Positive-strand RNA Research Articles

FHL1 promotes chikungunya and o’nyong-nyong virus infection and pathogenesis with implications for alphavirus vaccine design

Arthritogenic alphaviruses are positive-strand RNA viruses that cause debilitating musculoskeletal diseases affecting millions worldwide. A recent discovery identified the four-and-a-half-LIM domain protein 1 splice variant A (FHL1A) as a crucial host factor interacting with the hypervariable domain (HVD) of chikungunya virus (CHIKV) nonstructural protein 3 (nsP3). Here, we show that acute and chronic chikungunya disease in humans correlates with elevated levels of FHL1. We generated FHL1−/− mice, which when infected with CHIKV or o’nyong-nyong virus (ONNV) displayed reduced arthritis and myositis, fewer immune infiltrates, and reduced proinflammatory cytokine/chemokine outputs, compared to infected wild-type (WT) mice. Interestingly, disease signs were comparable in FHL1−/− and WT mice infected with arthritogenic alphaviruses Ross River virus (RRV) or Mayaro virus (MAYV). This aligns with pull-down assay data, which showed the ability of CHIKV and ONNV nsP3 to interact with FHL1, while RRV and MAYV nsP3s did not. We engineered a CHIKV mutant unable to bind FHL1 (CHIKV-ΔFHL1), which was avirulent in vivo. Following inoculation with CHIKV-ΔFHL1, mice were protected from disease upon challenge with CHIKV and ONNV, and viraemia was significantly reduced in RRV- and MAYV-challenged mice. Targeting FHL1-binding as an approach to vaccine design could lead to breakthroughs in mitigating alphaviral disease.

2-thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are causing significant morbidity and mortality worldwide. Furthermore, over 1 million cases of newly emerging or re-emerging viral infections, specifically dengue virus (DENV), are known to occur annually. Because no virus-specific and fully effective treatments against these or many other viruses have been approved, there is an urgent need for novel, effective therapeutic agents. Here, we identified 2-thiouridine (s2U) as a broad-spectrum antiviral ribonucleoside analogue that exhibited antiviral activity against several positive-sense single-stranded RNA (ssRNA+) viruses, such as DENV, SARS-CoV-2, and its variants of concern, including the currently circulating Omicron subvariants. s2U inhibits RNA synthesis catalyzed by viral RNA-dependent RNA polymerase, thereby reducing viral RNA replication, which improved the survival rate of mice infected with DENV2 or SARS-CoV-2 in our animal models. Our findings demonstrate that s2U is a potential broad-spectrum antiviral agent not only against DENV and SARS-CoV-2 but other ssRNA+ viruses.

Metagenomics analysis reveals presence of the Merida-like virus in Georgia.

Arbovirus surveillance is fundamental for the discovery of novel viruses and prevention of febrile vector-borne illnesses. Vector-borne pathogens can rapidly expand and adapt in new geographic and environmental conditions. In this study, metagenomic surveillance was conducted to identify novel viruses in the Country of Georgia. A total of 521 mosquitoes were captured near a military training facility and pooled from species Culex pipiens (Linnaeus) (87%) and Aedes albopictus (Skuse) (13%). We decided to further analyze the Culex pipiens mosquitoes, due to the more extensive number of samples collected. Our approach was to utilize an unbiased total RNA-seq for pathogen discovery in order to explore the mosquito virome. The viral reads from this analysis were mostly aligned to Insect-specific viruses from two main families, the Iflaviridae; a positive-stranded RNA virus and the Rhabdoviridae; a negative- and single-stranded RNA virus. Our pathogen discovery analysis revealed viral reads aligning to the Merida-like virus Turkey (MERDLVT) strain among the Rhabdoviridae. To further validate this result, we conducted a BLAST sequence comparison analysis of our samples with the MERDLVT strain. Our positive samples aligned to the MERDLVT strain with 96-100% sequence identity and 99.7-100% sequence coverage. A bootstrapped maximum-likelihood phylogenetic tree was used to evaluate the evolutionary relationships among these positive pooled specimens with the (MERDLVT) strain. The Georgia samples clustered most closely with two strains from Turkey, the Merida-like virus KE-2017a isolate 139-1-21 and the Merida-like virus Turkey isolate P431. Collectively, these results show the presence of the MERDLVT strain in Georgia.

MERS-CoV and SARS-CoV-2 membrane proteins are modified with polylactosamine chains.

Coronaviruses are positive-stranded RNA enveloped viruses. The helical nucleocapsid is surrounded by a lipid bilayer in which are anchored three viral proteins: the spike (S), membrane (M) and envelope (E) proteins. The M protein is the major component of the viral envelope and is believed to be its building block. The M protein of Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a short N-terminal domain with an N-glycosylation site. We investigated their N-glycosylation and show that polylactosamine chains are conjugated to SARS-CoV-2 and MERS-CoV M proteins in transfected and infected cells. Acidic residues present in the first transmembrane segments of the proteins are required for their glycosylation. No specific signal to specify polylactosamine conjugation could be identified and high mannose-conjugated protein was incorporated into virus-like particles.

Previous and potential mutations of Sars-cov-2 receptors and their interaction with known inhibitors

SARS-CoV-2, the seventh coronavirus known to infect humans after 229E, NL63, 0C43, HKU1, MERS-CoV, and the original SARS-CoV, has a large positive-stranded RNA genome of 29,891 nucleotides and 9,860 amino acids. It is a strain of coronavirus that causes COVID 19. It was first discovered in the Chinese city of Wuhan. It undergoes several mutations hence posing a great difficulty in finding a suitable inhibitor. This studies previous and potential mutations of Sars-cov-2 receptors and their interaction with known inhibitors. There are four measures to take in generating SARS-CoV-2 inhibitors that might surpass mutation should it arise which includes; a. finding inhibitors with two or more mechanism of inhibition in virus target inhibition b. employing host target inhibition c. complex forming compounds that will interfere with viral-host cell fusion independent of interaction with the host or organism d. human receptor cell lookalikes with higher affinity to the virus (proteins). Employing any of these measures might produce a good inhibitor however, drug resistance may emerge quickly after treatment as a result of the numerous mutations that RNA virus experience but host cell mutations are uncommon, treatments that target the host cells may delay the emergence of drug resistance.

A meta-analysis for vaccine protection rate of duck hepatitis a virus in mainland China in 2009–2021

BackgroundDuck hepatitis A virus (DHAV) is a single-stranded, positive-strand small RNA virus that causes a very high mortality rate in ducklings. The DHAV-3 subtype incidence rate has recently increased in China, causing great economic losses to the waterfowl breeding industry. We analyzed the protection rate of DHAV vaccines used in mainland China from 2009 to 2021 and evaluated the effectiveness of vaccine prevention and control to reduce the economic losses caused by DHAV to the waterfowl breeding industry. We screened five electronic research databases and obtained 14 studies and patents on the protection efficiency of DHAV-1 and DHAV-3 vaccines.ResultsMeta-analysis demonstrated that immunized ducklings produced higher antibody levels and had a significantly higher survival rate than non-immunized ducklings [relative risk (RR) = 12, 95% confidence interval (CI) 6–26, P < 0.01]. The age of the ducks and vaccine valence did not affect protection efficiency. Data source analysis of the vaccine protection rate demonstrated that the vaccines conferred immune protection for ducklings in both small-scale experiments and large-scale clinical conditions. The analysis results revealed that although the vaccines conferred protection, the immune protective effect differed between small-scale experimental conditions and large-scale clinical conditions. This might have been due to non-standard vaccination and environmental factors.ConclusionsDomestic DHAV vaccines can protect ducklings effectively. The subjects immunized (breeding ducks or ducklings) and vaccine valence had no effect on the protective effect. Both small-scale experiments and large-scale clinical conditions conferred immune protection on ducklings, but vaccine immunization under small-scale experimental conditions had slightly better protective effects than large-scale clinical immunization.

The 3' end of the coding region of senecavirus A contains a highly conserved sequence that potentially forms a stem-loop structure required for virus rescue.

Senecavirus A (SVA) can cause a vesicular disease in swine. It is a positive-strand RNA virus belonging to the genus Senecavirus in the family Picornaviridae. Positive-strand RNA viruses possess positive-sense, single-stranded genomes whose untranslated regions (UTRs) have been reported to contain cis-acting RNA elements. In the present study, a total of 100 SVA isolates were comparatively analyzed at the genome level. A highly conserved fragment (HCF) was found to be located in the 3D sequence and to be close to the 3' UTR. The HCF was computationally predicted to form a stem-loop structure. Eight synonymous mutations can individually disrupt the formation of a single base pair within the stem region. We found that SVA itself was able to tolerate each of these mutations alone, as evidenced by the ability to rescue all eight single-site mutants from their individual cDNA clones, and all of them were genetically stable during serial passaging. However, the replication-competent SVA could not be rescued from another cDNA clone containing all eight mutations. The failure to recover SVA might be attributed to disruption of the predicted stem-loop structure, whereas introduction of a wild-type HCF into the cDNA clone with eight mutations still had no effect on virus recovery. These results suggest that the putative stem-loop structure at the 3' end of the 3D sequence is a cis-acting RNA element that is required for SVA growth.

Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

Packaging defects in pestiviral NS4A can be compensated by mutations in NS2 and NS3.

The non-structural (NS) proteins of the Flaviviridae members play a dual role in genome replication and virion morphogenesis. For pestiviruses, like bovine viral diarrhea virus, the NS2-3 region and its processing by the NS2 autoprotease is of particular importance. While uncleaved NS2-3 in complex with NS4A is essential for virion assembly, it cannot replace free NS3/4A in the viral replicase. Furthermore, surface interactions between NS3 and the C-terminal cytosolic domain of NS4A were shown to serve as a molecular switch between RNA replication and virion morphogenesis. To further characterize the functionality of NS4A, we performed an alanine-scanning mutagenesis of two NS4A regions, a short highly conserved cytoplasmic linker downstream of the transmembrane domain and the C-terminal domain. NS4A residues critical for polyprotein processing, RNA replication, and/or virion morphogenesis were identified. Three double-alanine mutants, two in the linker region and one close to the C-terminus of NS4A, showed a selective effect on virion assembly. All three packaging defective mutants could be rescued by a selected set of two second-site mutations, located in NS2 and NS3, respectively. This phenotype was additionally confirmed by complementation studies providing the NS2-3/4A packaging molecules containing the rescue mutations in trans. This indicates that the linker region and the cytosolic C-terminal part of NS4A are critical for the formation of protein complexes required for virion morphogenesis. The ability of the identified sets of second-site mutations in NS2-3 to compensate for diverse NS4A defects highlights a surprising functional flexibility for pestiviral NS proteins. IMPORTANCE Positive-strand RNA viruses have a limited coding capacity due to their rather small genome size. To overcome this constraint, viral proteins often exhibit multiple functions that come into play at different stages during the viral replication cycle. The molecular basis for this multifunctionality is often unknown. For the bovine viral diarrhea virus, the non-structural protein (NS) 4A functions as an NS3 protease cofactor, a replicase building block, and a component in virion morphogenesis. Here, we identified the critical amino acids of its C-terminal cytosolic region involved in those processes and show that second-site mutations in NS2 and NS3 can compensate for diverse NS4A defects in virion morphogenesis. The ability to evolve alternative functional solutions by gain-of-function mutations highlights the astounding plasticity of the pestiviral system.

Astrovirus replication is dependent on induction of double-membrane vesicles through a PI3K-dependent, LC3-independent pathway.

Human astrovirus is a positive-sense, single-stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive-strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double-membrane vesicles (DMVs). Here, we show that astrovirus infection leads to an increase in DMV formation through a replication-dependent mechanism that requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Both chemical and genetic inhibition of the PI3K complex lead to significant reduction in DMVs, as well as viral replication. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. IMPORTANCE These studies provide critical new evidence that astrovirus replication requires formation of double-membrane vesicles, which utilize class III phosphatidylinositol 3-kinase (PI3K), but not LC3 conjugation autophagy machinery, for biogenesis. These results are consistent with replication mechanisms for other positive-sense RNA viruses suggesting that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive-sense RNA virus infections.

A sword or a buffet: plant endomembrane system in viral infections.

The plant endomembrane system is an elaborate collection of membrane-bound compartments that perform distinct tasks in plant growth and development, and in responses to abiotic and biotic stresses. Most plant viruses are positive-strand RNA viruses that remodel the host endomembrane system to establish intricate replication compartments. Their fundamental role is to create optimal conditions for viral replication, and to protect replication complexes and the cell-to-cell movement machinery from host defenses. In addition to the intracellular antiviral defense, represented mainly by RNA interference and effector-triggered immunity, recent findings indicate that plant antiviral immunity also includes membrane-localized receptor-like kinases that detect viral molecular patterns and trigger immune responses, which are similar to those observed for bacterial and fungal pathogens. Another recently identified part of plant antiviral defenses is executed by selective autophagy that mediates a specific degradation of viral proteins, resulting in an infection arrest. In a perpetual tug-of-war, certain host autophagy components may be exploited by viral proteins to support or protect an effective viral replication. In this review, we present recent advances in the understanding of the molecular interplay between viral components and plant endomembrane-associated pathways.

Genome-wide CRISPR screens identify noncanonical translation factor eIF2A as an enhancer of SARS-CoV-2 programmed −1 ribosomal frameshifting

Many positive-strand RNA viruses, including all known coronaviruses, employ programmed -1 ribosomal frameshifting (-1 PRF) to regulate the translation of polycistronic viral RNAs. However, only a few host factors have been shown to regulate -1 PRF. Through a genome-wide CRISPR-Cas9 knockout screen, we have identified host factors that either suppress or enhance severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-1 PRF. Among them, eukaryotic translation initiation factor 2A (eIF2A) specifically and directly enhances -1 PRF independent of changes in initiation. Consistent with the crucial role of efficient -1 PRF in transcriptase/replicase expression, loss of eIF2A reduces SARS-CoV-2 replication in cells. Furthermore, transcriptome-wide analysis shows that eIF2A preferentially binds CG-rich RNA motifs, including a region within 18S ribosomal RNA near the contacts between the SARS-CoV-2 frameshift-stimulatory element (FSE) and the ribosome. Thus, our results indicate a role for eIF2A in modulating the translation of specific RNAs independent of its role during initiation.

Structural basis for cloverleaf RNA-initiated viral genome replication.

The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5' end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CDpro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CDpro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem-loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.

Apis mellifera Solinvivirus-1, a Novel Honey Bee Virus That Remained Undetected for over a Decade, Is Widespread in the USA.

A metagenomic analysis of the virome of honey bees (Apis mellifera) from an apiary with high rates of unexplained colony losses identified a novel RNA virus. The virus, which was named Apis mellifera solinvivirus 1 (AmSV1), contains a 10.6 kb positive-strand genomic RNA with a single ORF coding for a polyprotein with the protease, helicase, and RNA-dependent RNA polymerase domains, as well as a single jelly-roll structural protein domain, showing highest similarity with viruses in the family Solinviviridae. The injection of honey bee pupae with AmSV1 preparation showed an increase in virus titer and the accumulation of the negative-strand of AmSV1 RNA 3 days after injection, indicating the replication of AmSV1. In the infected worker bees, AmSV1 was present in heads, thoraxes, and abdomens, indicating that this virus causes systemic infection. An analysis of the geographic and historic distribution of AmSV1, using over 900 apiary samples collected across the United States, showed AmSV1 presence since at least 2010. In the year 2021, AmSV1 was detected in 10.45% of apiaries (95%CI: 8.41-12.79%), mostly sampled in June and July in Northwestern and Northeastern United States. The diagnostic methods and information on the AmSV1 distribution will be used to investigate the connection of AmSV1 to honey bee colony losses.

Norovirus-associated neurological manifestations: summarizing the evidence

Norovirus, a positive-stranded RNA virus, is one of the leading causes of acute gastroenteritis among all age groups worldwide. The neurological manifestations of norovirus are underrecognized, but several wide-spectrum neurological manifestations have been reported among infected individuals in the last few years. Our objective was to summarize the features of norovirus-associated neurological disorders based on the available literature. We used the existing PRISMA consensus statement. Data were collected from PubMed, EMBASE, Web of Science, and Scopus databases up to Jan 30, 2023, using pre‐specified searching strategies. Twenty-one articles were selected for the qualitative synthesis. Among these, seven hundred and seventy-four patients with norovirus-associated neurological manifestations were reported. Most cases were seizure episodes, infection-induced encephalopathy, and immune-driven disorders. However, only a few studies have addressed the pathogenesis of norovirus-related neurological complications. The pathogenesis of these manifestations may be mediated by either neurotropism or aberrant immune-mediated injury, or both, depending on the affected system. Our review could help clinicians to recognize these neurological manifestations better and earlier while deepening the understanding of the pathogenesis of this viral infection.

Dengue Fever: Etiology, Diagnosis, Prevention and Treatment

Dengue fever (break-bone fever) is a viral disease transmitted by Aedes mosquitoes. It is caused by the Dengue virus, which is a single positive-stranded RNA virus belonging to the Flaviviridae family. Dengue fever is prevalent in tropical and subtropical areas and is a significant public health concern in many countries, including`g Nigeria. The disease is characterized by symptoms such as high fever, headache, body aches, nausea, vomiting, swollen glands, and rash. In severe cases, dengue fever can lead to complications such as bleeding, organ impairment, and dengue shock syndrome. Diagnosing dengue fever can be challenging, especially in areas where it is endemic. In endemic locations, diagnosis is often made clinically based on the patient's reported symptoms and a physical examination. Tourniquet testing, which involves applying a blood pressure cuff and counting any petechial hemorrhages, can help in the diagnosis. Laboratory methods, including full blood count, cell culture, nucleic acid identification (PCR), and serology, can be used to confirm the diagnosis. Preventing dengue fever involves controlling the mosquito vector and protecting oneself from mosquito bites. Measures such as eliminating mosquito breeding sites, using insect repellents, wearing protective clothing, and using bed nets can help prevent mosquito bites. Vaccination against dengue is also available, with the Dengvaxia vaccine being used in some countries. There is no specific antiviral treatment for dengue fever. Management focuses on supportive care, maintaining a healthy fluid balance, and relieving symptoms such as fever and pain. Severe cases may require hospitalization and intensive medical care. In Nigeria, dengue fever is often misdiagnosed or overlooked due to similarities with other febrile illnesses like malaria. This can lead to underreporting and inadequate management of dengue cases. Increasing awareness among healthcare professionals and the general population is crucial for early detection and appropriate management of dengue fever in Nigeria. In conclusion dengue fever is a viral disease transmitted by mosquitoes, primarily Aedes species. It is a significant global health concern, including in Nigeria. Early diagnosis, prevention measures, and supportive care are essential in managing dengue fever and reducing its impact on public health. This review is aimed at discussing the current issues of Dengue fever with focus on Nigeria.

A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of cis-acting elements in foot-and-mouth disease virus replication.

Foot-and-mouth-disease virus (FMDV), the aetiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus Aphthovirus within the family Picornavirus. In common with all picornaviruses, replication of the single-stranded positive-sense RNA genome involves synthesis of a negative-sense complementary strand that serves as a template for the synthesis of multiple positive-sense progeny strands. We have previously employed FMDV replicons to examine viral RNA and protein elements essential to replication, but the factors affecting differential strand production remain unknown. Replicon-based systems require transfection of high levels of RNA, which can overload sensitive techniques such as quantitative PCR, preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled in vivo with 5-ethynyl uridine. The modified base is then linked to a biotin tag using click chemistry, facilitating purification of newly synthesised viral genomes or anti-genomes from input RNA. This selected RNA can then be amplified by strand-specific quantitative PCR, thus enabling investigation of the consequences of defined mutations on the relative synthesis of negative-sense intermediate and positive-strand progeny RNAs. We apply this new approach to investigate the consequence of mutation of viral cis-acting replication elements and provide direct evidence for their roles in negative-strand synthesis.

Activation of MAPK-mediated immunity by phosphatidic acid in response to positive-strand RNA viruses

Increasing evidence suggests that mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant defense against viruses. However, the mechanisms that underlie the activation of MAPK cascades in response to viral infection remain unclear. In this study, we discovered that phosphatidic acid (PA) represents a major class of lipids that respond to Potato virus Y (PVY) at an early stage of infection. We identified NbPLDα1 (Nicotiana benthamiana phospholipase Dα1) as the key enzyme responsible for increased PA levels during PVY infection and found that it plays an antiviral role. 6K2 of PVY interacts with NbPLDα1, leading to elevated PA levels. In addition, NbPLDα1 and PA are recruited by 6K2 to membrane-bound viral replication complexes. On the other hand, 6K2 also induces activation of the MAPK pathway, dependent on its interaction with NbPLDα1 and the derived PA. PA binds to WIPK/SIPK/NTF4, prompting their phosphorylation of WRKY8. Notably, spraying with exogenous PA is sufficient to activate the MAPK pathway. Knockdown of the MEK2-WIPK/SIPK-WRKY8 cascade resulted in enhanced accumulation of PVY genomic RNA. 6K2 of Turnip mosaic virus and p33 of Tomato bushy stunt virus also interacted with NbPLDα1 and induced the activation of MAPK-mediated immunity. Loss of function of NbPLDα1 inhibited virus-induced activation of MAPK cascades and promoted viral RNA accumulation. Thus, activation of MAPK-mediated immunity by NbPLDα1-derived PA is a common strategy employed by hosts to counteract positive-strand RNA virus infection.

RNA-Protein Interactome at the Hepatitis E Virus Internal Ribosome Entry Site.

Multiple processes exist in a cell to ensure continuous production of essential proteins either through cap-dependent or cap-independent translation processes. Viruses depend on the host translation machinery for viral protein synthesis. Therefore, viruses have evolved clever strategies to use the host translation machinery. Earlier studies have shown that genotype 1 hepatitis E virus (g1-HEV) uses both cap-dependent and cap-independent translation machineries for its translation and proliferation. Cap-independent translation in g1-HEV is driven by an 87-nucleotide-long RNA element that acts as a noncanonical, internal ribosome entry site-like (IRESl) element. Here, we have identified the RNA-protein interactome of the HEV IRESl element and characterized the functional significance of some of its components. Our study identifies the association of HEV IRESl with several host ribosomal proteins, demonstrates indispensable roles of ribosomal protein RPL5 and DHX9 (RNA helicase A) in mediating HEV IRESl activity, and establishes the latter as a bona fide internal translation initiation site. IMPORTANCE Protein synthesis is a fundamental process for survival and proliferation of all living organisms. The majority of cellular proteins are produced through cap-dependent translation. Cells also use a variety of cap-independent translation processes to synthesize essential proteins during stress. Viruses depend on the host cell translation machinery to synthesize their own proteins. Hepatitis E virus (HEV) is a major cause of hepatitis worldwide and has a capped positive-strand RNA genome. Viral nonstructural and structural proteins are synthesized through a cap-dependent translation process. An earlier study from our laboratory reported the presence of a fourth open reading frame (ORF) in genotype 1 HEV, which produces the ORF4 protein using a cap-independent internal ribosome entry site-like (IRESl) element. In the current study, we identified the host proteins that associate with the HEV-IRESl RNA and generated the RNA-protein interactome. Through a variety of experimental approaches, our data prove that HEV-IRESl is a bona fide internal translation initiation site.

Gene expression of peripheral blood mononuclear cells and CD8+ T cells from gilts after PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus, which emerged in Europe and U.S.A. in the late 1980s and has since caused huge economic losses. Infection with PRRSV causes mild to severe respiratory and reproductive clinical symptoms in pigs. Alteration of the host immune response by PRRSV is associated with the increased susceptibility to secondary viral and bacterial infections resulting in more serious and chronic disease. However, the expression profiles underlying innate and adaptive immune responses to PRRSV infection are yet to be further elucidated. In this study, we investigated gene expression profiles of PBMCs and CD8+ T cells after PRRSV AUT15-33 infection. We identified the highest number of differentially expressed genes in PBMCs and CD8+ T cells at 7 dpi and 21 dpi, respectively. The gene expression profile of PBMCs from infected animals was dominated by a strong innate immune response at 7 dpi which persisted through 14 dpi and 21 dpi and was accompanied by involvement of adaptive immunity. The gene expression pattern of CD8+ T cells showed a strong adaptive immune response to PRRSV, leading to the formation of highly differentiated CD8+ T cells starting from 14 dpi. The hallmark of the CD8+ T-cell response was the increased expression of effector and cytolytic genes (PRF1, GZMA, GZMB, GZMK, KLRK1, KLRD1, FASL, NKG7), with the highest levels observed at 21 dpi. Temporal clustering analysis of DEGs of PBMCs and CD8+ T cells from PRRSV-infected animals revealed three and four clusters, respectively, suggesting tight transcriptional regulation of both the innate and the adaptive immune response to PRRSV. The main cluster of PBMCs was related to the innate immune response to PRRSV, while the main clusters of CD8+ T cells represented the initial transformation and differentiation of these cells in response to the PRRSV infection. Together, we provided extensive transcriptomics data explaining gene signatures of the immune response of PBMCs and CD8+ T cells after PRRSV infection. Additionally, our study provides potential biomarker targets useful for vaccine and therapeutics development.