Untranslated Region RNA Research Articles

Elongation factor 1A binds the 3’ translation element of the barley yellow dwarf virus (566.4)

The Barley Yellow Dwarf Virus (BYDV) is especially devastating due to its global widespread distribution and its infection of staple cereal crops. Our research aims to understand mechanisms of BYDV mRNA translation to gain insight into combating the disease and its dire economic implications. Previous studies have shown that BYDV initiates translation in a way that is unique to most eukaryotic messages. Almost all eukaryotic mRNA consist of a 5’ cap and initiates translation at the 5’ untranslated region (UTR). BYDV mRNA, however, consists of a cap‐independent translation element at the 3’ UTR (BTE). More interestingly, eukaryotic initiation factor 4F (eI4F) binds the 3’ end of BYDB mRNA, at the BTE, and translation occurs when the 3’ UTR interacts with the 5’ UTR. Pull‐down experiments using the BTE RNA as bait have revealed not only eIF4F binding but also a small array of other proteins not present when a nonfunctional BTE (BTEBF) or the 5’ UTR is used as bait. One prominent protein that appears to bind BTE but not the control BTEBF or 5’ UTR RNA is an unknown 50 kDa protein. Using Mass Spectrometry, we identify this protein to be elongation factor 1A (EF1A). EF1A has previously been shown to bind the 3’ UTR of Tobacco Etch Virus (TEV) mRNA and may have a role in in regulation and replication of the viral genes.Grant Funding Source: NSF MCB 1157632

Posttranscriptional Modulation of Cytokine Production in T Cells for the Regulation of Excessive Inflammation by TFL

Posttranscriptional machinery regulates inflammation and is associated with autoimmunity as well as tumorigenesis in collaboration with transcription factors. We previously identified the tumor suppressor gene transformed follicular lymphoma (TFL) on 6q25 in a patient with follicular lymphoma, which transformed into diffuse large B cell lymphoma. TFL families have a common RNase domain that governs macrophage-mediated inflammation. In human peripheral blood, TFL is dominantly expressed at the glycine- and tryptophan-rich cytoplasmic processing bodies of T lymphocytes, and it is persistently upregulated in activated T cells. To address its physiological role, we established TFL(-/-) mice in which TFL(-/-) lymphocytes proliferated more rapidly than TFL(+/+) upon stimulation with inappropriate cytokine secretion, including IL-2, IL-6, and IL-10. Moreover, TFL inhibited the synthesis of cytokines such as IL-2, IL-6, IL-10, TNF-α, and IL-17a by 3' untranslated region RNA degradation. Experimental autoimmune encephalitis induced in TFL(-/-) mice demonstrated persistent severe paralysis. CNS-infiltrated CD4(+) T cells in TFL(-/-) mice contained a higher proportion of Th17 cells than did those in TFL(+/+) mice during the resolution phase, and IL-17a mRNA levels were markedly increased in TFL(-/-) cells. These results suggest that TFL may play an important role in attenuating local inflammation by suppressing the infiltration of Th17 cells in the CNS during the resolution phase of experimental autoimmune encephalitis. TFL is a novel gradual and persistent posttranscriptional regulator, and the TFL-driven attenuation of excessive inflammation could contribute to recovery from T cell-mediated autoimmune diseases.

The Untranslated Regions of Classic Swine Fever Virus RNA Trigger Apoptosis

Classical swine fever virus (CSFV) causes a broad range of disease in pigs, from acute symptoms including high fever and hemorrhages, to chronic disease or unapparent infection, depending on the virus strain. CSFV belongs to the genus Pestivirus of the family Flaviviridae. It carries a single-stranded positive-sense RNA genome. An internal ribosomal entry site (IRES) in the 5′ untranslated region (UTR) drives the translation of a single open reading frame encoding a 3898 amino acid long polypeptide chain. The open reading frame is followed by a 3′ UTR comprising four highly structured stem-loops. In the present study, a synthetic RNA composed of the 5′ and 3′ UTRs of the CSFV genome devoid of any viral coding sequence and separated by a luciferase gene cassette (designated 5′UTR-Luc-3′UTR) triggered apoptotic cell death as early as 4 h post-transfection. The apoptosis was measured by DNA laddering analysis, TUNEL assay, annexin-V binding determined by flow cytometry, and by analysis of caspase activation. Contrasting with this, only trace DNA laddering was observed in cells transfected with the individual 5′ or 3′ UTR RNA; even when the 5′ UTR and 3′ UTR were co-transfected as separate RNA molecules, DNA laddering did not reach the level induced by the chimeric 5′UTR-Luc-3′UTR RNA. Interestingly, RNA composed of the 5′UTR and of stem-loop I of the 3′UTR triggered much stronger apoptosis than the 5′ or 3′UTR alone. These results indicate that the 5′ and 3′ UTRs act together in cis induce apoptosis. We furthered obtained evidence that the UTR-mediated apoptosis required double-stranded RNA and involved translation shutoff possibly through activation of PKR.

Dephosphorylation of HuR Protein during Alphavirus Infection Is Associated with HuR Relocalization to the Cytoplasm

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3' untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3' UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3' UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.

5'-UTR RNA G-quadruplexes: translation regulation and targeting

RNA structures in the untranslated regions (UTRs) of mRNAs influence post-transcriptional regulation of gene expression. Much of the knowledge in this area depends on canonical double-stranded RNA elements. There has been considerable recent advancement of our understanding of guanine(G)-rich nucleic acids sequences that form four-stranded structures, called G-quadruplexes. While much of the research has been focused on DNA G-quadruplexes, there has recently been a rapid emergence of interest in RNA G-quadruplexes, particularly in the 5′-UTRs of mRNAs. Collectively, these studies suggest that RNA G-quadruplexes exist in the 5′-UTRs of many genes, including genes of clinical interest, and that such structural elements can influence translation. This review features the progresses in the study of 5′-UTR RNA G-quadruplex-mediated translational control. It covers computational analysis, cell-free, cell-based and chemical biology studies that have sought to elucidate the roles of RNA G-quadruplexes in both cap-dependent and -independent regulation of mRNA translation. We also discuss protein trans-acting factors that have been implicated and the evidence that such RNA motifs have potential as small molecule target. Finally, we close the review with a perspective on the future challenges in the field of 5′-UTR RNA G-quadruplex-mediated translation regulation.

A luciferase reporter assay to investigate the differential selenium-dependent stability of selenoprotein mRNAs

The mechanisms regulating the differential selenium (Se)-dependent stability of selenoprotein mRNAs are partially characterized. To further study the Se-dependent regulation of selenoproteins, we developed a novel chemiluminescent reporter to monitor the steady-state mRNA level of an artificial selenoprotein. Our reporter is a fusion of the Renilla luciferase gene and of the β-globin gene, but contains features required for incorporation of selenocysteine (SEC), namely, a UGA-SEC codon and a 3′ untranslated region RNA stem loop called a SEC incorporation sequence (SECIS). At various levels of Se, the activity of reporters containing GPX1 or GPX4 SECIS elements is proportional to the steady-state mRNA level of the reporter construct and reflects the level of the corresponding endogenous mRNA. In a reporter containing a UGA codon and a functional GPX1 SECIS, Se-dependent nonsense-mediated decay (NMD) occurred in the cytoplasm, as opposed to the more typical nuclear location. To validate the reporter system, we used genetic and pharmacologic approaches to inhibit or promote NMD. Modulation of UPF1 by siRNA, overexpression, or by inhibition of SMG1 altered NMD in this system. Our reporter is derived from a Renilla luciferase reporter gene fused to an intron containing B-globin gene and is subject to degradation by NMD when a stop codon is inserted before the second intron.

Structural map of a microRNA-122: hepatitis C virus complex.

MicroRNA-122 (miR-122) enhances hepatitis C virus (HCV) fitness via targeting two sites in the 5'-untranslated region (UTR) of HCV. We used selective 2'-hydroxyl acylation analyzed by primer extension to resolve the HCV 5'-UTR's RNA secondary structure in the presence of miR-122. Nearly all nucleotides in miR-122 are involved in targeting the second site, beyond classic seed base pairings. These additional interactions enhance HCV replication in cell culture. To our knowledge, this is the first biophysical study of this complex to reveal the importance of 'tail' miR-122 nucleotide interactions.

Identification of the binding proteins of the 3' untranslated region of mouse Ard1b that may be responsible for its translational delay during spermatogenesis

Ard1b encodes the catalytic subunit of N(alpha)-acetyltransferase which is believed to play a role in spermatogenesis. In developing mouse testes, the Ard1b transcript level steadily increases before the appearance of Ard1b proteins. Therefore, translation of Ard1b is uncoupled from transcription. The Ard1b gene contains a long 3' untranslated region (UTR). Regulatory elements affecting the stability, localization and translational efficiency of RNA transcripts tend to localize on the 3'UTR of genes. We hypothesized that protein factors are involved in delaying Ard1b translation through their physical interaction with specific sequence motifs present in the 3'UTR of Ard1b transcripts. Our objective was to identify the proteins that bind to the Ard1b 3' UTR. Laboratory Study. The Ard1b 3'UTR was cloned into pCR4-TOPO vector, which was linearized for in vitro transcription in the presence of biotinylated ATP. The biotinylated Ard1b 3'UTR RNA was immobilized on streptavidin-coated magnetic agarose beads and the complex was incubated with mouse testicular lysate. Proteins captured by the Ard1b 3'UTR were subsequently eluted, separated by gel electrophoresis and analyzed by mass spectrometry (MS). The involvement of the candidate proteins in translational repression will be tested in a reporter assay. Three independent pull-down experiments were performed. Multiple proteins were captured by the Ard1b 3'UTR. Among them, 5 proteins (∼21, ∼33, ∼34, ∼35 and ∼ 97 kDa) were consistently revealed on protein gels. Of these, one was identified by MS as the heterogeneous nuclear ribonucleoprotein (hnRNP) C. These results suggest hnRNP C is a candidate repressor of Ard1b translation. Further studies will test if the 15-LOX-DICE regulatory element, located in the Ard1b 3'UTR, might bind hnRNP C and contribute to the delayed translation of Ard1b.

A conserved RNA pseudoknot in a putative molecular switch domain of the 3′-untranslated region of coronaviruses is only marginally stable

The 3'-untranslated region (UTR) of the group 2 coronavirus mouse hepatitis virus (MHV) genome contains a predicted bulged stem-loop (designated P0ab), a conserved cis-acting pseudoknot (PK), and a more distal stem-loop (designated P2). Base-pairing to create the pseudoknot-forming stem (P1(pk)) is mutually exclusive with formation of stem P0a at the base of the bulged stem-loop; as a result, the two structures cannot be present simultaneously. Herein, we use thermodynamic methods to evaluate the ability of individual subdomains of the 3' UTR to adopt a pseudoknotted conformation. We find that an RNA capable of forming only the predicted PK (58 nt; 3' nucleotides 241-185) adopts the P2 stem-loop with little evidence for P1(pk) pairing in 0.1 M KCl and the absence of Mg(2+); as Mg(2+) or 1 M KCl is added, a new thermal unfolding transition is induced and assignable to P1(pk) pairing. The P1(pk) helix is only marginally stable, ΔG(25) ≈ 1.2 ± 0.3 kcal/mol (5.0 mM Mg(2+), 100 mM K(+)), and unfolded at 37°C. Similar findings characterize an RNA 5' extended through the P0b helix only (89 nt; 294-185). In contrast, an RNA capable of forming either the P0a helix or the pseudoknot (97 nt; 301-185) forms no P1(pk) helix. Thermal unfolding simulations are fully consistent with these experimental findings. These data reveal that the PK forms weakly and only when the competing double-hairpin structure cannot form; in the UTR RNA, the double hairpin is the predominant conformer under all solution conditions.

FUSE Binding Protein 1 Interacts with Untranslated Regions of Japanese Encephalitis Virus RNA and Negatively Regulates Viral Replication

The untranslated regions (UTRs) located at the 5' and 3' ends of the Japanese encephalitis virus (JEV) genome, a positive-sense RNA, are involved in viral translation, the initiation of RNA synthesis, and the packaging of nascent virions. The cellular and viral proteins that participate in these processes are expected to interact with the UTRs. In this study, we used biotinylated RNA-protein pulldown and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses to identify that the far upstream element (FUSE) binding protein 1 (FBP1) binds with JEV 5' and 3' UTRs. The impact of FBP1 on JEV infection was determined in cells with altered FBP1 expression. JEV replication was enhanced by knockdown and reduced by the overexpression of FBP1, indicating a negative role for FBP1 in JEV infection. FBP1, a nuclear protein, was redistributed to the perinuclear region and appeared as cytoplasmic foci that partially colocalized with JEV RNA in the early stage of JEV infection. By using a JEV replicon reporter assay, FBP1 appeared to suppress JEV protein expression mediated by the 5' and 3' UTRs. Thus, we suggest that FBP1 binds with the JEV UTR RNA and functions as a host anti-JEV defense molecule by repressing viral protein expression.

A Biophysical Study of the Hepatitis C Virus 5’Untranslated Region Interactions with MIR-122

The liver-specific microRNA, miR-122 has been shown to be involved in positively regulating Hepatitis C Virus (HCV), which infects on average 3.2 million Americans. Emerging evidence in the literature suggests that miR-122 binds to two seed sequences (S1 and S2) located within the 5’ untranslated region (UTR) of HCV genomic RNA. Both sites S1 and S2 are important for the effect of miR122 in promoting an upregulation of HCV translation, whereas the S1 site plays a more crucial role in increasing the effect of miR122 upon the viral replication. In this study, we used biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy and native gel electrophoresis to analyze in vitro the direct interactions between miR-122 and the HCV 5’UTR RNA.

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

β(2)-adrenergic receptors (β(2)-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β(2)-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β(2)-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β(2)-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β(2)-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β(2)-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.

All three domains of the hepatitis C virus nonstructural NS5A protein contribute to RNA binding.

The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3' untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3' UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3' UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3' UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3' UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.

Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k(on)) and dissociation rate constant (k(off)) for eIF4F binding to IRES were 59 +/- 2.1 micro s(-1) and 12.9 +/- 0.3 s(-1), respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F approximately 3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.

Identification of in vivo interaction between Hepatitis C Virus core protein and 5′ and 3′ UTR RNA

Here, we investigated the ability of the Hepatitis C Virus (HCV) core protein to interact specifically with the 5′ and 3′ untranslated regions (UTRs) of HCV using an in vivo cell-based translation inhibition assay. HCV core protein interacts weakly but specifically with the SLIII stem loop in the 5′ UTR in which the SLIIIb subdomain is the major determinant and the SL2 loop in the X region of the 3′ UTR. These results revealed for the first time in vivo interaction of the core protein with 5′ and 3′ UTRs involved in the viral life cycle. This system provides a useful tool for further investigating interactions between the HCV core protein and 5′ and 3′ UTRs.

The optimization of viral vector translation improves the production of recombinant proteins in plants

One of the most convenient methods for the fast and efficient production of target proteins in plants involves self-replicating recombinant viral vectors. We have constructed a plant viral vector based on the genome of the potato X virus. This vector contains the sequence of the 5′-untranslated region of RNA 4 of the alfalfa mosaic virus immediately upstream of the target gene. The incorporation of this sequence into the viral vector increases the production of the target protein by the recipient plant three- to fourfold owing to the increased efficiency of translation of viral subgenomic RNA comprising the target gene. The new vector can be used for the production of recombinant proteins in plants.

The p38 MAPK pathway inhibits tristetraprolin-directed decay of interleukin-10 and pro-inflammatory mediator mRNAs in murine macrophages

p38 mitogen-activated protein kinase (MAPK) stabilises pro-inflammatory mediator mRNAs by inhibiting AU-rich element (ARE)-mediated decay. We show that in bone-marrow derived murine macrophages tristetraprolin (TTP) is necessary for the p38 MAPK-sensitive decay of several pro-inflammatory mRNAs, including cyclooxygenase-2 and the novel targets interleukin (IL)-6, and IL-1α. TTP −/− macrophages also strongly overexpress IL-10, an anti-inflammatory cytokine that constrains the production of the IL-6 despite its disregulation at the post-transcriptional level. TTP directly controls IL-10 mRNA stability, which is increased and insensitive to inhibition of p38 MAPK in TTP −/− macrophages. Furthermore, TTP enhances deadenylation of an IL-10 3′-untranslated region RNA in vitro.

G-quadruplexes: the beginning and end of UTRs.

Molecular mechanisms that regulate gene expression can occur either before or after transcription. The information for post-transcriptional regulation can lie within the sequence or structure of the RNA transcript and it has been proposed that G-quadruplex nucleic acid sequence motifs may regulate translation as well as transcription. Here, we have explored the incidence of G-quadruplex motifs in and around the untranslated regions (UTRs) of mRNA. We observed a significant strand asymmetry, consistent with a general depletion of G-quadruplex-forming RNA. We also observed a positional bias in two distinct regions, each suggestive of a specific function. We observed an excess of G-quadruplex motifs towards the 5′-ends of 5′-UTRs, supportive of a hypothesis linking 5′-UTR RNA G-quadruplexes to translational control. We then analysed the vicinity of 3′-UTRs and observed an over-representation of G-quadruplex motifs immediately after the 3′-end of genes, especially in those cases where another gene is in close proximity, suggesting that G-quadruplexes may be involved in the termination of gene transcription.

Y Box-binding Protein-1 Binds to the Dengue Virus 3′-Untranslated Region and Mediates Antiviral Effects

Dengue virus, a member of the family Flaviviridae, poses a serious public health threat worldwide. Dengue virus is a positive-sense RNA virus that harbors a genome of approximately 10.7 kb. Replication of dengue virus is mediated coordinately by cis-acting genomic sequences, viral proteins, and host cell factors. We have isolated and identified several host cell factors from baby hamster kidney cell extracts that bind with high specificity and high affinity to sequences within the untranslated regions of the dengue virus genome. Among the factors identified, Y box-binding protein-1 (YB-1) and the heterogeneous nuclear ribonucleoproteins (hnRNPs), hnRNP A1, hnRNP A2/B1, and hnRNP Q, bind to the dengue virus 3'-untranslated region. Further analysis indicated that YB-1 binds to the dengue virus 3' stem loop, a conserved structural feature located at the 3' terminus of the 3'-untranslated region of many flaviviruses. Analysis of the impact of YB-1 on replication of dengue virus in YB-1+/+ and YB-1-/- mouse embryo fibroblasts indicated that host YB-1 mediates an antiviral effect. Further studies demonstrated that this antiviral impact is due, at least in part, to a repressive role of YB-1 on dengue virus translation via a mechanism that requires viral genomic sequences. These results suggest a novel role for YB-1 as an antiviral host cell factor.

Role of CD151, A tetraspanin, in porcine reproductive and respiratory syndrome virus infection

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) is a RNA virus causing respiratory and reproductive diseases in swine. The susceptibility for PRRSV varies between the different breeds of swine. In cell culture, PRRSV virus can be propagated in primary porcine alveolar macrophages and some African green monkey kidney cell lines, such as MARC-145 cells. Previous studies have shown that 3' untranslated region (UTR) RNAs of the arteriviruses play an important role in the replication of the virus through interactions with cellular proteins. To better understand the differences in the replication capability of PRRSV in different cell lines, we sought to identify the host cellular proteins interacting with PRRSV 3' UTR RNA. We constructed a cDNA library of MARC-145 cell line in lambda ZAP Express vector and screened the library with the positive sense 3' UTR RNA of PRRSV.ResultsWe found that CD151, a host cellular protein, interacting with PRRSV 3' UTR RNA. The specificity of the interaction between CD151 and PRRSV 3' UTR RNA was examined by gel shift assay as well as North-Western hybridization. The transfection of CD151 expression clone into BHK-21 rendered these cells susceptible to PRRSV infection, and the transfection of siRNA against CD151 into MARC-145 significantly reduced the level of PRRSV infection. Also, anti-CD151 antibody treatment to MARC-145 completely blocked PRRSV infection.ConclusionBased on our results, we suggest that CD151 should cooperate in PRRSV infection in vitro in MARC-145 and BHK-21 cells.