Translation of Sindbis virus mRNA: effects of sequences downstream of the initiating codon

One incentive for developing the alphavirus Sindbis virus as a vector for the expression of heterologous proteins is the very high level of viral structural proteins that accumulates in infected cells. Although replacement of the structural protein genes by a heterologous gene should lead to an equivalent accumulation of the heterologous protein, the Sindbis virus capsid protein is produced at a level 10- to 20-fold higher than that of any foreign protein. Chimeric mRNAs which contain the first 275 nucleotides of the Sindbis virus 26S mRNA fused to the lacZ gene are also translated at the higher level. The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin-like structure; deletions in this region destroy the activity. These sequences enhance translation in infected cells but have the opposite effect in uninfected cells. Furthermore, translation of this RNA in infected cells is suppressed by a second viral RNA lacking the hairpin-like structure, but translation of the latter RNA is not affected. We propose that the hairpin-like structure presents a barrier to the movement of the ribosomes during translation of mRNA. In infected cells, under conditions in which this mRNA is essentially the only RNA being translated, a slowdown in the transit of the ribosomes gives factors present at low concentrations a chance to bind to the translation complex and permits a high level of functional complexes to be formed. In uninfected cells and in infected cells translating two different viral subgenomic mRNAs, a pause in the movement of the ribosomes along the RNA is no longer an advantage, because the required factors are now usurped by other translation complexes.     

Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells

We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40 degreesC selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.     

Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells

A short model genome RNA and also the genome RNA of influenza A virus bearing both 5'- and 3'-terminal common sequences activated the interferon-induced double-stranded-RNA-dependent protein kinase, PKR, by stimulating autophosphorylation in vitro. The activated PKR catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eIF2alpha). The NS1 protein efficiently eliminated the PKR-activating activity of these RNAs by binding to them. Two mutant NS1 proteins, each harboring a single amino acid substitution at different regions, exhibited temperature sensitivity in their RNA binding activity in the mutant virus-infected cell lysates as well as when they were prepared as fusion proteins expressed in bacteria. The virus strains carrying these mutant NS1 proteins exhibited temperature sensitivity in virus protein synthesis at the translational level, as reported previously, and could not repress the autophosphorylation of PKR developing during the virus growth, which is normally suppressed by a viral function(s). As a result, the level of eIF2alpha phosphorylation was elevated 2.5- to 3-fold. The defect in virus protein synthesis was well correlated with the level of phosphorylation of PKR and eIF2alpha.     

Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.     

Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions

During translation of bacteriophage T4 gene 60 mRNA, ribosomes bypass 50 nucleotides with high efficiency. One of the mRNA signals for bypass is a stem-loop in the first part of the coding gap. When the length of this stem-loop is extended by 36 nucleotides, bypass is reduced to 0.35% of the wild-type level. Bypass is partially restored by a mutation in the C-terminal domain of Escherichia coli large ribosomal subunit protein L9. Previous work has shown that L9 is an elongated protein with an alpha-helix that connects and orients the N and C-terminal domains that both contain a predicted RNA binding site. We have determined two binding sites of L9 on 23 S rRNA. A 778 nucleotide RNA fragment encompassing domain V (nucleotides 1999 to 2776) of the 23 S rRNA is retained on filters by L9 and contains both sites. The N and C-terminal domains of L9 were shown to interact with nucleotides just 5' to nucleotide 2231 and 2179 of the 23 S rRNA, respectively, using the toeprint assay. These L9 binding sites on 23 S rRNA suggest that L9 functions as a brace across helix 76 to position helices 77 and 78 relative to the peptidyl transferase center. In this study, bypass on a mutant gene 60 mRNA has been used as an assay to probe the importance of particular L9 amino acids for function. Amino acid substitutions in the C-terminal domain are shown to partially restore bypass. These mutant L9 proteins have reduced binding to a 23 S rRNA fragment (nucleotides 1999 to 2274) containing domain V, to which L9 binds. They partially retain both the N and C-terminal domain interactions. On the other hand, substitutions of amino acids in the N-terminal domain, which greatly reduce RNA binding, do not restore bypass. The latter mutants have completely lost the N-terminal domain interaction. Addition of an amino acid to the alpha-helix also restores gene 60 bypass. RNA binding by this mutant is similar to that observed for the C-terminal domain mutants that partially restore bypass.     

Yeast MAK3 N-acetyltransferase recognizes the N-terminal four amino acids of the major coat protein (gag) of the L-A double-stranded RNA virus

The MAK3 gene of Saccharomyces cerevisiae encodes an N-acetyltransferase whose acetylation of the N terminus of the L-A double-stranded RNA virus major coat protein (gag) is necessary for viral assembly. We show that the first 4 amino acids of the L-A gag protein sequence, MLRF, are a portable signal for N-terminal acetylation by MAK3. Amino acids 2, 3, and 4 are each important for acetylation by the MAK3 enzyme. In yeast cells, only three mitochondrial proteins are known to have the MAK3 acetylation signal, suggesting an explanation for the slow growth of mak3 mutants on nonfermentable carbon sources.     

Formation of a Sindbis virus nonstructural protein and its relation of 42S mRNA function

Chicken embryo fibroblasts infected with an RNA- temperature-sensitive mutant (ts24) of Sindbis virus accumulated a large-molecular-weight protein (p200) when cells were shifted from the permissive to nonpermissive temperature. Appearance of p200 was accompanied by a decrease in the synthesis of viral structural proteins, but [35S]methionine tryptic peptides from p200 were different from those derived from a 140,000-molecular-weight polypeptide that contains the amino acid sequences of viral structural proteins. Among three other RNA- ts mutants that were tested for p200 formation, only one (ts21) produced this protein. The accumulation of p200 in ts24- and ts21-infected cells could be correlated with a shift in the formation of 42S and 26S viral RNA that led to an increase in the relative amounts of 42S RNA. These data indicate that p200 is translated from the nonstructural genes of the virion 42S RNA and further suggest that this RNA does not function effectively in vivo as an mRNA for the Sindbis virus structural proteins.     

Structure and function of influenza virus NS1 and NS2 proteins

This review discusses the structure and function of the influenza virus NS1 and NS2 proteins. The NS1 is a phosphoprotein and has two nuclear localization signals. In the nucleus, the NS1 interferes with the splicing as well as the nuclear export of cellular mRNAs. In the later time of the infection, the NS1 is present in the cytoplasm and associates with the polysomes. The NS1 binds to the 5'UTR of some viral mRNAs and stimulates translation. The NS2 is a phosphoprotein and binds to the nucleoporin yRip1 and Rab/hRip as well as the M1 protein which associates with the vRNPs. Therefore, the NS2 protein plays an important role in the nuclear export of the vRNPs. Improved technique to genetically manipulate influenza virus allowed us to rescue NS1 and NS2 mutants which are useful for further study.     

Nuclear import and export of influenza virus nucleoprotein

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR)

Upon binding to double-stranded (ds) RNA, the dsRNA-dependent protein kinase (PKR) sequentially undergoes autophosphorylation and activation. Activated PKR may exist as a dimer and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (cIF-2 alpha) to inhibit polypeptide chain initiation. Transfection of COS-1 cells with a plasmid cDNA expression vector encoding a marker gene, activates endogenous PKR, and selectively inhibits translation of the marker mRNA, dihydrofolate reductase (DHFR). This system was used to study the dsRNA binding and dimerization requirements for over-expressed PKR mutants and subdomains to affect DHFR translation. DHFR translation was rescued by expression of either an ATP hydrolysis defective mutant PKR K296P, the amino-terminal 1-243 fragment containing two dsRNA binding motifs, or the isolated first RNA binding motif (amino acids 1-123). Mutation of K64E within the dsRNA binding motif 1 destroyed dsRNA binding and the ability to rescue DHFR translation. Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell lysates detected interaction between intact PKR and the amino-terminal 1-243 fragment as well as a 1-243 fragment harboring the K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of PKR activity, did not disrupt this interaction. In contrast, intact PKR did not interact with fragments containing the first dsRNA binding motif (1-123), the second dsRNA binding motif (98-243), or the isolated PKR kinase catalytic domain (228-551). These results demonstrate that the translational stimulation mediated by the dominant negative PKR mutant does not require dimerization, but requires the ability to bind dsRNA and indicate these mutants act by competition for binding to activators.     

RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA-binding sites. The 5' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5'-capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like motif, amino acids 249-256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA-chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.     

Effects of milk and milk products on rectal mucosal cell proliferation in humans

The interferon (IFN)-induced protein kinase (PKR) functions as a gatekeeper of mRNA translation initiation and is, therefore, a key mediator of the host IFN-induced antiviral defense system. Many viruses have invested countermeasures against PKR. Some apparently use more than one mechanism. The influenza virus can repress PKR activity through the use of at least two factors, the cellular P58IPK protein and the viral NS1 protein. The exact mode of action of the latter has not been established. Here, using a coprecipitation assay, we found that PKR could form a complex with NS1 in crude cell extracts prepared from influenza virus-infected HeLa cells. The NS1-PKR interaction was verified by using the yeast two-hybrid system and an in vitro binding assay. Deletion analysis mapped the NS1 binding site to the N-terminal 98 residues of PKR regulatory region. Furthermore, an NS1 mutant, which lacks PKR inhibitory activity, did not bind PKR. Finally, the functional role of NS1 in PKR inhibition was substantiated using an in vivo assay for PKR activity. These results support the role of NS1 in PKR modulation during viral infection that is mediated through a complex formation between the two proteins.     

Mutation of either of two cysteine residues or deletion of the amino or carboxy terminus of nonstructural protein NS1 of bluetongue virus abrogates virus-specified tubule formation in insect cells

Virus-specific tubules are characteristic of orbivirus infections and are likely to play an important role in virus morphogenesis. It has been shown that for bluetongue virus (BTV), the prototype orbivirus in the family Reoviridae, the virus-encoded NS1 protein forms tubules in insect cells when the BTV segment M6 gene is expressed by using a baculovirus vector. To understand the function of NS1 tubules and to identify the sequences involved in their polymerization, a series of mutant NS1 genes was generated and expressed in insect cell cultures by using baculovirus vectors. Three of the mutants were deletion mutants. One (AcNS1.dNT10) lacked 10 of the amino-terminal amino acids, and the other two mutants (AcNS1.dCT20 and AcNS1.dCT43) lacked 20 or 43 of the carboxy-terminal amino acids. In addition, site-directed mutants were constructed in which various single cysteines or pairs of cysteines were changed to serines. The ability of each mutant protein to form tubules was investigated. None of the deletion mutants formed tubules. The constructs in which the cysteines at amino acid positions 337 and/or 340 were replaced by serines (e.g., AcNS1.C337S,C340S) also did not form tubules. Instead, the NS1 protein of these and the deletion mutants made ribbon-like structures which formed large aggregates. Mutations involving six other cysteines (i.e., AcNS1.C37S,C43S,AcNS1.C462S,C465S, AcNS1.C104S, and AcNS1.C364S) produced tubules. The results show that both the amino and carboxy termini of the NS1 protein molecule and the cysteines at residues 337 and 340 are essential for tubule formation.     

Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses

Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.     

Synergistic stimulation of HIV-1 rev-dependent export of unspliced mRNA to the cytoplasm by hnRNP A1

The structural and accessory proteins of human immunodeficiency virus type 1 are expressed by unspliced or partially spliced mRNAs. Efficient transport of these mRNAs from the nucleus requires the binding of the viral nuclear transport protein Rev to an RNA stem-loop structure called the RRE (Rev response element). However, the RRE does not permit Rev to stimulate the export of unspliced mRNAs from the efficiently spliced beta-globin gene in the absence of additional cis-acting RNA regulatory signals. The p17gag gene instability (INS) element contains RNA elements that can complement Rev activity. In the presence of the INS element and the RRE, Rev permits up to 30 % of the total beta-globin mRNA to be exported to the cytoplasm as unspliced mRNA. Here, we show that a minimal sequence of 30 nt derived from the 5' end of the p17 gag gene INS element (5' INS) is functional and permits the export to the cytoplasm of 14% of the total beta-globin mRNA as unspliced pre-mRNA. Gel mobility shift assays and UV cross-linking experiments have shown that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and a cellular RNA-binding protein of 50 kDa form a complex on the 5' INS. Mutants in the 5' INS that prevent hnRNP A1 and 50 kDa protein binding are inactive in the transport assay. To confirm that the hnRNP A1 complex is responsible for INS activity, a synthetic high-affinity binding site for hnRNP A1 was also analysed. When the high affinity hnRNP A1 binding site was inserted into the beta-globin reporter, Rev was able to increase the cytoplasmic levels of unspliced mRNAs to 14%. In contrast, the mutant hnRNP A1 binding site, or binding sites for hnRNP C and L are unable to stimulate Rev-mediated RNA transport. We conclude that hnRNP A1 is able to direct unspliced globin pre-mRNA into a nuclear compartment where it is recognised by Rev and then transported to the cytoplasm.     

Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of Borna disease virus

Borna disease virus (BDV) is a nonsegmented negative-strand (NNS) RNA virus that is unusual because it replicates in the nucleus. The most abundant viral protein in infected cells is a 38/39-kDa doublet that is presumed to represent the nucleocapsid. Infectious particles also contain high levels of this protein, accounting for at least 50% of the viral proteins. The two forms of the protein differ by an additional 13 amino acids that are present at the amino terminus of the 39-kDa form and missing from the 38-kDa form. To examine whether this difference in amino acid content affects the localization of this protein in cells, the 39- and 38-kDa proteins were expressed in transfected cells. The 39-kDa form was concentrated in the nucleus, whereas the 38-kDa form was found in both the nucleus and cytoplasm. Inspection of the extra 13 amino acids present in the 39-kDa form revealed a sequence (Pro-Lys-Arg-Arg) that is very similar to the nuclear localization signals (in both sequence homology and amino-terminal location) of the VP1 proteins of simian virus 40 and polyomavirus. Primer extension analysis of total RNA from infected cells suggests that there are two mRNA species encoding the two forms of the nucleocapsid protein. In infected cells, the 39-kDa form is expressed at about twofold-higher levels than the 38-kDa form at both the RNA and protein levels. The novel nuclear localization of the 39-kDa nucleocapsid-like protein suggests that this form of the protein is targeted to the nucleus, the site for viral RNA replication, and that it may associate with genomic RNA.     

NS1-Binding protein (NS1-BP): a novel human protein that interacts with the influenza A virus nonstructural NS1 protein is relocalized in the nuclei of infected cells

We used the yeast interaction trap system to identify a novel human 70-kDa protein, termed NS1-binding protein (NS1-BP), which interacts with the nonstructural NS1 protein of the influenza A virus. The genetic interaction was confirmed by the specific coprecipitation of the NS1 protein from solution by a glutathione S-transferase-NS1-BP fusion protein and glutathione-Sepharose. NS1-BP contains an N-terminal BTB/POZ domain and five kelch-like tandem repeat elements of approximately 50 amino acids. In noninfected cells, affinity-purified antibodies localized NS1-BP in nuclear regions enriched with the spliceosome assembly factor SC35, suggesting an association of NS1-BP with the cellular splicing apparatus. In influenza A virus-infected cells, NS1-BP relocalized throughout the nucleoplasm and appeared distinct from the SC35 domains, which suggests that NS1-BP function may be disturbed or altered. The addition of a truncated NS1-BP mutant protein to a HeLa cell nuclear extract efficiently inhibited pre-mRNA splicing but not spliceosome assembly. This result could be explained by a possible dominant-negative effect of the NS1-BP mutant protein and suggests a role of the wild-type NS1-BP in promoting pre-mRNA splicing. These data suggest that the inhibition of splicing by the NS1 protein may be mediated by binding to NS1-BP.     

The economics of treating tibia fractures. The cost of delayed unions

We cloned a gene (topA) encoding DNA topoisomerase II from Dictyostelium discoideum nuclear DNA using oligo probes corresponding to the consensus amino acid sequences found in the gene in other eukaryotes. The gene encoding a predicted polypeptide of 1282 amino acids with M(r) of about 146 kDa, is a single copy that is expressed as a polyadenylated 4.5 kb RNA. The predicted amino acid sequence shares similarity with those of other eukaryotes with identity between 32 and 46%. The protein is 260-300 amino acids shorter in the C-terminal region and 50-80 longer in the N-terminal region than those of other eukaryotes. In TopA of D. discoideum, the N-terminal region with stretches of charged and hydrophilic amino acids is predicted to fold into an amphiphilic alpha-helix which is characteristic of a mitochondrial targeting signal presequence. Four independent polyclonal antibodies against bacterially expressed GST fusion proteins containing four portions of the polypeptide detected a single band on Western blots at about 135 kDa. Western blots analysis of subcellular fractions revealed that this protein is localized in mitochondria. The protein and the mRNA are present in growth phase and during development, although levels of both declined as development proceeded.