Foot-and-Mouth Disease Virus Leader Protease Cleaves G3BP1 and G3BP2 and Inhibits Stress Granule Formation.

Like other viruses, the picornavirus foot-and-mouth disease virus (FMDV; genus Aphthovirus), one of the most notorious pathogens in the global livestock industry, needs to navigate antiviral host responses to establish an infection. There is substantial insight into how FMDV suppresses the type I interferon (IFN) response, but it is largely unknown whether and how FMDV modulates the integrated stress response. Here, we show that the stress response is suppressed during FMDV infection. Using a chimeric recombinant encephalomyocarditis virus (EMCV), in which we functionally replaced the endogenous stress response antagonist by FMDV leader protease (Lpro) or 3Cpro, we demonstrate an essential role for Lpro in suppressing stress granule (SG) formation. Consistently, infection with a recombinant FMDV lacking Lpro resulted in SG formation. Additionally, we show that Lpro cleaves the known SG scaffold proteins G3BP1 and G3BP2 but not TIA-1. We demonstrate that the closely related equine rhinitis A virus (ERAV) Lpro also cleaves G3BP1 and G3BP2 and also suppresses SG formation, indicating that these abilities are conserved among aphthoviruses. Neither FMDV nor ERAV Lpro interfered with phosphorylation of RNA-dependent protein kinase (PKR) or eIF2α, indicating that Lpro does not affect SG formation by inhibiting the PKR-triggered signaling cascade. Taken together, our data suggest that aphthoviruses actively target scaffolding proteins G3BP1 and G3BP2 and antagonize SG formation to modulate the integrated stress response.IMPORTANCE The picornavirus foot-and-mouth disease virus (FMDV) is a notorious animal pathogen that puts a major economic burden on the global livestock industry. Outbreaks have significant consequences for animal health and product safety. Like many other viruses, FMDV must manipulate antiviral host responses to establish infection. Upon infection, viral double-stranded RNA (dsRNA) is detected, which results in the activation of the RNA-dependent protein kinase (PKR)-mediated stress response, leading to a stop in cellular and viral translation and the formation of stress granules (SG), which are thought to have antiviral properties. Here, we show that FMDV can suppress SG formation via its leader protease (Lpro). Simultaneously, we observed that Lpro can cleave the SG scaffolding proteins G3BP1 and G3BP2. Understanding the molecular mechanisms of the antiviral host response evasion strategies of FMDV may help to develop countermeasures to control FMDV infections in the future.

E6AP/UBE3A catalyzes encephalomyocarditis virus 3C protease polyubiquitylation and promotes its concentration reduction in virus-infected cells.

The encephalomyocarditis virus (EMCV) 3C protease (3Cpro) is one of a small number of viral proteins whose concentration is known to be regulated by the cellular ubiquitin-proteasome system. Here we report that the ubiquitin-conjugating enzyme UbcH7/UBE2L3 and the ubiquitin-protein ligase E6AP/UBE3A are components of a previously unknown EMCV 3Cpro-polyubiquitylating pathway. Following the identification of UbcH7/UBE2L3 as a participant in 3Cpro ubiquitylation, we purified a UbcH7-dependent 3Cpro-ubiquitylating activity from mouse cells, which we identified as E6AP. In vitro reconstitution assays demonstrated that E6AP catalyzes the synthesis of 3Cpro-attached Lys48-linked ubiquitin chains, known to be recognized by the 26S proteasome. We found that the 3Cpro accumulates to higher levels in EMCV-infected E6AP knockdown cells than in control cells, indicating a role for E6AP in in vivo 3Cpro concentration regulation. We also discovered that ARIH1 functions with UbcH7 to catalyze EMCV 3Cpro monoubiquitylation, but this activity does not influence the in vivo 3Cpro concentration.

Encephalomyocarditis virus 3C protease attenuates type I interferon production through disrupting the TANK-TBK1-IKKε-IRF3 complex.

TRAF family member-associated NF-κB activator (TANK) is a scaffold protein that assembles into the interferon (IFN) regulator factor 3 (IRF3)-phosphorylating TANK-binding kinase 1 (TBK1)-(IκB) kinase ε (IKKε) complex, where it is involved in regulating phosphorylation of the IRF3 and IFN production. However, the functions of TANK in encephalomyocarditis virus (EMCV) infection-induced type I IFN production are not fully understood. Here, we demonstrated that, instead of stimulating type I IFN production, the EMCV-HB10 strain infection potently inhibited Sendai virus- and polyI:C-induced IRF3 phosphorylation and type I IFN production in HEK293T cells. Mechanistically, EMCV 3C protease (EMCV 3C) cleaved TANK and disrupted the TANK-TBK1-IKKε-IRF3 complex, which resulted in the reduction in IRF3 phosphorylation and type I IFN production. Taken together, our findings demonstrate that EMCV adopts a novel strategy to evade host innate immune responses through cleavage of TANK.

Encephalomyocarditis Virus 3C Protease Relieves TRAF Family Member-associated NF-κB Activator (TANK) Inhibitory Effect on TRAF6-mediated NF-κB Signaling through Cleavage of TANK.

TRAF family member-associated NF-κB activator (TANK) is a negative regulator of canonical NF-κB signaling in the Toll-like receptor- and B-cell receptor-mediated signaling pathways. However, functions of TANK in viral infection-mediated NF-κB activation remain unclear. Here, we reported that TANK was cleaved by encephalomyocarditis virus 3C at the 197 and 291 glutamine residues, which depends on its cysteine protease activity. In addition, encephalomyocarditis virus 3C impaired the ability of TANK to inhibit TRAF6-mediated NF-κB signaling. Interestingly, we found that several viral proteases encoded by the foot and mouth disease virus, porcine reproductive and respiratory syndrome virus, and equine arteritis virus also cleaved TANK. Our results suggest that TANK is a novel target of some viral proteases, indicating that some positive RNA viruses have evolved to utilize their major proteases to regulate NF-κB activation.

Site-specific cleavage of the host poly(A) binding protein by the encephalomyocarditis virus 3C proteinase stimulates viral replication.

Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3C(pro) cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3C(pro) cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication.

Promyelocytic leukemia isoform IV confers resistance to encephalomyocarditis virus via the sequestration of 3D polymerase in nuclear bodies.

Promyelocytic leukemia (PML) protein is the organizer of nuclear matrix-associated nuclear bodies (NBs), and its conjugation to the small ubiquitin-like modifier (SUMO) is required for the formation of these structures. Several alternatively spliced PML transcripts from a single PML gene lead to the production of seven PML isoforms (PML isoform I [PMLI] to VII [PMLVII]), which all share a N-terminal region that includes the RBCC (RING, B boxes, and a α-helical coiled-coil) motif but differ in the C-terminal region. This diversity of PML isoforms determines the specific functions of each isoform. There is increasing evidence implicating PML in host antiviral defense and suggesting various strategies involving PML to counteract viral production. We reported that mouse embryonic fibroblasts derived from PML knockout mice are more sensitive than wild-type cells to infection with encephalomyocarditis virus (EMCV). Here, we show that stable expression of PMLIV or PMLIVa inhibited viral replication and protein synthesis, leading to a substantial reduction of EMCV multiplication. This protective effect required PMLIV SUMOylation and was not observed with other nuclear PML isoforms (I, II, III, V, and VI) or with the cytoplasmic PMLVII. We demonstrated that only PMLIV interacted with EMCV 3D polymerase (3Dpol) and sequestered it within PML NBs. The C-terminal region specific to PMLIV was required for both interaction with 3Dpol and the antiviral properties. Also, depletion of PMLIV by RNA interference significantly boosted EMCV production in interferon-treated cells. These findings indicate the mechanism by which PML confers resistance to EMCV. They also reveal a new pathway mediating the antiviral activity of interferon against EMCV.

Phosphatidylinositol 3-kinase regulates macrophage responses to double-stranded RNA and encephalomyocarditis virus.

Virus infection of macrophages stimulates the expression of proinflammatory and antiviral genes interleukin-1 (IL-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In this study, we show that phosphatidylinositol 3-kinase (PI3K) is required for the inflammatory response of macrophages to virus infection. When macrophages are infected with encephalomyocarditis virus (EMCV) there is a rapid and transient activation of PI3K and phosphorylation of its downstream target Akt. Inhibitors of PI3K attenuate EMCV- and double-stranded RNA-induced iNOS, COX-2 and IL-1 beta expression in RAW264.7 cells and mouse peritoneal macrophages. The attenuation of inflammatory gene expression in response to PI3K inhibition correlates with the induction of macrophage apoptosis. The morphology of macrophages shifts from activation in response to EMCV infection to apoptosis in the cells treated with PI3K inhibitors and EMCV. These morphological changes are accompanied by the activation of caspase-3. These findings suggest that PI3K plays a central role in the regulation of macrophage responses to EMCV infection. When PI3K is activated, it participates in the regulation of inflammatory gene expression; however, if PI3K is inhibited macrophages are unable to mount an inflammatory antiviral response and die by apoptosis.

TRIM22 E3 ubiquitin ligase activity is required to mediate antiviral activity against encephalomyocarditis virus.

The interferon (IFN) system is a major effector of the innate immunity that allows time for the subsequent establishment of an adaptive immune response against a wide-range of pathogens. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. Ubiquitin ligase members of the tripartite motif (TRIM) protein family have emerged as IFN-induced proteins involved in both innate and adaptive immunity. In this report, we provide evidence that TRIM22 is a functional E3 ubiquitin ligase that is also ubiquitinated itself. We demonstrate that TRIM22 expression leads to a viral protection of HeLa cells against encephalomyocarditis virus infections. This effect is dependent upon its E3 ubiquitinating activity, since no antiviral effect was observed in cells expressing a TRIM22-deletion mutant defective in ubiquitinating activity. Consistent with this, TRIM22 interacts with the viral 3C protease (3C(PRO)) and mediates its ubiquitination. Altogether, our findings demonstrate that TRIM22 E3 ubiquitin ligase activity represents a new antiviral pathway induced by IFN against picornaviruses.

Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo.

We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination.

Kinetic analysis of the conjugation of ubiquitin to picornavirus 3C proteases catalyzed by the mammalian ubiquitin-protein ligase E3alpha.

The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3alpha. The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3alpha and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E2(14Kb), and human E3alpha. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K(m) = 42 +/- 11 microm and V(max) = 0.051 +/- 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are K(m) = 20 +/- 5 microm and V(max) = 0.018 +/- 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3alpha conjugates ubiquitin to the altered 3C protease proteins. The K(m) and V(max) values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily a k(cat) effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the K(m) and V(max) values for these reactions are both higher than for the wild type protein. The ability of E3alpha to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3alpha that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases.

Deletion mapping of the encephalomyocarditis virus primary cleavage site.

The cotranslational, primary self-cleavage reaction of cardiovirus polyprotein relies on a highly conserved, short segment of amino acids at the 2A-2B protein boundary. The amino terminus of the required element for encephalomyocarditis virus has now been mapped to include Tyr(126) of the 2A protein, the 18th amino acid before the cleavage site.

Characterization and transduction of a retroviral vector encoding human interleukin-4 and herpes simplex virus-thymidine kinase for glioma tumor vaccine therapy.

Vaccination with cytokine-transduced tumor cells represents a potentially important approach to the treatment of central nervous system tumors. We have recently demonstrated the therapeutic efficacy of tumor cell vaccines expressing the murine interleukin 4 (IL-4) and the herpes simplex virus-thymidine kinase in a rat brain tumor model in which nonirradiated vaccine cells can be eliminated by the subsequent administration of ganciclovir. In this report, we demonstrate the construction and characterization of a retroviral vector that encodes human IL-4, neomycin phosphotransferase, and herpes simplex virus-thymidine kinase genes for use in human clinical trials. An MFG-based retroviral vector was used to generate the recombinant retrovirus, TFG-hIL4-Neo-Tk, in which a long terminal repeat-driven polycistronic transcript encodes three cDNAs that are linked and coexpressed using two intervening internal ribosome entry site fragments from the encephalomyocarditis virus. The amphotropic retroviral vector TFG-hIL4-Neo-Tk was then used to infect human primary glioma cultures and skin-derived fibroblasts. After infection and G418 selection, cells produced 89-131 ng/10(6) cells/48 hours of human IL-4, which was determined to be biologically active. Transduced glioma cells were highly sensitive to the cytotoxic effect of ganciclovir. These data demonstrate the suitability of the TFG-hIL4-Neo-Tk vector for therapeutic studies of cytokine-transduced autologous tumor vaccination in patients with malignant gliomas.

Quantification of endogenous viral polymerase, 3D(pol), in preparations of Mengo and encephalomyocarditis viruses.

Measurement of an antigenic response to the aphthovirus infection-associated antigen (VIA), the viral RNA polymerase 3D(pol), is frequently used as a discriminating assay for the extent of viral replication in animals. In practice, animals seropositive for VIA are assumed to have been exposed to live virus, although in fact it is suspected that endogenous 3D(pol) in commercial inactivated vaccines may occasionally stimulate analogous responses and result in false-positive tests for virus exposure. Cardiovirus infections in mice produce similar anti-VIA antibodies, and in view of recently developed attenuated Mengo vaccines and live Mengo vectors, these VIA responses are also under investigation as potential correlates of vaccine efficacy. We have purified recombinant Mengo 3D(pol), developed monoclonal antibodies to the protein, and used these reagents in highly sensitive Western blot assays to quantify the levels of endogenous 3D(pol) in Mengo and encephalomyocarditis virus (EMCV) preparations. The presence of 3D(pol) was detected at all stages of standard vaccine purification procedures, including materials purified by CsCl. Clarified suspensions of Mengo- or encephalomyocarditis virus-infected HeLa cells were found to contain very high quantities of 3D(pol), averaging approximately 1.2-1.5 micrograms of protein/micrograms of virus. Pelleting through 30% sucrose or purification by CsCl removed much of this material, but even these samples retained approximately 0.2-0.4 ng of 3D(pol)/micrograms virus. These ratios represent approximately 1 3D(pol) molecule/20 virus particles in the most highly purified materials and probably indicate that 3D(pol) is a contaminant on the particle surface rather than an intrinsically packaged molecule. In clarified cell lysates, which are commonly used as vaccine inocula, the protein to virus ratio was approximately 210:1, a level that could represent serious contamination problems for future VIA detection if such inocula are used without further purification.

Identification and characterization of a protein destruction signal in the encephalomyocarditis virus 3C protease.

The amino acid sequence LLVRGRTLVV, which is probably located in a strand-turn-strand structure, has been identified as a protein destruction signal in the rapidly degraded encephalomyocarditis virus 3C protease. Mutations within this sequence reduced the susceptibility of the 3C protease toward ubiquitination and degradation in rabbit reticulocyte lysate. This signal is transferable, since poliovirus 3C protease, which is a poor ubiquitin-mediated proteolytic system substrate, was found to be ubiquitinated and degraded when the signal sequence was either generated at an internal location in the protein or fused to the N terminus. An evaluation of the behavior of 3C protease proteins containing mutations in the signal region indicates that considerable variability in the primary structure is tolerated, although the conservation of certain features appears to be required for signal function. Two E3 ubiquitin-protein ligases that recognize the encephalomyocarditis virus 3C protease as a substrate were also partially purified. One of these was identified as the previously described E3alpha, and this was shown to require the destruction signal sequence to catalyze efficiently the ubiquitination of the 3C protease. The other is a Ubc5-dependent E3 that appears to recognize a different, unidentified feature of the 3C protease.

Localization of binding site for encephalomyocarditis virus RNA polymerase in the 3'-noncoding region of the viral RNA.

We previously showed that encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) binds specifically to 3'-terminal segments of EMC virus RNA. This binding, which depends on both the 3'-noncoding region (3'-NCR) and 3'-poly (A) tail [together denoted 3'-NCR(A)], may be an important step in the initiation of virus replication. In this paper, the 3'-NCR and 3'-poly(A) were separately transcribed then mixed, but no complex with 3Dpol was obtained, showing that covalent attachment of the 3'-poly(A) to the 3'-NCR is essential for complex formation. Mutational and deletion analyses localized a critical determinant of 3Dpol binding to a U-rich sequence located 38-49 nucleotides upstream of the 3'-poly(A). Similar analyses led to the identification of a sequence of A residues between positions +10 and +15 of the 3'-poly(A) which are also critical for 3Dpol binding. As U-rich and A-rich regions are important for 3Dpol binding, a speculative model is proposed in which 3Dpol induces and stabilizes the base-pairing of the 3'-poly(A) with the adjacent U-rich sequence to form an unusual pseudoknot structure to which 3Dpol binds with high affinity.

The encephalomyocarditis virus 3C protease is a substrate for the ubiquitin-mediated proteolytic system.

The encephalomyocarditis virus 3C protease has been shown to be rapidly degraded in infected cells and in vitro in rabbit reticulocyte lysate. The in vitro degradation, at least, is accomplished by a virus-independent, ATP-dependent proteolytic system. Here we identify this proteolytic system as the ubiquitin-mediated system. Incubation of the 3C protease in rabbit reticulocyte or cultured mouse cell lysate preparations, alone or in the presence of added ubiquitin or methylated ubiquitin, resulted in the generation of new higher molecular weight species. These new products were shown to be 3C protease-ubiquitin conjugates by their ability to bind antibodies against both the 3C protease and ubiquitin. Supplemental ubiquitin also stimulated the degradation of the 3C protease in these preparations. Large 3C protease-polyubiquitin conjugates were observed to accumulate in reticulocyte lysate in the presence of adenosine 5'-O-(3-thiotriphosphate), an inhibitor of the 26 S multicatalytic protease. This, combined with the fact that the proteolytic activity could be removed from the lysate by sedimentation, implicates the multicatalytic protease in the degradation of the 3C protease-ubiquitin conjugates. It was also found that the slow rate of degradation of a model polyprotein, which resembles the stable viral 3CD diprotein produced in vivo, is likely due to the fact that the polyprotein is a poor substrate for the ubiquitin-conjugating system.

Binding of encephalomyocarditis virus RNA polymerase to the 3'-noncoding region of the viral RNA is specific and requires the 3'-poly(A) tail.

An in vitro RNA bandshift assay has been developed to demonstrate the binding of purified recombinant encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) to the 3'-noncoding region (NCR) and 30 nucleotides of the adjacent 3'-terminal poly(A) tail (3'-NCR(A)) of EMC virus RNA. The binding of 3Dpol to the 3'-NCR(A) fragment was specific since four other unrelated proteins including an RNA polymerase did not bind, and unlabeled 3'-NCR(A), but not alpha-globin mRNA, tRNA, or pure poly(A), competed with radiolabeled 3'-NCR(A) for binding. Surprisingly, 3Dpol failed to bind to the 3'-NCR of EMC virus RNA lacking the poly(A) tail. The results together show that EMC virus RNA template specificity depends only on 3Dpol, the 3'-NCR, and the poly(A) tail. This suggests that 3'-poly(A) is essential for viral RNA template selection by the EMC virus RNA polymerase.

The encephalomyocarditis virus 3C protease is rapidly degraded by an ATP-dependent proteolytic system in reticulocyte lysate.

The encephalomyocarditis virus 3C protease has been observed to undergo rapid degradation, both in vivo in mouse cells and in vitro in reticulocyte lysate. Experiments were carried out to characterize the turnover of the 3C protease in reticulocyte lysate. 3C protease prepared in reticulocyte lysate by in vitro translation and processing of a precursor polyprotein could be separated from the proteolytic activity responsible for its degradation. This implies the 3C protease is not directly involved in its own proteolysis. Active 3C protease flanked by only a few amino acids was degraded at a rate identical to that of a similar protein containing an inactivated catalytic site. This indicates that 3C protease activity is not indirectly required for the proteolytic process. Other viral proteins, including the 3D polymerase and capsid proteins, were relatively stable in the lysate. In addition, polyprotein precursors containing 3C protease with an inactive catalytic site and various flanking proteins displayed distinctly different stabilities. These results suggest that the reticulocyte proteolytic system functions in a selective manner toward the viral proteins. The effects of several proteolytic inhibitors on the lysate proteolytic system were evaluated. The results of these experiments indicate that the rapid degradation of the EMC virus 3C protease requires the hydrolysis of ATP.

Point mutations which drastically affect the polymerization activity of encephalomyocarditis virus RNA-dependent RNA polymerase correspond to the active site of Escherichia coli DNA polymerase I.

The inhibitor sensitivity and functional domains of recombinant encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) have been extensively analyzed. The inhibitor profiles of EMC virus 3Dpol and Escherichia coli DNA-dependent RNA polymerase are distinct, and experiments with substrate analogs indicate that EMC virus 3Dpol lacks reverse transcriptase activity. Twenty amino acid substitutions were engineered in EMC virus 3Dpol based on sequence alignments of viral RNA-dependent RNA polymerases that identified conserved amino acid residues within motifs. Ten out of 17 conservative substitutions within the four most conserved motifs reduced the RNA polymerase activity of the mutants to 0-6% of the activity of the wild-type enzyme, demonstrating the importance of these amino acids in the structure and/or function of EMC virus 3Dpol. Remarkably, 5 of the 10 mutations in EMC virus 3Dpol which had the most drastic effect on its RNA polymerase activity (D240E, S293T, N302Q, G332A, and D333E) were found to correspond to active site residues in E. coli DNA-dependent DNA polymerase I (Klenow). Our results reveal that a basic structural and functional framework is conserved in the most distantly related classes of nucleic acid polymerases and demonstrate the validity of modeling the active site of an RNA-dependent RNA polymerase on the known structure of a DNA polymerase.

Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase.

Encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which allowed easy purification of the fusion protein by affinity chromatography on immobilized glutathione. Inclusion of a thrombin cleavage site between the GST carrier and the viral enzyme facilitated the release of purified mature EMC virus RNA polymerase from the GST carrier by proteolysis with thrombin. The purified recombinant enzyme has a molecular mass of about 52 kDa and is recognized by polyclonal immune serum raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme comigrates with immunoprecipitated EMC virus RNA polymerase from infected mouse L929 cell extracts when run in parallel lanes on a sodium dodecyl sulfate-polyacrylamide gel. The enzyme exhibits rifampin-resistant, poly(A)-dependent poly(U) polymerase activity and RNA polymerase activity, which are both oligo(U) dependent. Template-size products are synthesized in in vitro reactions with EMC virus genomic RNA or globin mRNA. The availability of recombinant EMC virus RNA polymerase in a purified form will allow biochemical analysis of its role in the replication of the virus as well as structure-function studies of this unique class of enzyme.