Remdesivir is presently been considered as 'molecule of hope' to curb the menace of COVID19. Non-availability of any USFDA approved drug has led to several attempt of drug-repurposing and development of new therapeutic molecules. However, Remdesivir has been found to be effective against a broad range of virus including SARS, MERS and COVID 19 through in-vitro studies. Several clinical research attempt are presently being conducted showing promising result yet not conclusive. This review summarized all such clinical trials to critically appraise the usage of Remdesivir against COVID 19 along with the publications related to the results of the clinical studies. The present regulatory aspect i. e. Emergency Use Authorization (EYA) and information of molecule and plausible mechanism is also dealt.

RNA-dependent RNA polymerase: Structure, mechanism, and drug discovery for COVID-19.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global pandemic. Although great efforts have been made to develop effective therapeutic interventions, only the nucleotide analog remdesivir was approved for emergency use against COVID-19. Remdesivir targets the RNA-dependent RNA polymerase (RdRp), an essential enzyme for viral RNA replication and a promising drug target for COVID-19. Recently, several structures of RdRp in complex with substrate RNA and remdesivir were reported, providing insights into the mechanisms of RNA recognition by RdRp. These structures also reveal the mechanism of RdRp inhibition by nucleotide inhibitors and offer a molecular template for the development of RdRp-targeting drugs. This review discusses the recognition mechanism of RNA and nucleotide inhibitor by RdRp, and its implication in drug discovery.

Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase.

Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2'-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2'-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

Transcriptome Profiling of Human Monocyte-Derived Macrophages Upon CCL2 Neutralization Reveals an Association Between Activation of Innate Immune Pathways and Restriction of HIV-1 Gene Expression.

Macrophages are key targets of human immunodeficiency virus type 1 (HIV-1) infection and main producers of the proinflammatory chemokine CC chemokine ligand 2 (CCL2), whose expression is induced by HIV-1 both in vitro and in vivo. We previously found that CCL2 neutralization in monocyte-derived macrophages (MDMs) strongly inhibited HIV-1 replication affecting post-entry steps of the viral life cycle. Here, we used RNA-sequencing to deeply characterize the cellular factors and pathways modulated by CCL2 blocking in MDMs and involved in HIV-1 replication restriction. We report that exposure to CCL2 neutralizing antibody profoundly affected the MDM transcriptome. Functional annotation clustering of up-regulated genes identified two clusters enriched for antiviral defense and immune response pathways, comprising several interferon-stimulated, and restriction factor coding genes. Transcripts in the clusters were enriched for RELA and NFKB1 targets, suggesting the activation of the canonical nuclear factor κB pathway as part of a regulatory network involving miR-155 up-regulation. Furthermore, while HIV-1 infection caused small changes to the MDM transcriptome, with no evidence of host defense gene expression and type I interferon signature, CCL2 blocking enabled the activation of a strong host innate response in infected macrophage cultures, and potently inhibited viral genes expression. Notably, an inverse correlation was found between levels of viral transcripts and of the restriction factors APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 A), ISG15, and MX1. These findings highlight an association between activation of innate immune pathways and HIV-1 restriction upon CCL2 blocking and identify this chemokine as an endogenous factor contributing to the defective macrophage response to HIV-1. Therapeutic targeting of CCL2 may thus strengthen host innate immunity and restrict HIV-1 replication.

Eukaryotic Translation Elongation Factor 1 Delta Inhibits the Nuclear Import of the Nucleoprotein and PA-PB1 Heterodimer of Influenza A Virus.

The viral ribonucleoprotein (vRNP) of the influenza A virus (IAV) is responsible for the viral RNA transcription and replication in the nucleus, and its functions rely on host factors. Previous studies have indicated that eukaryotic translation elongation factor 1 delta (eEF1D) may associate with RNP subunits, but its roles in IAV replication are unclear. Herein, we showed that eEF1D was an inhibitor of IAV replication because knockout of eEF1D resulted in a significant increase in virus yield. eEF1D interacted with RNP subunits polymerase acidic protein (PA), polymerase basic 1 (PB1), polymerase basic 2 (PB2), and also with nucleoprotein (NP) in an RNA-dependent manner. Further studies revealed that eEF1D impeded the nuclear import of NP and PA-PB1 heterodimer of IAV, thereby suppressing the vRNP assembly, viral polymerase activity, and viral RNA synthesis. Together, our studies demonstrate eEF1D negatively regulating the IAV replication by inhibition of the nuclear import of RNP subunits, which not only uncovers a novel role of eEF1D in IAV replication but also provides new insights into the mechanisms of nuclear import of vRNP proteins.IMPORTANCE Influenza A virus is the major cause of influenza, a respiratory disease in humans and animals. Different from most other RNA viruses, the transcription and replication of IAV occur in the cell nucleus. Therefore, the vRNPs must be imported into the nucleus for viral transcription and replication, which requires participation of host proteins. However, the mechanisms of the IAV-host interactions involved in nuclear import remain poorly understood. Here, we identified eEF1D as a novel inhibitor for the influenza virus life cycle. Importantly, eEF1D impaired the interaction between NP and importin α5 and the interaction between PB1 and RanBP5, which impeded the nuclear import of vRNP. Our studies not only reveal the molecular mechanisms of the nuclear import of IAV vRNP but also provide potential anti-influenza targets for antiviral development.