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1.
FASEB J ; 36(10): e22537, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36070077

RESUMO

Influenza A viruses (IAVs) rely on viral ribonucleoprotein (vRNP) complexes to control transcription and replication. Each vRNP consists of one viral genomic RNA segment associated with multiple nucleoproteins (NP) and a trimeric IAV RNA polymerase complex. Previous studies showed that post-translational modifications of vRNP components, such as NP, by host factors would in turn affect the IAV life cycle or modulate host anti-viral response. In this study, we found host E3 ubiquitin ligase Pirh2 interacted with NP and mediated short-chain ubiquitination of NP at lysine 351, which suppressed NP-PB2 interaction and vRNP formation. In addition, we showed that knockdown of Pirh2 promoted IAV replication, whereas overexpression of Pirh2 inhibited IAV replication. However, Pirh2-ΔRING lacking E3 ligase activity failed to inhibit IAV infection. Moreover, we showed that Pirh2 had no effect on the replication of a rescued virus, WSN-K351R, carrying lysine-to-arginine substitution at residue 351. Interestingly, by analyzing human and avian IAVs from 2011 to 2020 in influenza research databases, we found that 99.18% of 26 977 human IAVs encode lysine, but 95.3% of 9956 avian IAVs encode arginine at residue 351 of NP protein. Consistently, knockdown of Pirh2 failed to promote propagation of two avian-like influenza viruses, H9N2-W1 and H9N2-C1, which naturally encode arginine at residue 351 of NP. Taken together, we demonstrated that Pirh2 is a host factor restricting IAV infection by modulating short-chain ubiquitination of NP. Meanwhile, it is noteworthy that residue 351 of NP targeted by Pirh2 may associate with the evasion of human anti-viral response against avian-like influenza viruses.


Assuntos
Vírus da Influenza A Subtipo H9N2 , Ribonucleoproteínas , Ubiquitina-Proteína Ligases , Replicação Viral , Arginina/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Humana/virologia , Lisina/metabolismo , RNA Viral/metabolismo , Ribonucleoproteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
2.
Sci Rep ; 12(1): 14972, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36100631

RESUMO

During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure.


Assuntos
COVID-19 , Edição de RNA , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , COVID-19/genética , Células CACO-2 , Citidina Desaminase , Humanos , Mutação , Pandemias , Proteínas , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética
3.
PLoS Pathog ; 18(9): e1010752, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36048900

RESUMO

Positive-strand RNA viruses assemble their viral replication complexes (VRCs) on specific host organelle membranes, yet it is unclear how viral replication proteins recognize and what motifs or domains in viral replication proteins determine their destinations. We show here that an amphipathic helix, helix B in replication protein 1a of brome mosaic virus (BMV), is necessary for 1a's localization to the nuclear endoplasmic reticulum (ER) membrane where BMV assembles its VRCs. Helix B is also sufficient to target soluble proteins to the nuclear ER membrane in yeast and plant cells. We further show that an equivalent helix in several plant- and human-infecting viruses of the Alsuviricetes class targets fluorescent proteins to the organelle membranes where they form their VRCs, including ER, vacuole, and Golgi membranes. Our work reveals a conserved helix that governs the localization of VRCs among a group of viruses and points to a possible target for developing broad-spectrum antiviral strategies.


Assuntos
Bromovirus , RNA Viral , Retículo Endoplasmático/metabolismo , Humanos , RNA Viral/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Virais/metabolismo , Replicação Viral
4.
Proc Natl Acad Sci U S A ; 119(39): e2206292119, 2022 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-36122222

RESUMO

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)-for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.


Assuntos
Bromovirus , Vírus de RNA , Bromovirus/genética , Bromovirus/metabolismo , Capsídeo/metabolismo , Vírus de RNA/genética , RNA Viral/genética , RNA Viral/metabolismo , Vírion/genética , Vírion/metabolismo
5.
Sci Adv ; 8(36): eabp8655, 2022 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-36083899

RESUMO

During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVGs) and mini viral RNAs (mvRNAs). Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid-inducible gene I (RIG-I) to viral RNAs. However, it is not clear what factors determine which influenza A virus RNAs are RIG-I agonists. Here, we provide evidence that RNA structures, called template loops (t-loops), stall the viral RNA polymerase and contribute to innate immune activation by mvRNAs during influenza A virus infection. Impairment of replication by t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and this effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings are suggestive of a mechanism involving polymerase stalling that links aberrant viral replication to the activation of the innate immune response.


Assuntos
Influenza Humana , Linhagem Celular , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Humanos , Imunidade Inata , Influenza Humana/genética , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral/genética
6.
Front Immunol ; 13: 989298, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36119073

RESUMO

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that are implicated in RNA metabolism, such as alternative splicing, mRNA stabilization and translational regulation. According to their different cellular localization, hnRNPs display multiple functions. Most hnRNPs were predominantly located in the nucleus, but some of them could redistribute to the cytoplasm during virus infection. HnRNPs consist of different domains and motifs that enable these proteins to recognize predetermined nucleotide sequences. In the virus-host interactions, hnRNPs specifically bind to viral RNA or proteins. And some of the viral protein-hnRNP interactions require the viral RNA or other host factors as the intermediate. Through various mechanisms, hnRNPs could regulate viral translation, viral genome replication, the switch of translation to replication and virion release. This review highlights the common features and the distinguish roles of hnRNPs in the life cycle of positive single-stranded RNA viruses.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas , Vírus de RNA de Cadeia Positiva , Animais , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Estágios do Ciclo de Vida , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA , Proteínas Virais/metabolismo
7.
J Virol ; 96(17): e0091922, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-35938871

RESUMO

Alphavirus infection induces the expression of type I interferons, which inhibit the viral replication by upregulating the expression of interferon-stimulated genes (ISGs). Identification and mechanistic studies of the antiviral ISGs help to better understand how the host controls viral infection and help to better understand the viral replication process. Here, we report that the ISG product TMEM45B inhibits the replication of Sindbis virus (SINV). TMEM45B is a transmembrane protein that was detected mainly in the trans-Golgi network, endosomes, and lysosomes but not obviously at the plasma membrane or endoplasmic reticulum. TMEM45B interacted with the viral nonstructural proteins Nsp1 and Nsp4 and inhibited the translation and promoted the degradation of SINV RNA. TMEM45B overexpression rendered the intracellular membrane-associated viral RNA sensitive to RNase treatment. In line with these results, the formation of cytopathic vacuoles (CPVs) was dramatically diminished in TMEM45B-expressing cells. TMEM45B also interacted with Nsp1 and Nsp4 of chikungunya virus (CHIKV), suggesting that it may also inhibit the replication of other alphaviruses. These findings identified TMEM45B as an antiviral factor against alphaviruses and help to better understand the process of the viral genome replication. IMPORTANCE Alphaviruses are positive-stranded RNA viruses with more than 30 members. Infection with Old World alphaviruses, which comprise some important human pathogens such as chikungunya virus and Ross River virus, rarely results in fatal diseases but can lead to high morbidity in humans. Infection with New World alphaviruses usually causes serious encephalitis but low morbidity in humans. Alphavirus infection induces the expression of type I interferons, which subsequently upregulate hundreds of interferon-stimulated genes. Identification and characterization of host antiviral factors help to better understand how the viruses can establish effective infection. Here, we identified TMEM45B as a novel interferon-stimulated antiviral factor against Sindbis virus, a prototype alphavirus. TMEM45B interacted with viral proteins Nsp1 and Nsp4, interfered with the interaction between Nsp1 and Nsp4, and inhibited the viral replication. These findings provide insights into the detailed process of the viral replication and help to better understand the virus-host interactions.


Assuntos
Infecções por Alphavirus , Interferon Tipo I , Proteínas de Membrana , Vírus Sindbis , Proteínas não Estruturais Virais , Fatores de Restrição Antivirais , Vírus Chikungunya/genética , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , RNA Viral/metabolismo , Vírus Sindbis/genética , Vírus Sindbis/fisiologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
8.
Cell Death Dis ; 13(8): 707, 2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35970851

RESUMO

RIG-I-like receptors (RLRs), protein kinase R (PKR), and endosomal Toll-like receptor 3 (TLR3) sense viral non-self RNA and are involved in cell fate determination. However, the mechanisms by which intracellular RNA induces apoptosis, particularly the role of each RNA sensor, remain unclear. We performed cytoplasmic injections of different types of RNA and elucidated the molecular mechanisms underlying viral dsRNA-induced apoptosis. The results obtained revealed that short 5'-triphosphate dsRNA, the sole ligand of RIG-I, induced slow apoptosis in a fraction of cells depending on IRF-3 transcriptional activity and IFN-I production. However, intracellular long dsRNA was sensed by PKR and TLR3, which activate distinct signals, and synergistically induced rapid apoptosis. PKR essentially induced translational arrest, resulting in reduced levels of cellular FLICE-like inhibitory protein and functioned in the TLR3/TRIF-dependent activation of caspase 8. The present results demonstrated that PKR and TLR3 were both essential for inducing the viral RNA-mediated apoptosis of infected cells and the arrest of viral production.


Assuntos
Antivirais , Receptor 3 Toll-Like , Antivirais/farmacologia , Apoptose , Interferon beta/genética , RNA de Cadeia Dupla/genética , RNA Viral/metabolismo , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
9.
Antiviral Res ; 206: 105398, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35985406

RESUMO

A marked reorganization of internal membranes occurs in the cytoplasm of cells infected by single stranded positive-sense RNA viruses. Most cell compartments change their asset to provide lipids for membrane rearrangement into replication organelles, where to concentrate viral proteins and enzymes while hiding from pathogen pattern recognition molecules. Because the endoplasmic reticulum is a central hub for lipid metabolism, when viruses hijack the organelle to form their replication organelles, a cascade of events change the intracellular environment. This results in a marked increase in lipid consumption, both by lipolysis and lipophagy of lipid droplets. In addition, lipids are used to produce energy for viral replication. At the same time, inflammation is started by signalling lipids, where lysosomal processing plays a relevant role. This review is aimed at providing an overview on what takes place after human class IV viruses have released their genome into the host cell and the consequences on lipid metabolism, including lysosomes.


Assuntos
Vírus de RNA de Cadeia Positiva , Vírus de RNA , Retículo Endoplasmático/metabolismo , Humanos , Lipídeos , Lisossomos/metabolismo , RNA Viral/metabolismo , Replicação Viral
10.
J Virol ; 96(17): e0069922, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-35993738

RESUMO

Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of plus-stranded RNA viruses. VPg is covalently linked to the 5' end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses have multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use cricket paralysis virus (CrPV), a dicistrovirus that has four nonidentical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses contain two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1 to VPg4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, mutations of which in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant negative effect. Using viral minigenome reporters that monitor dicistrovirus 5' untranslated (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of distinct IRES translation activities. We uncovered a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote infection. IMPORTANCE Genetic duplication is exceedingly rare in small RNA viral genomes, as there is selective pressure to prevent RNA genomes from expanding. However, some small RNA viruses encode multiple copies of a viral protein, most notably an unusual viral protein that is linked to the viral RNA genome. Here, we investigate a family of viruses that contains multiple viral protein genome-linked proteins and reveal a novel viral strategy whereby viral protein copy number counterbalances differences in viral protein synthesis mechanisms.


Assuntos
Infecções por Vírus de RNA , Vírus de RNA , Viroses , Animais , Drosophila/genética , Sítios Internos de Entrada Ribossomal/genética , Vírus de RNA/genética , RNA Viral/metabolismo , Serina/genética , Proteínas Virais/metabolismo
11.
J Virol ; 96(17): e0115122, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36000838

RESUMO

Viruses have evolved different strategies to overcome their recognition by the host innate immune system. The addition of caps at their 5' RNA ends is an efficient mechanism not only to ensure escape from detection by the innate immune system but also to ensure the efficient synthesis of viral proteins. Rotavirus mRNAs contain a type 1 cap structure at their 5' end that is added by the viral capping enzyme VP3, which is a multifunctional protein with all the enzymatic activities necessary to add the cap and also functions as an antagonist of the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. Here, the relative abundances of capped and noncapped viral RNAs during the replication cycle of rotavirus were determined. We found that both classes of rotaviral plus-sense RNAs (+RNAs) were encapsidated and that they were present in a 1:1 ratio in the mature infectious particles. The capping of viral +RNAs was dynamic, since different ratios of capped and noncapped RNAs were detected at different times postinfection. Similarly, when the relative amounts of capped and uncapped viral +RNAs produced in an in vitro transcription system were determined, we found that the proportions were very similar to those in the mature viral particles and in infected cells, suggesting that the capping efficiency of VP3, both in vivo and in vitro, might be close to 50%. Unexpectedly, when the effect of simultaneously knocking down the expression of VP3 and RNase L on the cap status of viral +RNAs was evaluated, we found that, even though at late times postinfection there was an increased proportion of capped viral RNAs in infected cells, the viral particles isolated from this condition contained equal ratios of capped and noncapped viral RNA, suggesting that there might be selective packaging of capped and noncapped RNAs. IMPORTANCE Rotaviruses have a genome composed of 11 segments of double-stranded RNA. Whether all 5' ends of the positive-sense genomic RNAs contained in the mature viral particles are modified by a cap structure is unknown. In this work, we characterized the relative proportions of capped and noncapped viral RNAs in rotavirus-infected cells and in viral particles by using a direct quantitative assay. We found that, independent of the relative proportions of capped/noncapped RNAs present in rotavirus-infected cells, there were similar proportions of these two kinds of 5'-modified positive-sense RNAs in the viral particles.


Assuntos
Rotavirus , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Rotavirus/metabolismo , Vírion/metabolismo
12.
Viruses ; 14(8)2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-36016411

RESUMO

The on-going global pandemic of COVID-19 is caused by SARS-CoV-2, which features a proofreading mechanism to facilitate the replication of its large RNA genome. The 3'-to-5' exoribonuclease (ExoN) activity of SARS-CoV-2 non-structural protein 14 (nsp14) removes nucleotides misincorporated during RNA synthesis by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and thereby compromises the efficacy of antiviral nucleoside/nucleotide analogues. Here we show biochemically that SARS-CoV-2 nsp14 can excise the natural antiviral chain-terminating nucleotide, 3'-deoxy-3',4'-didehydro-cytidine 5'-monophosphate (ddhCMP), incorporated by RdRp at the 3' end of an RNA strand. Nsp14 ExoN processes an RNA strand terminated with ddhCMP more efficiently than that with a non-physiological chain terminator 3'-deoxy-cytidine monophosphate (3'-dCMP), whereas RdRp is more susceptible to chain termination by 3'-dCTP than ddhCTP. These results suggest that nsp14 ExoN could play a role in protecting SARS-CoV-2 from ddhCTP, which is produced as part of the innate immune response against viral infections, and that the SARS-CoV-2 enzymes may have adapted to minimize the antiviral effect of ddhCTP.


Assuntos
COVID-19 , Exorribonucleases , Antivirais/farmacologia , Citidina/farmacologia , Exorribonucleases/metabolismo , Humanos , Mutação , Nucleotídeos , RNA , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2 , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
13.
Life Sci Alliance ; 5(10)2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35995566

RESUMO

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin ß (Impß), but how Rev associates with Impß is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impß/Rev complex. We show that Impß binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impß cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impß-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impß with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impß and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impß cargos.


Assuntos
HIV-1 , beta Carioferinas , HIV-1/metabolismo , Modelos Estruturais , Simulação de Acoplamento Molecular , RNA Viral/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismo
14.
J Virol ; 96(16): e0067122, 2022 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-35924919

RESUMO

Positive-strand RNA viruses replicate their genomes using virally encoded RNA-dependent RNA polymerases (RdRP) with a common active-site structure and closure mechanism upon which replication speed and fidelity can evolve to optimize virus fitness. Coronaviruses (CoV) form large multicomponent RNA replication-transcription complexes containing a core RNA synthesis machine made of the nsp12 RdRP protein with one nsp7 and two nsp8 proteins as essential subunits required for activity. We show that assembly of this complex can be accelerated 5-fold by preincubation of nsp12 with nsp8 and further optimized with the use of a novel nsp8L7 heterodimer fusion protein construct. Using rapid kinetics methods, we measure elongation rates of up to 260 nucleotides (nt)/s for the core replicase, a rate that is unusually fast for a viral polymerase. To address the origin of this fast rate, we examined the roles of two CoV-specific residues in the RdRP active site: Ala547, which replaces a conserved glutamate above the bound NTP, and Ser759, which mutates the palm domain GDD sequence to SDD. Our data show that Ala547 allows for a doubling of replication rate, but this comes at a fidelity cost that is mitigated by using a SDD sequence in the palm domain. Our biochemical data suggest that fixation of mutations in polymerase motifs F and C played a key role in nidovirus evolution by tuning replication rate and fidelity to accommodate their large genomes. IMPORTANCE Replicating large genomes represents a challenge for RNA viruses because fast RNA synthesis is needed to escape innate immunity defenses, but faster polymerases are inherently low-fidelity enzymes. Nonetheless, the coronaviruses replicate their ≈30-kb genomes using the core polymerase structure and mechanism common to all positive-strand RNA viruses. The classic explanation for their success is that the large-genome nidoviruses have acquired an exonuclease-based repair system that compensates for the high polymerase mutation rate. In this work, we establish that the nidoviral polymerases themselves also play a key role in maintaining genome integrity via mutations at two key active-site residues that enable very fast replication rates while maintaining typical mutation rates. Our findings further demonstrate the evolutionary plasticity of the core polymerase platform by showing how it has adapted during the expansion from short-genome picornaviruses to long-genome nidoviruses.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/química , Vírus da SARS , Domínio Catalítico , Genoma Viral , RNA/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , Vírus da SARS/fisiologia , Replicação Viral
15.
Protein Sci ; 31(9): e4395, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36040262

RESUMO

SARS-CoV-2 nsp10-nsp16 complex is a 2'-O-methyltransferase (MTase) involved in viral RNA capping, enabling the virus to evade the immune system in humans. It has been considered a valuable target in the discovery of antiviral therapeutics, as the RNA cap formation is crucial for viral propagation. Through cross-screening of the inhibitors that we previously reported for SARS-CoV-2 nsp14 MTase activity against nsp10-nsp16 complex, we identified two compounds (SS148 and WZ16) that also inhibited nsp16 MTase activity. To further enable the chemical optimization of these two compounds towards more potent and selective dual nsp14/nsp16 MTase inhibitors, we determined the crystal structure of nsp10-nsp16 in complex with each of SS148 and WZ16. As expected, the structures revealed the binding of both compounds to S-adenosyl-L-methionine (SAM) binding pocket of nsp16. However, our structural data along with the biochemical mechanism of action determination revealed an RNA-dependent SAM-competitive pattern of inhibition for WZ16, clearly suggesting that binding of the RNA first may help the binding of some SAM competitive inhibitors. Both compounds also showed some degree of selectivity against human protein MTases, an indication of great potential for chemical optimization towards more potent and selective inhibitors of coronavirus MTases.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/tratamento farmacológico , Humanos , Metiltransferases/química , RNA Viral/metabolismo , Proteínas não Estruturais Virais/química
16.
Nat Commun ; 13(1): 4725, 2022 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-35953468

RESUMO

Ample evidence indicates that codon usage bias regulates gene expression. How viruses, such as the emerging mosquito-borne Chikungunya virus (CHIKV), express their genomes at high levels despite an enrichment in rare codons remains a puzzling question. Using ribosome footprinting, we analyze translational changes that occur upon CHIKV infection. We show that CHIKV infection induces codon-specific reprogramming of the host translation machinery to favor the translation of viral RNA genomes over host mRNAs with an otherwise optimal codon usage. This reprogramming was mostly apparent at the endoplasmic reticulum, where CHIKV RNAs show high ribosome occupancy. Mechanistically, it involves CHIKV-induced overexpression of KIAA1456, an enzyme that modifies the wobble U34 position in the anticodon of tRNAs, which is required for proper decoding of codons that are highly enriched in CHIKV RNAs. Our findings demonstrate an unprecedented interplay of viruses with the host tRNA epitranscriptome to adapt the host translation machinery to viral production.


Assuntos
Febre de Chikungunya , Vírus Chikungunya , Animais , Vírus Chikungunya/genética , Códon/genética , Códon/metabolismo , Humanos , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
17.
Nat Commun ; 13(1): 4802, 2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35970826

RESUMO

Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with a non-segmented genome, closely related to rabies virus. Both have characteristic bullet-like shapes. We report the structure of intact, infectious VSV particles determined by cryogenic electron microscopy. By compensating for polymorphism among viral particles with computational classification, we obtained a reconstruction of the shaft ("trunk") at 3.5 Å resolution, with lower resolution for the rounded tip. The ribonucleoprotein (RNP), genomic RNA complexed with nucleoprotein (N), curls into a dome-like structure with about eight gradually expanding turns before transitioning into the regular helical trunk. Two layers of matrix (M) protein link the RNP with the membrane. Radial inter-layer subunit contacts are fixed within single RNA-N-M1-M2 modules, but flexible lateral and axial interactions allow assembly of polymorphic virions. Together with published structures of recombinant N in various states, our results suggest a mechanism for membrane-coupled self-assembly of VSV and its relatives.


Assuntos
Estomatite Vesicular , Animais , RNA , RNA Viral/genética , RNA Viral/metabolismo , Ribonucleoproteínas , Vírus da Estomatite Vesicular Indiana/genética , Vesiculovirus/genética , Vírion/metabolismo , Montagem de Vírus
18.
Int J Mol Sci ; 23(15)2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35955844

RESUMO

In microbiological research, it is important to understand the time course of each step in a pathogen's lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3-4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5' UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.


Assuntos
Hepacivirus , Hepatite C , Regiões 5' não Traduzidas , Hepatite C/genética , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Replicon/genética , Replicação Viral
19.
Proc Natl Acad Sci U S A ; 119(32): e2201453119, 2022 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-35914138

RESUMO

Because multipartite viruses package their genome segments in different viral particles, they face a potentially huge cost if the entire genomic information, i.e., all genome segments, needs to be present concomitantly for the infection to function. Previous work with the octapartite faba bean necrotic stunt virus (FBNSV; family Nanoviridae, genus Nanovirus) showed that this issue can be resolved at the within-host level through a supracellular functioning; all viral segments do not need to be present within the same host cell but may complement each other through intercellular trafficking of their products (protein or messenger RNA [mRNA]). Here, we report on whether FBNSV can as well decrease the genomic integrity cost during between-host transmission. Using viable infections lacking nonessential virus segments, we show that full-genome infections can be reconstituted and function through separate acquisition and/or inoculation of complementary sets of genome segments in recipient hosts. This separate acquisition/inoculation can occur either through the transmission of different segment sets by different individual aphid vectors or by the sequential acquisition by the same aphid of complementary sets of segments from different hosts. The possibility of a separate between-host transmission of different genome segments thus offers a way to at least partially resolve the genomic maintenance problem faced by multipartite viruses.


Assuntos
Afídeos , Genoma Viral , Interações entre Hospedeiro e Microrganismos , Insetos Vetores , Nanovirus , Vicia faba , Animais , Afídeos/virologia , Genoma Viral/genética , Insetos Vetores/virologia , Nanovirus/genética , Doenças das Plantas/virologia , Transporte Proteico , Transporte de RNA , RNA Viral/genética , RNA Viral/metabolismo , Vicia faba/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
Viral Immunol ; 35(7): 491-502, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35930238

RESUMO

Lymphocytes are the main orchestrators that regulate the immune response in SARS-COV-2 infection. The exhaustion of T lymphocytes is a contributing factor to lymphopenia, which is responsible for the COVID-19 adverse outcome. However, it is still not demonstrated on a large scale, including cancer patients. Peripheral blood samples were obtained from 83 SARS-CoV2 infected cancer patients, and 29 COVID-19 infected noncancer patients compared to 28 age-matched healthy controls. Lymphocyte subsets were assessed for CD3, CD4, CD8, CD56, PD-1, and CD95 using flow cytometry. The data were correlated to the patients' clinical features, COVID-19 severity and outcomes. Lymphopenia, and decreased CD4+ T cells and CD8+ T cells were significantly observed in COVID-19 cancer and noncancer patients compared to the control group (p < 0.001, for all). There was a significantly increased expression of CD95 and PD-1 on the NK cells, CD4+ T cells, and CD8+ T cells in COVID-19 cancer and noncancer patients in comparison to the control group. The increased expression of CD95 on CD8+ T cells, as well as the increased expression of PD-1 on CD8+ T cells and NK cells are significantly associated with the severity of COVID-19 infection in cancer patients. The increased expression of CD95 and PD-1 on the CD4+ T cells, CD8+ T cells, and NK cells was observed significantly in nonsurviving patients and those who were admitted to the intensive care unit in COVID-19 cancer and noncancer patients. The increased expression of PD-1 and CD95 could be possible prognostic factors for COVID-19 severity and adverse outcomes in COVID-19 cancer and noncancer patients.


Assuntos
COVID-19 , Linfopenia , Neoplasias , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Humanos , Subpopulações de Linfócitos , Linfopenia/metabolismo , Neoplasias/complicações , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1 , RNA Viral/metabolismo , SARS-CoV-2 , Subpopulações de Linfócitos T
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