Viral Messenger RNA Research Articles

Proteolytic Disassembly Is a Critical Determinant for Reovirus Oncolysis

See page 1406Mammalian ortheoreoviruses are currently being investigated as novel cancer therapeutics, but the cellular mechanisms that regulate susceptibility to reovirus oncolysis remain poorly understood. In this study, we present evidence that virion disassembly is a key determinant of reovirus oncolysis. To penetrate cell membranes and initiate infection, the outermost capsid proteins of reovirus must be proteolyzed to generate a disassembled particle called an infectious subviral particle (ISVP). In fibroblasts, this process is mediated by the endo/lysosomal proteases cathepsins B and L. We have analyzed the early events of infection in reovirus-susceptible and -resistant cells. We find that, in contrast to susceptible glioma cells and Ras-transformed NIH3T3 cells, reovirus-resistant cancer cells and untransformed NIH3T3 cells restrict virion uncoating and subsequent gene expression. Disassembly-restrictive cells support reovirus infection, as in vitro-generated ISVPs establish productive infection, and pretreatment with poly(I:C) does not prevent infection in cancer cells. We find that the level of active cathepsin B and L is increased in tumors and that disassembly-restrictive glioma cells support reovirus oncolysis when grown as a tumor in vivo. Together, these results provide a model in which proteolytic disassembly of reovirus is a critical determinant of susceptibility to reovirus oncolysis.

Quand une structure particulière de l’ARN du VIH-1 conduit à un décalage de phase : perspectives pour le traitement de l’infection par le VIH

Ribosomal frameshift is used by HIV-1 to synthesize the precursor of its enzymes. The frameshift stimulator is a peculiar structure in the viral messenger RNA coding for this precursor, which increases the probability that this frameshift occurs. It was proposed to be either a triplex structure or an irregular stem-loop. Recently, two NMR groups independently showed that the frameshift stimulatory signal of HIV-1 is an extended stem-loop, with an internal three-purine bulge separating two helical regions. However, it remains unclear how such a structure promotes frameshifting. It is proposed that frameshifting results from a specific interaction between the stimulatory signal and either a hypothetical protein factor or the ribosome. The characterization of the structure of the frameshift stimulatory signal paves the way to the rational design of novel antiviral drugs, which, by binding to this signal, could interfere with frameshifting and viral replication.

RNA Interference with Measles Virus N, P, and L mRNAs Efficiently Prevents and with Matrix Protein mRNA Enhances Viral Transcription

In contrast to studies with genetically modified viruses, RNA interference allows the analysis of virus infections with identical viruses and posttranscriptional ablation of individual gene functions. Using RNase III-generated multiple short interfering RNAs (siRNAs) against the six measles virus genes, we found efficient downregulation of viral gene expression in general with siRNAs against the nucleocapsid (N), phosphoprotein (P), and polymerase (L) mRNAs, the translation products of which form the ribonucleoprotein (RNP) complex. Silencing of the RNP mRNAs was highly efficient in reducing viral messenger and genomic RNAs. siRNAs against the mRNAs for the hemagglutinin (H) and fusion (F) proteins reduced the extent of cell-cell fusion. Interestingly, siRNA-mediated knockdown of the matrix (M) protein not only enhanced cell-cell fusion but also increased the levels of both mRNAs and genomic RNA by a factor of 2 to 2.5 so that the genome-to-mRNA ratio was constant. These findings indicate that M acts as a negative regulator of viral polymerase activity, affecting mRNA transcription and genome replication to the same extent.

Impaired Control of IRES-Mediated Translation in X-Linked Dyskeratosis Congenita

The DKC1 gene encodes a pseudouridine synthase that modifies ribosomal RNA (rRNA). DKC1 is mutated in people with X-linked dyskeratosis congenita (X-DC), a disease characterized by bone marrow failure, skin abnormalities, and increased susceptibility to cancer. How alterations in ribosome modification might lead to cancer and other features of the disease remains unknown. Using an unbiased proteomics strategy, we discovered a specific defect in IRES (internal ribosome entry site)-dependent translation in Dkc1(m) mice and in cells from X-DC patients. This defect results in impaired translation of messenger RNAs containing IRES elements, including those encoding the tumor suppressor p27(Kip1) and the antiapoptotic factors Bcl-xL and XIAP (X-linked Inhibitor of Apoptosis Protein). Moreover, Dkc1(m) ribosomes were unable to direct translation from IRES elements present in viral messenger RNAs. These findings reveal a potential mechanism by which defective ribosome activity leads to disease and cancer.

Characterization of RNA in Cytologic Samples Preserved in a Methanol-Based Collection Solution.

Background: ThinPrep is a fluid-based technique for collection and processing of cytologic specimens. The present study was designed to determine whether the collection solution preserved RNA for molecular analysis. Methods and Results: Cervical cancer cell lines and cord blood lymphocytes were used to test the efficacy of various protocols for fixation, storage, and extraction of RNA. Total RNA was extracted and analyzed by denaturing gel electrophoresis. Preserved cells stored for 24 hours at room temperature or 4 degreesC had intact 28S and 18S ribosomal RNA. Both cellular and viral messenger RNAs were amplified from preserved samples by reverse transcription polymerase chain reaction (RT-PCR). Viral messenger RNA (mRNA) could be detected in a mixture of preserved cells containing 10% human papillomavirus (HPV) positive cells. RNA preservation in clinical samples was adequate for RT-PCR of cellular mRNA. Conclusions: Both experimental samples and clinical samples collected in the preservation media had intact total RNA. Amplification of both cellular and HPV ad mRNA was sucessful.

780. Inhibition of Respiratory Syncytial Virus Gene Expression and Replication by 27mer Dicer Substrate siRNAs

Respiratory Syncytial Virus (RSV) is a leading cause of infant hospitalization worldwide. There is currently no vaccine for RSV, and treatment of acute infections is limited to supportive measures. Recent reports also implicate RSV as an important pathogen in elderly and immunocompromised populations. Because of the need for improved treatments for RSV, we are evaluating siRNA-based approaches to inhibit viral replication in vitro and in vivo. We evaluated a series of 27mer dicer substrate small interfering RNAs (siRNAs) that target the RSV A2 strain nucleocapsid, phosphoprotein, and large polymerase. The nucleocapsid and large polymerase represent novel siRNA antiviral targets. These 27mer siRNAs are asymmetrically designed to preferentially target viral messenger RNAs rather than the viral genome. Following incubation with recombinant Dicer, they are predictably cleaved to 21mer or 22mer siRNAs. We have observed a dose-dependent reduction in viral gene expression following administration of siRNAs in cell culture, including decreased viral mRNAs, viral protein, and an approximately 40-fold reduction in viral titer. These siRNAs are active over a wide dose range in cell culture, with greatest efficacy observed between 10 and 100 nM. Since siRNAs against RNA viruses could theoretically target either the positive or negative sense strand of the virus, we screened for activity against portions of the viral genome cloned non-directionally into a psiCheck2 dual luciferase vector. We observed preferential targeting of the positive sense (mRNA) of the virus by our lead siRNAs. We are also evaluating siRNAs with the opposite polarity to determine the optimal targeting method for antiviral activity. We delivered fluorescently labeled oligos to evaluate in vitro and in vivo delivery. Unformulated oligos applied to the apical surface of well-differentiated primary cultures of human airway epithelia were visualized in the cytoplasm. Similarly labeled oligos delivered intratracheally to mice were visualized in respiratory epithelia in a dose-dependent pattern. Alveolar macrophages recovered by bronchoalveolar lavage contained a significant fluorescent signal. We are applying these techniques for in vivo delivery of siRNAs targeting RSV in a mouse model of infection.

The virion-associated Gag–Pol is decreased in chimeric Moloney murine leukemia viruses in which the readthrough region is replaced by the frameshift region of the human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) requires a programmed −1 translational frameshift event to synthesize the precursor of its enzymes, Gag–Pol, when ribosomes from the infected cells translate the full-length viral messenger RNA. Translation of the same RNA according to conventional translational rules produces Gag, the precursor of the structural proteins of the virus. The efficiency of the frameshift controls the ratio of Gag–Pol to Gag, which is critical for viral infectivity. The Moloney murine leukemia virus (MoMuLV) uses a different strategy, the programmed readthrough of a stop codon, to synthesize Gag–Pol. In this study, we investigated whether different forms of the HIV-1 frameshift region can functionally replace the readthrough signal in MoMuLV. Chimeric proviral DNAs were obtained by inserting into the MoMuLV genome the HIV-1 frameshift region encompassing the slippery sequence where the frameshift occurs, followed by the frameshift stimulatory signal. The inserted signal was either a simple stem-loop, previously considered as the stimulatory signal, or a longer bulged helix, now shown to be the complete stimulatory signal, or a mutated version of the complete signal with a three-nucleotide deletion. Although the three chimeric viruses can propagate essentially as the wild-type virus in NIH 3T3 cells, single-round infectivity assays revealed that the infectivity of the chimeric virions is about three to fivefold lower than that of the wild-type virions, depending upon the nature of the frameshift signal. It was also observed that the Gag–Pol to Gag ratio was decreased about two to threefold in chimeric virions. Comparison of the readthrough efficiency of MoMuLV to the HIV-1 frameshift efficiency, by monitoring the expression of a luciferase reporter in cultured cells, revealed that the frameshift efficiencies were only 30–60% of the readthrough efficiency. Altogether, these observations indicate that replacement of the readthrough region of MoMuLV with the frameshift region of HIV-1 results in virions that are replication competent, although less infectious than wild-type MoMuLV. This type of chimera could provide an interesting tool for in vivo studies of novel drugs targeted against the HIV-1 frameshift event.

Clinical and Pathological Features of Posttransplantation Lymphoproliferative Disorders Presenting With Skin Involvement in 4 Patients

Posttransplantation lymphoproliferative disorders (PTLDs) are lymphoid proliferations that can develop in recipients of solid organ or allogeneic bone marrow transplants. They are clinically and pathologically heterogeneous and range from polyclonal hyperplastic lesions to malignant lymphomas. Although extranodal involvement in PTLD is common, cutaneous presentation is rare, with only 19 cases reported previously. We describe 4 patients with cutaneous presentations of PTLD. All patients had relatively late-onset PTLD (>1 year after transplantation) with a median of 8 years from organ allograft to tumor diagnosis. The extent, number, and anatomic location of skin lesions varied from a localized patch to widespread nodules. None of the patients exhibited systemic symptoms at the time of PTLD diagnosis. Pathological findings ranged from plasmacytic hyperplasia to monomorphic PTLD. In situ hybridization detected Epstein-Barr virus messenger RNA in all 3 cases with evaluable tissue. All patients underwent reduction in immunosuppressive therapy and received other individualized treatments. Median follow-up was 2.5 years. At the most recent follow-up, 3 patients were in complete remission and 1 had residual disease. In this study, PTLD lesions presenting in the skin responded to therapy. Despite their relatively late occurrence after transplantation, most of these cases were positive for Epstein-Barr virus. As observed with other cutaneous lymphomas, the PTLDs with predominant skin involvement had a relatively favorable outcome.

Cryo-EM Visualization of a Viral Internal Ribosome Entry Site Bound to Human Ribosomes: The IRES Functions as an RNA-Based Translation Factor

Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in cricket paralysis virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.

Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus.

Transcriptionally active high-risk human papilloma viruses (HPVs), particularly HPV type 16 (HPV16), are found in a subset of head and neck squamous-cell carcinomas (HNSCCs). HPV16-associated carcinogenesis is mediated by expression of the viral E6 and E7 oncoproteins, which cause deregulation of the cell cycle by inactivating p53 and pRb, respectively. We tested the hypothesis that HPV-associated HNSCCs display a pattern of genetic alterations different from those of HNSCCs without HPV DNA. Polymerase chain reaction-based assays were used to examine 143 consecutive HNSCCs (106 of the oral cavity and 37 of the oropharynx) for the presence of HPV DNA and for viral E6 and/or E7 messenger RNA (mRNA) expression. The HPV DNA-and E6 and E7 mRNA-positive HNSCCs and an equal number of HPV DNA-negative HNSCCs were further analyzed for mutations in TP53, the gene encoding p53, and for allelic loss of 28 microsatellite markers at chromosome arms 3p, 6q, 8p, 9p, 13q, 17p, and 18q, including markers located in regions of chromosome arms 9p and 17p that harbor genes involved the p53 and pRb pathways. All statistical tests were two-sided. Twenty-four (16.7%) of the 143 HNSCCs were positive for HPV16 DNA, and 12 of these HNSCCs (8.4% of total number) expressed E6 and E7 mRNAs. None of the HPV DNA-and E6/E7 mRNA-positive tumors had TP53 gene mutations, whereas nine (75%) of the 12 HPV DNA-negative tumors had such mutations (P<.001). Compared with the HPV DNA-negative HNSCCs, the E6/E7 mRNA-positive HNSCCs had statistically significantly lower levels of allelic loss for 13 of the 15 markers on 3p, 9p, and 17p. HNSCCs with transcriptionally active HPV16 DNA are characterized by occasional chromosomal loss, whereas HNSCCs lacking HPV DNA are characterized by gross deletions that involve whole or large parts of chromosomal arms and that already occur early in HNSCC development. These distinct patterns of genetic alterations suggest that HPV16 infection is an early event in HNSCC development.

Chemical Route to the Capped RNAs

Eukaryotic and viral messenger RNAs contain a CAP structure that plays an important role in the initiation of translation and several other cellular processes that involve mRNAs. In this paper, we report a convenient chemical approach to the preparation of milligram quantities of short, capped RNA oligonucleotides, which overcomes some of the limitations of previous approaches. The method is based on the use of a reactive precursor, m7GppQ [P1‐7‐methylguanosine‐5′‐O‐yl, P2‐O‐8‐(5‐chloroquinolyl) pyrophosphate]. The precursor reacts smoothly with 5′‐phosphorylated unprotected short RNA in the presence of CuCl2 in organic media. The feasibility of this approach was demonstrated by the synthesis of the capped pentaribonucleotide m7GpppGpApCpU. The synthesized capped oligonucleotide was isolated and purified by reverse phase and ion exchange HPLC with a final yield of 37%. The structure of the m7GpppGpApCpU was confirmed by 31P NMR, mass‐spectrometry and enzymatic hydrolysis.

Deficiency in virion secretion and decreased stability of the hepatitis B virus immune escape mutant G145R

Hepatitis B virus with a G145R mutation in the small surface protein is considered the quintessential immune escape mutant because it frequently is found in vaccinated individuals with breakthrough infections and liver transplant recipients under anti-hepatitis B surface antigen (HBsAg) immunoglobulin prophylaxis. Nowadays the prevalence of the variant progressively increases. However, because spread of a virus depends not only on immune pressure but also on the viral phenotype, we investigated the biologic properties of the G145R variant. The G145R mutation was introduced into wild-type (Wt) virus genome by in vitro mutagenesis. After transfection into human hepatoma cells, the DNA, RNA, and protein synthesis and viral secretion ability of the mutant were studied. Furthermore, cotransfection studies were performed with the G145R variant and a Wt virus S-protein expressing construct and vice versa. Production and stability of viral messenger RNAs (mRNAs), DNA, and proteins were not affected by the G145R mutation. In contrast, secretion of mutant virions was reduced significantly. Only 20% of virions were found in the medium of G145R variant-transfected cells compared with Wt virus. Furthermore, mutant virions were more sensitive to detergent treatment suggesting a diminished stability. In cotransfection studies, Wt virus S-protein rescued secretion of mutant virions, whereas mutant S-protein had a transdominant negative effect on secretion of Wt virus. Both mechanisms may support persistence of the defective mutant in a mixed population with Wt virus. In conclusion, the significant defect of the G145R mutant for secretion of infectious virions and the diminished stability of mutant virions may limit global spread of the mutant.

Sequence Specificity in the Interaction of Bluetongue Virus Non-structural Protein 2 (NS2) with Viral RNA

The non-structural protein NS2 of Bluetongue virus (BTV) is synthesized abundantly in virus-infected cells and has been suggested to be involved in virus replication. The protein, with a high content of charged residues, possesses a strong affinity for single-stranded RNA species but, to date, all studies have failed to identify any specificity in the NS2-RNA interaction. In this report, we have examined, through RNA binding assays using highly purified NS2, the specificity of interaction with different single-stranded RNA (ssRNA) species in the presence of appropriate competitors. The data obtained show that NS2 indeed has a preference for BTV ssRNA over nonspecific RNA species and that NS2 recognizes a specific region within the BTV10 segment S10. The secondary structure of this region was determined and found to be a hairpin-loop with substructures within the loop. Modification-inhibition experiments highlighted two regions within this structure that were protected from ribonuclease cleavage in the presence of NS2. Overall, these data imply that a function of NS2 may be to recruit virus messenger RNAs (that also act as templates for synthesis of genomic RNAs) selectively from other RNA species within the infected cytosol of the cell during virus replication.

The Frameshift Stimulatory Signal of Human Immunodeficiency Virus Type 1 Group O is a Pseudoknot

Human immunodeficiency virus type 1 (HIV-1) requires a programmed −1 ribosomal frameshift to produce Gag–Pol, the precursor of its enzymatic activities. This frameshift occurs at a slippery sequence on the viral messenger RNA and is stimulated by a specific structure, downstream of the shift site. While in group M, the most abundant HIV-1 group, the frameshift stimulatory signal is an extended bulged stem-loop, we show here, using a combination of mutagenesis and probing studies, that it is a pseudoknot in group O. The mutagenesis and probing studies coupled to an in silico analysis show that group O pseudoknot is a hairpin-type pseudoknot with two coaxially stacked stems of eight base-pairs (stem 1 and stem 2), connected by single-stranded loops of 2 nt (loop 1) and 20 nt (loop 2). Mutations impairing formation of stem 1 or stem 2 of the pseudoknot reduce frameshift efficiency, whereas compensatory changes that allow re-formation of these stems restore the frameshift efficiency to near wild-type level. The difference between the frameshift stimulatory signal of group O and group M supports the hypothesis that these groups originate from a different monkey to human transmission.

IRESdb: the Internal Ribosome Entry Site database.

Internal Ribosome Entry Sites (IRES) are cis-acting RNA sequences able to mediate internal entry of the 40S ribosomal subunit on some eukaryotic and viral messenger RNAs upstream of a translation initiation codon. These sequences are very diverse and are present in a growing list of mRNAs. Novel IRES sequences continue to be added to public databases every year and the list of unknown IRESes is certainly still very large. The IRES database is a comprehensive WWW resource for internal ribosome entry sites and presents currently available general information as well as detailed data for each IRES. It is a searchable, periodically updated collection of IRES RNA sequences. Sequences are presented in FASTA form and hotlinked to NCBI GenBank files. Several subsets of data are classified according to the viral taxon (for viral IRESes), to the gene product function (for cellular IRESes), to the possible cellular regulation or to the trans-acting factor that mediates IRES function. This database is accessible at http://ifr31w3.toulouse.inserm.fr/IRESdatabase/.

Characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1.

Synthesis of the Gag-Pol protein of the human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshifting when ribosomes translate the unspliced viral messenger RNA. This frameshift occurs at a slippery sequence followed by an RNA structure motif that stimulates frameshifting. This motif is commonly assumed to be a simple stem-loop for HIV-1. In this study, we show that the frameshift stimulatory signal is more complex than believed and consists of a two-stem helix. The upper stem-loop corresponds to the classic stem-loop, and the lower stem is formed by pairing the spacer region following the slippery sequence and preceding this classic stem-loop with a segment downstream of this stem-loop. A three-purine bulge interrupts the two stems. This structure was suggested by enzymatic probing with nuclease V1 of an RNA fragment corresponding to the gag/pol frameshift region of HIV-1. The involvement of the novel lower stem in frameshifting was supported by site-directed mutagenesis. A fragment encompassing the gag/pol frameshift region of HIV-1 was inserted in the beginning of the coding sequence of a reporter gene coding for the firefly luciferase, such that expression of luciferase requires a -1 frameshift. When the reporter was expressed in COS cells, mutations that disrupt the capacity to form the lower stem reduced frameshifting, whereas compensatory changes that allow re-formation of this stem restored the frameshift efficiency near wild-type level. The two-stem structure that we propose for the frameshift stimulatory signal of HIV-1 differs from the RNA triple helix structure recently proposed.

Les différents interférons : Pharmacologie, mécanismes d'action, tolérance et effets secondaires

Background. -Interferons are a recombinant form of endogenous interferon. By inducing the release of intracellular enzymes such as 2′5′ oligoadenylate synthetase and double stranded RNA dependent protein kinase, the drug causes degradation of viral messenger RNA and inhibits protein synthesis. Main points. -Interferons are cytokines efficient in the treatment of infections diseases. Three main types are described as alpha, beta and gamma depending on their antigenic specificity. They have antiviral activity, antiproliferative and immunomodulation properties in response to a viral infection or other enzyme inducers. Furthermore, they increase expression of major histo-compatible complex antigens, increase natural killer and cytotoxic T cell activity, cytokine induction and production of endogenous interferon. Adverse effects frequently occur but are generally wild and reversible at current dosages. The pharmacokinetics of interferons have been fairly well described. The decline in serum concentrations is rapid after intravenous administration. The volume of distribution approximates 20–60% of bodyweight. Terminal elimination half lives range from 4–16 hrs, 1–2 hrs and 25–35 min for alpha, beta, gamma respectively. Intra-muscular and subcutaneous administration of interferons alpha and beta results in protracted but fairly good absorption > 80% for interferon alpha to 70% for interferon gamma. Pegylated interferon safety and pharmacodynamic profiles were comparable. Pegylated interferon demonstrated delayed clearance compared with non-pegylated interferon, consistent with once-weekly administration. The severity of adverse effects is dose dependent. Most patients treated have an influenza like syndrome within 2–8 hrs of drug administration. Other effects such as fatigue, lethargy, anorexia are usually dose limiting. Neuropsychiatric reactions may also become dose limiting. Interferon induced the formation of serum neutralizing antibodies in approximately 10–20% of treated patients. Perspectives. -The development of pegylated forms, a greater understanding of action mechanisms, and the combination with othertherapies should reinforce the place of interferons in the therapeutic arsenal.

RNA interference--a new weapon against HIV and beyond.

A recently discovered type of RNA, small interfering RNA, silences messenger RNA (the transcript of an active gene) by binding to specific sequences in the messenger RNA. Small interfering RNAs can block the replication of HIV in vitro by triggering the degradation of specific viral messenger RNAs. This work reveals new possibilities for controlling not only HIV infection but also the growth of cancer cells.

Inhibition of retroviral RNA production by ZAP, a CCCH-type zinc finger protein.

Cells have evolved multiple mechanisms to inhibit viral replication. To identify previously unknown antiviral activities, we screened mammalian complementary DNA (cDNA) libraries for genes that prevent infection by a genetically marked retrovirus. Virus-resistant cells were selected from pools of transduced clones, and an active antiviral cDNA was recovered. The gene encodes a CCCH-type zinc finger protein designated ZAP. Expression of the gene caused a profound and specific loss of viral messenger RNAs (mRNAs) from the cytoplasm without affecting the levels of nuclear mRNAs. The finding suggests the existence of a previously unknown machinery for the inhibition of virus replication, targeting a step in viral gene expression.

NSP5 phosphorylation regulates the fate of viral mRNA in rotavirus infected cells.

Elucidation of the function of the non-structural rotavirus proteins during infection is difficult in the absence of a reverse genetic system. To study the role of NSP5, nonstructural phosphoprotein NSP5, we constructed a reassortant strain (SACC11) in the SA11 background that harbours a heterologous segment 11 encoding a variant protein (h-NSP5). Cells infected by SACC11 produced viral polypeptides at earlier times than SA11 infected cells while showing less accumulation of genomic dsRNA. These changes suggested that NSP5 might direct viral messenger RNA to protein synthesis or genome replication. Distinct patterns of proteins were shown to form complexes with NSP5 in co-immunoprecipitation studies with SA11 and SACC11 infected cells. Recombinant h-NSP5 from either bacteria or eucaryotic cells migrated faster in PAGE suggesting that it was hypophosphorylated. Indeed, the kinase inhibitor H-7 enhanced translation of viral proteins in SA11 but not SACC11 infected cells suggesting that NSP5 function in the regulation of the fate of viral positive strand RNA is mediated by phosphorylation.