Stability Of Viral RNA Research Articles

Identification and structural studies of natural inhibitors against SARS-CoV-2 viral RNA methyltransferase (NSP16)

Pathogenic RNA viruses are emerging as one of the major threats and posing challenges to human community. RNA viruses have an exceptionally shorter generation time and easy to adapt in host cells. The recent emergence of SARS-CoV-2, a long RNA virus, has shown us how difficult it is to overcome this kind of pandemic without understanding the viral infection and replication mechanisms. It is essential to comprehend replications of the viral genome, including RNA polymerization and the final capping process. The mRNAs of SARS-CoV-2 coronaviruses are protected at their 5′-ends by cap structure. The cap-like system plays a significant role in viral translational process, viral RNA stability, and scatting in detecting innate immune recognition in host cells. Two coronavirus enzymes, Nsp14 and Nsp16, critically help in the formation of capping and are considered as potential drug targets for antiviral therapy. Natural and herbal medicines have a past record of treating various acute respiratory diseases. In this work, we have exploited 56000 natural compounds to screen potential inhibitors against NSP16. In silico virtual screening, docking and Molecular Dynamics (MD) simulation studies were performed to understand how these potential inhibitors are bound to NSP16. We observed that the most highly screened compound binds to protein molecules with a high dock score, primarily through hydrophobic interactions and hydrogen bonding, as previously reported for NSP16. Compound-13 (2-hydroxy-N-({1-[2-hydroxy-1-(hydroxymethyl)ethyl]piperidin-3-yl}methyl)-5-methylbenzamide) and compound-51 (N-(2-isobutoxybenzyl)-N,2-dimethyl-2,8-diazaspiro[4.5]decane-3-carboxamide) occupied in active site along with good pharmokinetices properties. In conclusion, the selected compounds could be used as a novel therapeutic against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

Detection and infectivity of SARS-CoV-2 in exhumated corpses

Postmortem detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) after the exhumation of a corpse can become important, e.g. in the case of subsequent medical malpractice allegations. To date, data on possible detection periods [e.g. by reverse transcription polymerase chain reaction (RT-PCR)] or on the potential infectivity of the virus after an exhumation are rare. In the present study, these parameters were examined in two cases with a time span of approximately 4 months between day of death and exhumation. Using SARS-CoV-2 RT-PCR on swabs of both lungs and the oropharynx detection was possible with cycle threshold (Ct) values of about 30 despite signs of beginning decay. RT-PCR testing of perioral and perinasal swabs and swabs collected from the inside of the body bag, taken to estimate the risk of infection of those involved in the exhumation, was negative. Cell culture-based infectivity testing was negative for both, lung and oropharyngeal swabs. In one case, RT-PCR testing at the day of death of an oropharyngeal swab showed almost identical Ct values as postmortem testing of an oropharyngeal swab, impressively demonstrating the stability of viral RNA in the intact corpse. However, favorable climatic conditions in the grave have to be taken into account, as it was wintertime with constant low temperatures. Nevertheless, it was possible to demonstrate successful postmortem detection of SARS-CoV-2 infection following exhumation even after months in an earth grave.

Peptide inhibitors against SARS-CoV-2 2′-O-methyltransferase involved in RNA capping: A computational approach

The COVID-19 pandemic is still evolving and is caused by SARS-CoV-2. The 2′-O-methyltransferase (nsp16) enzyme is crucial for maintaining the stability of viral RNA for effective translation of viral proteins and its life cycle. Another protein, nsp10, is important for enzymatic activity of nsp16. Any disturbance in the interaction between nsp16 and nsp10 may affect viral replication fidelity. Here, five peptide inhibitors, derived from nsp16, were designed and assessed for their effectiveness in binding to nsp10 using molecular dynamics simulation. The inhibitors were derived from the nsp10/nsp16 binding interface. Post-simulation analysis showed that inhibitors 2 and 5 were stable and bind to the nsp16 interacting region of nsp10 which could potentially prevent the interaction between the two proteins. The proposed peptides are useful starting points for the development of therapeutics to manage the spread of COVID-19.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

A method for detection of SARS-CoV-2 RNA in healthy human stool: a validation study

BackgroundFaecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool.MethodsIn this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at −80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing.FindingsThe lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at −80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at −80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p<0·0001), at 4°C storage in DNA/RNA Shield (from 28·5 [0·15] to 29·8 [0·09]; p=0·0019), and at ambient temperature in STAR buffer (from 30·4 [0·24] to 32·4 [0·62]; p=0·0083). 30 contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, SARS-CoV-2 RNA was detected using this assay in the stool and anal swab specimens of 11 of 23 individuals known to be positive for SARS-CoV-2.InterpretationThis is a sensitive and reproducible assay for detection of SARS-CoV-2 RNA in human stool, with potential uses in faecal microbiota transplantation donor screening, sewage monitoring, and further research into the effects of faecal shedding on the epidemiology of the COVID-19 pandemic.FundingNational Institute of Allergy and Infectious Diseases, US National Institutes of Health; Center for Biologics Evaluation and Research, US Food and Drug Administration.

A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection

SUMMARYHepatitis A virus (HAV) is a positive-sense RNA virus causing acute inflammation of the liver. Here, using a genome-scale CRISPR screen, we provide a comprehensive picture of the cellular factors that are exploited by HAV. We identify genes involved in sialic acid/ganglioside biosynthesis and members of the eukaryotic translation initiation factor complex, corroborating their putative roles for HAV. Additionally, we uncover all components of the cellular machinery for UFMylation, a ubiquitin-like protein modification. We show that HAV translation specifically depends on UFM1 conjugation of the ribosomal protein RPL26. Furthermore, we find that components related to the yeast Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex are required for viral translation independent of controlling viral poly(A) tails or RNA stability. Finally, we demonstrate that pharmacological inhibition of the TRAMP-like complex decreases HAV replication in hepatocyte cells and human liver organoids, thus providing a strategy for host-directed therapy of HAV infection.

Examining the persistence of human Coronavirus 229E on fresh produce

Human coronaviruses (HCoVs) are mainly associated with respiratory infections. However, there is evidence that highly pathogenic HCoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome (MERS-CoV), infect the gastrointestinal (GI) tract and are shed in the fecal matter of the infected individuals. These observations have raised questions regarding the possibility of fecal-oral route as well as foodborne transmission of SARS-CoV-2 and MERS-CoV. Studies regarding the survival of HCoVs on inanimate surfaces demonstrate that these viruses can remain infectious for hours to days, however, there is limited data regarding the viral survival on fresh produce, which is usually consumed raw or with minimal heat processing. To address this knowledge gap, we examined the persistence of HCoV-229E, as a surrogate for highly pathogenic HCoVs, on the surface of commonly consumed fresh produce, including: apples, tomatoes, cucumbers and lettuce. Herein, we demonstrated that viral infectivity declines within a few hours post-inoculation (p.i) on apples and tomatoes, and no infectious virus was detected at 24h p.i, while the virus persists in infectious form for 72h p.i on cucumbers and lettuce. The stability of viral RNA was examined by droplet-digital RT-PCR (ddRT-PCR), and it was observed that there is no considerable reduction in viral RNA within 72h p.i.

Postmortem Stability of SARS-CoV-2 in Nasopharyngeal Mucosa.

Analyses of infection chains have demonstrated that severe acute respiratory syndrome coronavirus 2 is highly transmissive. However, data on postmortem stability and infectivity are lacking. Our finding of nasopharyngeal viral RNA stability in 79 corpses showed no time-dependent decrease. Maintained infectivity is supported by virus isolation up to 35 hours postmortem.

Identification of the RNase-binding site of SARS-CoV-2 RNA for anchor primer-PCR detection of viral loading in 306 COVID-19 patients

The pandemic of coronavirus disease 2019 (COVID-19) urgently calls for more sensitive molecular diagnosis to improve sensitivity of current viral nuclear acid detection. We have developed an anchor primer (AP)-based assay to improve viral RNA stability by bioinformatics identification of RNase-binding site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and implementing AP dually targeting the N gene of SARS-CoV-2 RNA and RNase 1, 3, 6. The arbitrarily primed polymerase chain reaction (AP-PCR) improvement of viral RNA integrity was supported by (a) the AP increased resistance of the targeted gene (N gene) of SARS-CoV-2 RNA to RNase treatment; (b) the detection of SARS-CoV-2 RNA by AP-PCR with lower cycle threshold values (−2.7 cycles) compared to two commercially available assays; (c) improvement of the viral RNA stability of the ORF gene upon targeting of the N gene and RNase. Furthermore, the improved sensitivity by AP-PCR was demonstrated by detection of SARS-CoV-2 RNA in 70–80% of sputum, nasal, pharyngeal swabs and feces and 36% (4/11) of urine of the confirmed cases (n = 252), 7% convalescent cases (n = 54) and none of 300 negative cases. Lastly, AP-PCR analysis of 306 confirmed and convalescent cases revealed prolonged presence of viral loading for >20 days after the first positive diagnosis. Thus, the AP dually targeting SARS-CoV-2 RNA and RNase improves molecular detection by preserving SARS-CoV-2 RNA integrity and reveals the prolonged viral loading associated with older age and male gender in COVID-19 patients.

Stability of MERS-CoV RNA on spin columns of RNA extraction kit at room temperature

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus causing a highly fatal respiratory disease in humans. Confirmation of MERS-CoV infection and molecular study on the virus may require transportation of samples to specialized laboratories. While freezing at −80 °C is the gold standard method for RNA preservation, maintaining the integrity of viral RNA during transport will require additional precautions and, as a result, increase transport costs. We aimed at testing the stability of MERS-CoV RNA on spin columns of RNA extraction kit at room temperature for 16 weeks. Respiratory samples spiked with stock culture of MERS-CoV were extracted and loaded on QIAamp Viral RNA Mini Kit spin columns and preserved at room temperature. Amount of viral RNA was evaluated periodically by real-time quantitative reverse-transcription polymerase chain reaction. Minimal changes in cycle threshold values over the study period were noted, suggesting stability of viral RNA by this preservation method.

Structural analysis of the SARS-CoV-2 methyltransferase complex involved in RNA cap creation bound to sinefungin

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2′-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.

N6-methyladenosine regulates PEDV replication and host gene expression

Methylation of the N6 position of adenosine (m6A) is a widespread RNA modification that is critical for various physiological and pathological processes. Although this modification was also found in the RNA of several viruses almost 40 years ago, its biological functions during viral infection have been elucidated recently. Here, we investigated the effects of viral and host RNA methylation during porcine epidemic diarrhea virus (PEDV) infection. The results demonstrated that the m6A modification was abundant in the PEDV genome and the host methyltransferases METTL3 and METTL14 and demethylase FTO were involved in the regulation of viral replication. The knockdown of the methyltransferases increased PEDV replication while silencing the demethylase decreased PEDV output. Moreover, the proteins of the YTHDF family regulated the PEDV replication by affecting the stability of m6A-modified viral RNA. In particular, PEDV infection could trigger an increasement of m6A in host RNA and decrease the expression of FTO. The m6A modification sites in mRNAs and target genes were also altered during PEDV infection. Additionally, part of the host responses to PEDV infection was controlled by m6A modification, which could be reversed by the expression of FTO. Taken together, our results identified the role of m6A modification in PEDV replication and interactions with the host.

Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability

Epitranscriptomic RNA modifications, including methylation of adenine and cytidine residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of the N4 position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression. HIV-1 transcripts are modified with ac4C at multiple discrete sites, and silent mutagenesis of these ac4C sites led to decreased HIV-1 gene expression. Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. Interestingly, the NAT10 inhibitor remodelin could inhibit HIV-1 replication at concentrations that have no effect on cell viability, thus identifying ac4C addition as a potential target for antiviral drug development.

Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability

Epitranscriptomic RNA modifications, including methylation of A and C residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of the N4 position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression. HIV-1 transcripts are modified with ac4C at multiple discreet sites, and silent mutagenesis of these ac4C sites led to decreased HIV-1 gene expression. Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. Interestingly, the NAT10 inhibitor Remodelin could inhibit HIV-1 replication at levels that have no effect on cell viability, thus identifying ac4C addition as a potential novel target for antiviral drug development.

Negatively charged amino acids at the foot-and-mouth disease virus capsid reduce the virion-destabilizing effect of viral RNA at acidic pH

Elucidation of the molecular basis of the stability of foot-and-mouth disease virus (FMDV) particles is relevant to understand key aspects of the virus cycle. Residue N17D in VP1, located at the capsid inner surface, modulates the resistance of FMDV virion to dissociation and inactivation at acidic pH. Here we have studied whether the virion-stabilizing effect of amino acid substitution VP1 N17D may be mediated by the alteration of electrostatic charge at this position and/or the presence of the viral RNA. Substitutions that either introduced a positive charge (R,K) or preserved neutrality (A) at position VP1 17 led to increased sensitivity of virions to inactivation at acidic pH, while replacement by negatively charged residues (D,E) increased the resistance of virions to acidic pH. The role in virion stability of viral RNA was addressed using FMDV empty capsids that have a virtually unchanged structure compared to the capsid in the RNA-filled virion, but that are considerably more resistant to acidic pH than WT virions, supporting a virion-destabilizing effect of the RNA. Remarkably, no differences were observed in the resistance to dissociation at acidic pH between the WT empty capsids and those harboring replacement N17D. Thus, the virion-destabilizing effect of viral RNA at acidic pH can be partially restored by introducing negatively charged residues at position VP1 N17.

The Functional Role of the 3' Untranslated Region and Poly(A) Tail of Duck Hepatitis A Virus Type 1 in Viral Replication and Regulation of IRES-Mediated Translation.

The duck hepatitis A virus type 1 (DHAV-1) is a member of Picornaviridae family, the genome of the virus contains a 5′ untranslated region (5′ UTR), a large open reading frame that encodes a polyprotein precursor and a 3′ UTR followed by a poly(A) tail. The translation initiation of virus proteins depends on the internal ribosome-entry site (IRES) element within the 5′ UTR. So far, little information is known about the role of the 3′ UTR and poly(A) tail during the virus proliferation. In this study, the function of the 3′ UTR and poly(A) tail of DHAV-1 in viral replication and IRES-mediated translation was investigated. The results showed that both 3′ UTR and poly(A) tail are important for maintaining viral genome RNA stability and viral genome replication. During DHAV-1 proliferation, at least 20 adenines were required for the optimal genome replication and the virus replication could be severely impaired when the poly (A) tail was curtailed to 10 adenines. In addition to facilitating viral genome replication, the presence of 3′ UTR and poly(A) tail significantly enhance IRES-mediated translation efficiency. Furthermore, 3′ UTR or poly(A) tail could function as an individual element to enhance the DHAV-1 IRES-mediated translation, during which process, the 3′ UTR exerts a greater initiation efficiency than the poly(A)25 tail.

Exploitation of nuclear functions by human rhinovirus, a cytoplasmic RNA virus.

Protein production, genomic RNA replication, and virion assembly during infection by picornaviruses like human rhinovirus and poliovirus take place in the cytoplasm of infected human cells, making them the quintessential cytoplasmic pathogens. However, a growing body of evidence suggests that picornavirus replication is promoted by a number of host proteins localized normally within the host cell nucleus. To systematically identify such nuclear proteins, we focused on those that appear to re-equilibrate from the nucleus to the cytoplasm during infection of HeLa cells with human rhinovirus via quantitative protein mass spectrometry. Our analysis revealed a highly selective re-equilibration of proteins with known mRNA splicing and transport-related functions over nuclear proteins of all other functional classes. The multifunctional splicing factor proline and glutamine rich (SFPQ) was identified as one such protein. We found that SFPQ is targeted for proteolysis within the nucleus by viral proteinase 3CD/3C, and a fragment of SFPQ was shown to migrate to the cytoplasm at mid-to-late times of infection. Cells knocked down for SFPQ expression showed significantly reduced rhinovirus titers, viral protein production, and viral RNA accumulation, consistent with SFPQ being a pro-viral factor. The SFPQ fragment that moved into the cytoplasm was able to bind rhinovirus RNA either directly or indirectly. We propose that the truncated form of SFPQ promotes viral RNA stability or replication, or virion morphogenesis. More broadly, our findings reveal dramatic changes in protein compartmentalization during human rhinovirus infection, allowing the virus to systematically hijack the functions of proteins not normally found at its cytoplasmic site of replication.

Roles of the 5′ Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication

The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5'-terminal domain (I); three additional domains (I', II, and III), which are homologous to domains I, II, and III, respectively, of HCV; and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5' UTR was lower than that of the HCV 5' UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5' UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domains I and I' of EHcV (the 5'-proximal EHcV-specific region) improved RNA stability and provided the HCV SGR with microRNA 122 (miR-122)-independent replication capability, while EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domains I and I' improves RNA stability and viral replication regardless of miR-122 expression. The 5'-proximal EHcV-specific region may represent an inherent mechanism to facilitate viral replication in nonhepatic tissues.IMPORTANCE EHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs in horses, donkeys, and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5' untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5'-proximal EHcV-specific region enhances viral replication and RNA stability in a miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5' UTR contributes to the differences in tissue tropism observed between these hepaciviruses.

Plant RNA Regulatory Network and RNA Granules in Virus Infection.

Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual components contribute to these processes. In this review we discuss the roles of cellular RNA regulatory mechanisms and RNA granules in plant virus infection in the light of current knowledge and compare the findings to those made in animal virus studies.

Assessing the Effects of Medium Chain Fatty Acids and Fat Sources on Porcine Epidemic Diarrhea Virus Viral RNA Stability and Infectivity

Assessing the Effects of Medium Chain Fatty Acids and Fat Sources on Porcine Epidemic Diarrhea Virus Viral RNA Stability and Infectivity