Functional characterization of cleavage-defective mutants of encephalomyocarditis virus.

We characterized seven temperature-sensitive capsid cleavage (cleavage-defective) mutants of encephalomyocarditis virus. Our experimental approach was to monitor in vitro proteolysis reactions of either wild-type or cleavage-defective mutant capsid precursors mixed with cell-free translation products (containing the viral protease) of either wild-type or mutant viral RNA. The cell-free translation reactions and in vitro proteolysis reactions were done at 38 degrees C, because at this temperature cleavage of the capsid precursors was restricted in reactions containing cleavage-defective mutant viral RNA as the message, relative to those reactions containing wild-type viral RNA as the message. Wild-type or cleavage-defective mutant capsid precursors were prepared by adding cycloheximide to cell-free translation reactions primed with wild-type or mutant viral RNA, respectively, 12 min after the initiation of translation. In vitro proteolysis of wild-type capsid precursors with cell-free translation products of either wild-type or cleavage-defective mutant viral RNA led to similar products at 38 degrees C, indicating that the cleavage-defective mutant viral protease was not temperature sensitive. As a corollary to this, at 38 degrees C cleavage-defective mutant capsid precursors were not cleaved as completely as were wild-type capsid precursors by products of cell-free translation of wild-type viral RNA. The results from these in vitro proteolysis experiments indicate that all seven of the cleavage-defective mutants have capsid precursors with a temperature-sensitive configuration.     

Cowpea Mosaic Virus-Encoded Protease Does Not Recognize Primary Translation Products of M RNAs from Other Comoviruses

The protease encoded by the large (B) RNA segment of cowpea mosaic virus was tested for its ability to recognize the in vitro translation products of the small (M) RNA segment from the comoviruses squash mosaic virus, red clover mottle virus, and cowpea severe mosaic virus (CPsMV, strains Dg and Ark), and from the nepovirus tomato black ring virus. Like M RNA from cowpea mosaic virus, the M RNAs from squash mosaic virus, red clover mottle virus, CPsMV-Dg, and CPsMV-Ark were all translated into two large polypeptides with apparent molecular weights which were different for each virus and even for the two CPsMV strains. Neither the in vitro products from squash mosaic virus, red clover mottle virus, and CPsMV M RNAs nor the in vitro product from tomato black ring virus RNA-2 were processed by the cowpea mosaic virus-encoded protease, indicating that the activity of this enzyme is highly specific.     

Rubella virus nonstructural protein protease domains involved in trans- and cis-cleavage activities

Rubella virus (RV) genomic RNA contains two large open reading frames (ORFs): a 5'-proximal ORF encoding nonstructural proteins (NSPs) that function primarily in viral RNA replication and a 3'-proximal ORF encoding the viral structural proteins. Proteolytic processing of the RV NSP ORF translation product p200 is essential for viral replication. Processing of p200 to two mature products (p150 and p90) in the order NH(2)-p150-p90-COOH is carried out by an RV-encoded protease residing in the C-terminal region of p150. The RV nonstructural protease (NS-pro) belongs to a viral papain-like protease family that cleaves the polyprotein both in trans and in cis. A conserved X domain of unknown function was found from previous sequence analysis to be associated with NS-pro. To define the domains responsible for cis- and trans-cleavage activities and the function of the X domain in terms of protease activity, an in vitro translation system was employed. We demonstrated that the NSP region from residue 920 to 1296 is necessary for trans-cleavage activity. The domain from residue 920 to 1020 is not required for cis-cleavage activity. The X domain located between residues 834 and 940, outside the regions responsible for both cis- and trans-cleavage activities of NS-pro, was found to be important for NS-pro trans-cleavage activity but not for cis-cleavage activity. Analysis of sequence homology and secondary structure of the RV NS-pro catalytic region reveals a folding structure similar to that of papain.     

Identification of a Packaged Cellular mRNA in Virions of Rous Sarcoma Virus

A novel messenger activity has been identified by in vitro translation of the 70S virion RNAs of a variety of avian leukosis and avian sarcoma viruses. When the 70S virion RNA complex was heat dissociated and the polyadenylated RNA was fractionated on neutral sucrose gradients, a polypeptide of 34,000 daltons (34K) was observed in the translation products of 18S polyadenylic acid-containing virion RNA. Aside from the p60(src)-related subgenomic messenger activities, this was the only prominent messenger activity that sedimented at <20S. It was determined that the 34K protein was not virally coded because (i) messenger activity for the 34K protein was not generated by mild alkaline hydrolysis of 35S genomic RNA, (ii) the 34K proteins synthesized in response to different virion RNAs had identical tryptic peptide maps, and (iii) the tryptic peptide map of the 34K protein coded for by virion RNA was identical to that of a major in vitro translation product of 34,000 daltons made from 18S uninfected chick cell polyadenylated RNA. The 18S RNA was shown to be contained within virion particles, rather than part of a cellular structure copurifying with virus preparations, by demonstrating the presence of 34K messenger activity in virion cores made from detergent-disrupted virus. This cellular mRNA, however, was not observed in the virion RNAs of Rous-associated virus types 0 and 2 avian leukosis viruses and therefore is not packaged by all avian retroviruses. Since no other cellular message has been detected by this assay, it seems likely that the 34K mRNA found in 70S virion RNA is the result of selective packaging of an abundant host cell mRNA by certain avian retroviruses.     

A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects.

Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells.     

Translation of Sindbis virus 26S mRNA does not require intact eukariotic initiation factor 4G

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.     

Cell-free translation of exogenous mRNA in extracts from dry pea primary axes

Extracts from the primary axes of dry pea (Pisum sativum L.) seeds are able to perform an initiation-dependent translation of exogenous mRNA. SDS polyacrylamide gel electrophoresis of the products synthesized under direction of alfalfa mosaic virus RNA (AMV-RNA) and tobacco mosaic virus RNA (TMV-RNA) shows that the fidelity of translation in this pea system is at least as high as in a wheat embryo cell-free protein synthesizing system. The endogenous messengers are also efficiently translated in extracts from the primary axes of pea seeds. The direct translation of these messengers in a homologous cell-free system may be of interest for a study of the products coded for by the long-lived messengers present in this plant.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - SDS sodium dodecyl sulphate - mRNP messenger ribonucleoprotein - AMV-RNA alfalfa mosaic virus RNA - TMV-RNA tobacco mosaic virus RNA - ATA aurin tricarboxylic acid - TCA trichloroacetic acid - S.A. specific activity     

Removal of the genome-linked protein of foot-and-mouth disease virus by rabbit reticulocyte lysate.

Rabbit reticulocyte lysate cleaves the genome-linked protein VPg from foot-and-mouth disease virus (FMDV) RNA. This activity could be reliably monitored since removal of the protein resulted in a change in migration in polyacrylamide gels of the small specific 5' and fragment of the RNA (S fragment). The unlinking activity cleaved the bond between the tyrosine residue of VPg and the RNA to leave a 5' phosphate on the RNA. The 5' sequence of the RNA from which VPg had been removed by rabbit reticulocyte lysate was the same as that of FMDV mRNA isolated from infected cells. VPg released from the RNA was rapidly degraded by the rabbit reticulocyte lysate to material which eluted with the inclusion volume of a Sepharose 6B column and partitioned to the aqueous phase during phenol extraction. The unlinking activity was inhibited by heating the lysate to 56 degrees C, by sodium dodecyl sulfate (SDS), EDTA, and Zn2+ ions but was unaffected by reducing agents, a translation inhibitor, and a number of protease and RNase inhibitors.     

Characterization of ribosomal frameshifting for expression of pol gene products of human T-cell leukemia virus type I.

For study of the pol gene expression of human T-cell leukemia virus type I (HTLV-I), RNA was transcribed in vitro from proviral DNA and translated in rabbit reticulocyte lysates. This cell-free translation resulted in two major translation products representing the Gag and Gag-Pro polyproteins. By contrast, the Gag-Pro-Pol polyprotein could be readily observed only when translation was performed with mutant mRNA in which the protease (pro) reading frame was aligned to gag to eliminate the frameshifting event in the gag-pro overlap. The results indicated that two independent ribosomal frameshifting events are required for expression of the HTLV-I pol gene product. Studies with mutant DNAs facilitated the characterization of the primary structure of the HTLV-I mRNA responsible for the ribosomal frameshift in the pro-pol overlap and demonstrated that the frameshift occurs at the signal sequence UUUAAAC. Direct amino acid sequencing of the transframe protein localized the site of the frameshift to the asparagine codon AAC.     

A phylogenetically conserved RNA structure in the poliovirus open reading frame inhibits the antiviral endoribonuclease RNase L

RNase L is an antiviral endoribonuclease that cleaves viral mRNAs after single-stranded UA and UU dinucleotides. Poliovirus (PV) mRNA is surprisingly resistant to cleavage by RNase L due to an RNA structure in the 3C(Pro) open reading frame (ORF). The RNA structure associated with the inhibition of RNase L is phylogenetically conserved in group C enteroviruses, including PV type 1 (PV1), PV2, PV3, coxsackie A virus 11 (CAV11), CAV13, CAV17, CAV20, CAV21, and CAV24. The RNA structure is not present in other human enteroviruses (group A, B, or D enteroviruses). Coxsackievirus B3 mRNA and hepatitis C virus mRNA were fully sensitive to cleavage by RNase L. HeLa cells expressing either wild-type RNase L or a dominant-negative mutant RNase L were used to examine the effects of RNase L on PV replication. PV replication was not inhibited by RNase L activity, but rRNA cleavage characteristic of RNase L activity was detected late during the course of PV infection, after assembly of intracellular virus. Rather than inhibiting PV replication, RNase L activity was associated with larger plaques and better cell-to-cell spread. Mutations in the RNA structure associated with the inhibition of RNase L did not affect the magnitude of PV replication in HeLa cells expressing RNase L, consistent with the absence of observed RNase L activity until after virus assembly. Thus, PV carries an RNA structure in the 3C protease ORF that potently inhibits the endonuclease activity of RNase L, but this RNA structure does not prevent RNase L activity late during the course of infection, as virus assembly nears completion.     

Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.     

Features of the autonomous function of the translational enhancer domain of satellite tobacco necrosis virus.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a cap and a poly(A) tail. Translation of STNV RNA in vitro is promoted by a 120-nt translational enhancer domain (TED) in the 3'-untranslated region. TED also stimulates translation of heterologous mRNAs. In this study, we show that TED stimulates translation of a cat mRNA by increasing translation efficiency to the level of capped mRNA. This stimulatory activity is not impaired by translation through TED. TED stimulates translation efficiency from different positions within the mRNA, varying from the 5' end to 940 nt downstream of the coding region. Duplication of TED has an additive effect on translation stimulation only when located at both ends of the mRNA. On dicistronic RNAs, TED stimulates translation of both cistrons to the same extent. These data suggest that TED acts primarily by recruiting the translational machinery to the RNA.     

Preparation and characteristics of a wheat embryo cell-free extract active in the synthesis of high molecular weight viral polypeptides

Wheat embryo extracts are commonly used in cell-free protein synthesis studies. Soluble inhibitor(s) that diminish aminoacylation of tRNA exist in such extracts. These inhibitors can be effectively removed by prolonged dialysis, provided that proper salt conditions are maintained to ensure stability of the extract. The system, when only partially depleted of inhibitors, does not effectively support translation of certain classes of messengers, i.e., brome mosaic virus RNA 1 and RNA 2. All four proteins expected as the products of translation of BMV RNA, including peptides of molecular weight approaching 120,000, are formed in the fully active system. At limiting concentrations of template, the fully activated system incorporates the following amounts of [¹⁴C]leucine per microgram of template: Qβ RNA, 4.5 pmol; reovirus mRNA, 200 pmol, tobacco mosaic virus RNA, 227 pmol, and brome mosaic virus, up to 350 pmol.     

Cell-free translation of purified virion-associated high-molecular-weight RNA synthesized in vitro by vaccinia virus.

Virion-associated high-molecular-weight (HMW) RNA synthesized in vitro by purified vaccinia virus particles has been translated in a wheat germ cell-free protein synthesizing system. Purified HMW RNA directs the synthesis of translation products which are identical to the translation products made in response to in vitro-synthesized, virion-released 8 to 12S mRNA. The translation of HMW RNA proceeds exclusively through a 5'-terminal cap-mediated initiation step. Furthermore, only one coding sequence is translated per HMW RNA molecule, and that sequence is probably located near the 5' end of the molecule. These conclusions are based on the following results. (i) Sodium dodecyl sulfate--polyacrylamide gel electrophoresis patterns of translation products synthesized in response to HMW RNA and in response to 8 to 12S mRNA were qualitatively identical. (ii) On an equal weight basis, HMW RNA was 25 to 30% as active as 8 to 12S mRNA in stimulating in vitro protein synthesis. (iii) Unmethylated HMW RNA was translated at 10% the efficiency of the methylated form of this RNA. (iv) m7pG inhibited the translation of fully methylated HMW RNA by 90%. (v) After the initiation step of translation was blocked by aurintricarboxylic acid, the rate with which amino acids were incorporated into individual polypeptides decreased in a similar manner for the translation of both HMW RNA and 8 to 12S mRNA. Virion-released 8 to 12S mRNA derived from virion-associated HMW RNA during a chase in the presence of ATP, GTP, and S-adenosylmethionine was also translated. At low RNA concentrations, the derived RNA appeared to stimulate amino acid incorporation more efficiently than the HMW RNA precursor. However, at higher concentrations of this RNA, protein synthesis was severely inhibited.     

Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus.

Poliovirus RNA polymerase and infectious virus particles were synthesized by translation of virion RNA in vitro in HeLa S10 extracts. The in vitro translation reactions were optimized for the synthesis of the viral proteins found in infected cells and in particular the synthesis of the viral polymerase 3Dpol. There was a linear increase in the amount of labeled protein synthesized during the first 6 h of the reaction. The appearance of 3Dpol in the translation products was delayed because of the additional time required for the proteolytic processing of precursor proteins. 3Dpol was first observed at 1 h in polyacrylamide gels, with significant amounts being detected at 6 h and later. Initial attempts to assay for polymerase activity directly in the translation reaction were not successful. Polymerase activity, however, was easily detected by adding a small amount (3 microliters) of translation products to a standard polymerase assay containing poliovirion RNA. Full-length minus-strand RNA was synthesized in the presence of an oligo(U) primer. In the absence of oligo(U), product RNA about twice the size of virion RNA was synthesized in these reactions. RNA stability studies and plaque assays indicated that a significant fraction of the input virion RNA in the translation reactions was very stable and remained intact for 20 h or more. Plaque assays indicated that infectious virus was synthesized in the in vitro translation reactions. Under optimal conditions, the titer of infectious virus produced in the in vitro translation reactions was greater than 100,000 PFU/ml. Virus was first detected at 6 h and increased to maximum levels by 12 h. Overall, the kinetics of poliovirus replication (protein synthesis, polymerase activity, and virus production) observed in the HeLa S10-initiation factor in vitro translation reactions were similar to those observed in infected cells.