Virus nsP2 Research Articles

Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2

The C-terminal region of Venezuelan equine encephalitis virus (VEEV) nsP2 is responsible for proteolytic processing of the VEEV polyprotein replication complex. This action regulates the activity of the replication complex and is essential for viral replication, thus making nsP2 a very attractive target for development of VEEV therapeutics. The 338-amino-acid C-terminal region of VEEV nsP2 has been overexpressed in Escherichia coli, purified and crystallized. Crystals diffract to beyond 2.5 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1). Isomorphous heavy-atom derivatives suitable for phase analysis have been obtained and work on building a complete structural model is under way.

Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis

The function of Semliki Forest Virus nsP2 protease was investigated by site-directed mutagenesis. Mutations were introduced in its protease domain, Pro39, and the mutated proteins were expressed in Escherichia coli, purified and their activity in vitro was compared to that of the wild type Pro39. Mutations M781T, A662T and G577R, found in temperature-sensitive virus strains, rendered the enzyme temperature-sensitive in vitro as well. Five conserved residues were required for the proteolytic activity of Pro39. Changes affecting Cys 478, His 548, and Trp 549 resulted in complete inactivation of the enzyme, whereas the replacements N600D and N605D significantly impaired its activity. The importance of Trp 549 for the proteolytic cleavage specificity is discussed and a new structural motif involved in substrate recognition by cysteine proteases is proposed.

Phage display of the Equine arteritis virus nsp1 ZF domain and examination of its metal interactions

Phage display of the Equine arteritis virus nsp1 ZF domain and examination of its metal interactions

Identification of the amino acid sequence in Sindbis virus nsP4 that binds to the promoter for the synthesis of the subgenomic RNA.

A gel mobility-shift assay was used to demonstrate the binding of the Sindbis virus transcriptase to the promoter for the synthesis of subgenomic (SG) RNA. The assay made use of a P15 fraction (the cell fraction that is pelleted at 15,000 x g) from cells infected with recombinant vaccinia virions expressing various Sindbis virus nonstructural proteins (nsPs) and a (32)P-labeled 24-mer oligoribonucleotide representing the minimal sequence with SG promoter activity. By itself, nsP4, the viral RNA-dependent RNA polymerase, did not bind to the SG promoter; rather, all four nsPs were required for the binding of the transcriptase to the promoter. UV crosslinking of the transcriptase to a thiouridine-containing SG promoter, followed by V8 protease digestion of the complex, generated a peptide fragment that was bound to the SG promoter. This peptide fragment contained a sequence that corresponded to residues 329-334 of nsP4. This peptide may be in the fingers domain of nsP4. The peptide that was identified contained Arg residues at positions 331 and 332. Another Arg is present at position 327. By changing each of the Arg residues to Ala, we demonstrated that only the Arg residues at positions 331 and 332 were required for binding nsP4 to the SG promoter.

Phage display of the Equine arteritis virus nsp1 ZF domain and examination of its metal interactions

A putative zinc finger (ZF) domain in the Equine arteritis virus (EAV) nsp1 protein was described recently to be required for viral transcription. The nsp1 ZF (50 aa) was expressed on the surface of M13KE gIII phage, fused to the N terminus of the phage pIII protein. To evaluate the functionality of the ZF domain, a binding assay was developed, based on the use of immobilized Ni 2+ ions (Ni-NTA). Phages displaying ZF bound significantly better to Ni-NTA than did phages displaying negative-control peptides, which also contained metal-coordinating residues. Also, binding of ZF-displaying phages could be inhibited by an anti-nsp1 serum, or by mutation of residues predicted to be important for zinc coordination. Finally, binding was abolished by low concentrations (0.1%) Tween 20, and rescued by including Zn 2+, Ni 2+ or Cu 2+, but not Mg 2+, in the binding buffer, suggesting that formation of secondary structure was involved in binding of the ZF to Ni-NTA. These findings provide the first experimental evidence that the putative nsp1 ZF domain can coordinate divalent metal ions, and that this property is associated with the secondary structure of the domain. The Ni-NTA binding assay developed in the present study may have general applications in the study of other ZF domains.

Modification of Asn374 of nsP1 Suppresses a Sindbis Virus nsP4 Minus-Strand Polymerase Mutant

Our recent study (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76:8632-8640, 2002) found minus-strand synthesis to be temperature sensitive in vertebrate and invertebrate cells when the Arg183 residue of the Sindbis virus nsP4 polymerase was changed to Ser, Ala, or Lys. Here we report the results of studies identifying an interacting partner of the region of the viral polymerase containing Arg183 that suppresses the Ser183 codon mutation. Large-plaque revertants were observed readily following growth of the nsP4 Ser183 mutant at 40 degrees C. Fifteen revertants were characterized, and all had a mutation in the Asn374 codon of nsP1 that changed it to either a His or an Ile codon. When combined with nsP4 Ser183, substitution of either His374 or Ile374 for Asn374 restored wild-type growth in chicken embryo fibroblast (CEF) cells at 40 degrees C. In Aedes albopictus cells at 34.5 degrees C, neither nsP1 substitution suppressed the nsP4 Ser183 defect in minus-strand synthesis. This argued that the nsP4 Arg183 residue itself is needed for minus-strand replicase assembly or function in the mosquito environment. The nsP1 His374 suppressor when combined with the wild-type nsP4 gave greater than wild-type levels of viral RNA synthesis in CEF cells at 40 degrees C ( approximately 140%) and in Aedes cells at 34.5 degrees C (200%). Virus producing nsP1 His374 and wild-type nsP4 Arg183 made more minus strands during the early period of infection and before minus-strand synthesis ceased at about 4 h postinfection. Shirako et al. (Y. Shirako, E. G. Strauss, and J. H. Strauss, Virology 276:148-160, 2000) identified amino acid substitutions in nsP1 and nsP4 that suppressed mutations that changed the N-terminal Tyr of nsP4. The nsP4 N-terminal mutants were defective also in minus-strand synthesis. Our study implicates an interaction between another conserved nsP1 region and an internal region, predicted to be in the finger domain, of nsP4 for the formation or activity of the minus-strand polymerase. Finally, the observation that a single point mutation in nsP1 results in minus-strand synthesis at greater than wild-type levels supports the concept that the wild-type nsP sequences are evolutionary compromises.

Elimination of Phosphorylation Sites of Semliki Forest Virus Replicase Protein nsP3

nsP3 is one of the four RNA replicase subunits encoded by alphaviruses. The specific essential functions of nsP3 remain unknown, but it is known to be phosphorylated on serine and threonine residues. Here we have completed mapping of the individual phosphorylation sites on Semliki Forest virus nsP3 (482 amino acids) by point mutational analysis of threonine residues. This showed that threonines 344 and 345 represented the major threonine phosphorylation sites in nsP3. Experiments with deletion variants suggested that nsP3 itself had no kinase activity; instead, it was likely to be phosphorylated by multiple cellular kinases. Phosphorylation was not necessary for the peripheral membrane association of nsP3, which was mediated by the N-terminal region preceding the phosphorylation sites. Two deletion variants of nsP3 with either reduced or undetectable phosphorylation were studied in the context of virus infection. Cells infected with mutant viruses produced close to wild type levels of infectious virions; however, the rate of viral RNA synthesis was significantly reduced in the mutants. A virus totally defective in nsP3 phosphorylation and exhibiting a decreased rate of RNA synthesis also exhibited greatly reduced pathogenicity in mice.

Phosphorylation Site Analysis of Semliki Forest Virus Nonstructural Protein 3

Nonstructural protein 3 (Nsp3) is an essential subunit of the alphavirus RNA replication complex, although its specific function(s) has yet to be well defined. Previously, it has been shown that Semliki Forest virus Nsp3 (482 amino acids) is a phosphoprotein, and, in the present study, we have mapped its major phosphorylation sites. Mass spectrometric methods utilized included precursor ion scanning, matrix-assisted laser desorption/ionization mass spectrometry used in conjunction with on-target alkaline phosphatase digestions, and tandem mass spectrometry. Two-dimensional peptide mapping was applied to separate tryptic (32)P-labeled phosphopeptides of Nsp3. Radiolabeled peptides were then subjected to Edman sequencing, and phosphoamino acid analysis. In addition, radiolabeled Nsp3 was cleaved successively with cyanogen bromide and trypsin, and microscale iron-chelate affinity chromatography was used to enrich phosphopeptides. By combining these methods, we showed that Nsp3 is phosphorylated on serine residues 320, 327, 332, 335, 356, 359, 362, and 367, and is heavily phosphorylated on peptide Gly(338)-Lys(415), which carries 7-12 phosphates distributed over its 13 potential phosphorylation sites. These analytical findings were corroborated by constructing a Nsp3 derivative devoid of phosphorylation. The results represent the first determination of phosphorylation sites of an alphavirus nonstructural protein, but the approach can be utilized in phosphoprotein analysis in general.

Effects of palmitoylation of replicase protein nsP1 on alphavirus infection.

The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.

Identification of a Novel Function of the AlphavirusCapping Apparatus: RNA 5′-TRIPHOSPHATASE ACTIVITY OF Nsp2

Both genomic and subgenomic RNAs of the Alphavirus have m(7)G(5')ppp(5')N (cap0 structure) at their 5' end. Previously it has been shown that Alphavirus-specific nonstructural protein Nsp1 has guanine-7N-methyltransferase and guanylyltransferase activities needed in the synthesis of the cap structure. During normal cap synthesis the 5' gamma-phosphate of the nascent viral RNA chain is removed by a specific RNA 5'-triphosphatase before condensation with GMP, delivered by the guanylyltransferase. Using a novel RNA triphosphatase assay, we show here that nonstructural protein Nsp2 (799 amino acids) of Semliki Forest virus specifically cleaves the gamma,beta-triphosphate bond at the 5' end of RNA. The same activity was demonstrated for Nsp2 of Sindbis virus, as well as for the amino-terminal fragment of Semliki Forest virus Nsp2-N (residues 1-470). The carboxyl-terminal part of Semliki Forest virus Nsp2-C (residues 471-799) had no RNA triphosphatase activity. Replacement of Lys-192 by Asn in the nucleotide-binding site completely abolished RNA triphosphatase and nucleoside triphosphatase activities of Semliki Forest virus Nsp2 and Nsp2-N. Here we provide biochemical characterization of the newly found function of Nsp2 and discuss the unique properties of the entire Alphavirus-capping apparatus.

Guanosine nucleotide analogs as inhibitors of alphavirus mRNA capping enzyme

The two virus-specific reactions in the capping of alphavirus RNAs, catalyzed by the replicase protein nsP1, are promising targets for developing virus-specific inhibitors. In this report, we have studied the effect of over 50 cap analogs on the guanine-7-methyltransferase and guanylyltransferase activities of Semliki Forest virus nsP1. Recombinant nsP1 was expressed in Escherichia coli and partially purified by flotation in a discontinuous sucrose gradient. The methyltransferase activity had a pH optimum between pH 6.5 and 7.1, and the apparent K m values were 1.9 mM for GTP, 6.0 μM for S-adenosyl- l-methionine and 170 μM for Mg 2+. NsP1 methyltransferase was able to methylate efficiently GTP (relative activity 100%), GDP (16%), GpppG (35%), GppppG (50%) and less efficiently GpppA (12%), m 2GTP (9%), and m 2,2GTP (25%), but not m 7GppG. The most potent inhibitors for nsP1 methyltransferase were et 2m 7GMP ( K i value 42 μM), m 2,7GMP, (64 μM), m 2,7GpppG (82 μM), m2et7GMP (105 μM), m 2(2-phet) 7GMP (194 μM) and m 2GMP (386 μM). Of these compounds, m 2GMP, m 2et 7GMP and m 2(2-phet) 7GMP showed competitive inhibition, whereas the others showed mixed type inhibition. All compounds that inhibited the methyltransferase activity inhibited also the guanylyltransferase activity of nsP1.

Regulation of alphavirus 26S mRNA transcription by replicase component nsP2.

Semliki Forest virus (SFV) mutant ts4 has a reversible temperature-sensitive defect in the synthesis the subgenomic 26S mRNA. The viral nonstructural protein nsP2 was identified as a regulator of 26S synthesis by transferring nsP2 coding sequences from ts4 into the infectious SFV cDNA clone (SFoto) to create SFots4. Sequencing identified the causal mutation as C4038U, predicting the amino acid change M781T in nsP2. A revertant was isolated in which a back mutation of U to C restored the wild-type phenotype. Compared to Sindbis virus nsP2 mutants ts15, ts17, ts18, ts24 and ts133, which also exhibit temperature-sensitive 26S RNA synthesis, ts4 and SFots4 reduced 26S RNA synthesis faster and to lower levels after temperature shift. Under these conditions, ts4 and SFots4 also displayed complete conversion of RFII+RFIII into RFI and reactivated minus-strand synthesis. After shift to 39 degrees C, ts4 nsP2 was released from a crude RNA polymerase preparation consisting of membranes sedimenting at 15,000 g (P15) and the remaining, unreleased nsP2 was capable of being cross-linked in almost equimolar ratio with nsP1 and nsP3. This supports the hypothesis that nsP2 binds directly or undirectly to the promoter for 26S RNA and that it is also an essential component of the viral replicase synthesizing 42S RNA plus strands. Only the former activity is temperature-sensitive in ts4 mutant.

Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2.

A panel of monoclonal antibodies (MAbs) was raised against Semliki Forest virus (SFV) nonstructural protein nsP2, which is a protease, an NTPase, a putative RNA helicase, and a regulator of the synthesis of the subgenomic 26S mRNA encoding the structural proteins. nsP2, used for immunization, was expressed as a histidine fusion protein in Escherichia coli and purified by metal affinity chromatography. Dot-blot assay, using a membrane fraction from SFV-infected cells as antigen, gave 33 positive clones. Of these, 30 MAbs recognized nsP2 in Western immunoblotting, and 25 showed positive indirect immunofluorescence (IFAT) in SFV-infected cells; 15 MAbs stained the cytoplasmic vacuoles (CPVI), which are the sites of viral RNA synthesis in alphavirus-infected cells. MAb 3B5 recognized only CPVIs, as shown by double immunofluorescence staining with polyclonal anti-nsP3 antiserum. Most of the MAbs (20/33) recognized the nuclear form of nsP2, which may be associated with SFV neurovirulence. Immunoprecipitation with MAbs revealed that the SFV nonstructural proteins are associated with each other. None of the MAbs recognized Sindbis virus nsP2 in immunoblotting, indicating that they were directed to non-conserved sequences specific for SFV. Interestingly, these epitopes were located mostly within the N-terminal half of nsP2. Unexpectedly, the anti-nsP2 MAb 1E9 cross-reacted strongly with a host protein of 78 kDa from uninfected human, murine, avian and insect cells. This protein was identified as the immunoglobulin binding protein, BiP, by 2-D gel mapping and reaction with anti-BiP antiserum.

Synthesis of Semliki Forest virus RNA polymerase components nsP1 through nsP4 in Saccharomyces cerevisiae by expression of cDNA encoding the nonstructural polyprotein

A Semliki Forest virus nonstructural polyprotein, P1234, expressed in the yeast Saccharomyces cerevisiae in the absence of a replicative RNA template appeared to be properly cleaved into nsP1 to nsP4. All nsPs were membrane associated, and nsP2 was also transported to the nucleus. The membrane fraction containing nsPs showed guanine-7-methyltransferase and guanylyltransferase-like activities, typical for Semliki Forest virus nsP1.

Mutagenesis of the Sindbis Virus nsP1 Protein: Effects on Methyltransferase Activity and Viral Infectivity

It has been suggested that four amino acids which are absolutely conserved in the nsP1 nonstructural proteins encoded by togaviruses and in the homologous proteins encoded by plant viruses in the Sindbis virus (SV) superfamily may constitute a “methyltransferase motif.” In the Sindbis virus nsP1 protein (540 amino acids) these four amino acids are represented by His39, Arg91, Asp94, and Tyr249. Earlier, in assays of methyltransferase (MTase) activity generated in SV-infected cells, we had shown that amino acid changes at positions 87 and 88 of SV nsP1 resulted in a 10-fold lowerKmfor S-adenosyl methionine, the methyl donor in MTase reactions. Using site-directed mutagenesis we now report the expression of nsP1 inEscherichia coli,and in the infectious clone of Sindbis virus, Toto/1101, in which His39, Arg91, Asp94, and Tyr249were changed one at a time to Ala. We also expressed nsP1 with C-terminal deletions of varying size, as well as with internal deletions in the C-terminal portion of the protein, inE. coli.Changing His39, Arg91, Asp94, or Tyr249to Ala led to a loss of both MTase activity and viral infectivity; however, changing Ile369to Val, a conservative change in the carboxy-terminal half of nsP1, had no effect on either MTase activity or viral infectivity. With respect to the deleted forms of nsP1, a carboxy-terminal deletion of 48 amino acids was still compatible with MTase activityin vitro.However, larger deletions including those in which the amino acids between positions 442 and 492 were deleted abolished MTase activity.

Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP.

After the start of transcription, the 5' ends of eukaryotic mRNA molecules are modified by the addition of a guanylyl residue to form a cap structure, G(5')ppp(5')N. The guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) reaction responsible for cap formation usually proceeds via a covalent enzyme-GMP intermediate. We have studied the alphavirus-specific guanylyltransferase by incubating lysates from Semliki Forest and Sindbis virus-infected cells with [alpha-32P]GTP, using vaccinia virus and mock-infected cells as controls. One additional 32P-labeled protein was detected in alphavirus-infected cells but only in the presence of S-adenosylmethionine. This protein was identified as the nonstructural protein nsP1. The properties of the covalent enzyme-guanylate complex were studied with Semliki Forest virus nsP1 expressed in recombinant baculovirus-infected cells. S-Adenosylmethionine and divalent cations were required for the complex formation. The reaction was specific for guanylate nucleotides (GTP, dGTP) and was inhibited by pyrophosphate. nsP1 could be labeled with S-adenosyl[methyl-3H]methionine but only under conditions in which the nsP1-guanylate complex was formed. 7-Methyl-GMP was released from the nsP1-guanylate complex by treatment with acid or acidic hydroxylamine. Similar treatment of vaccinia virus capping enzyme released GMP. These findings suggest that in the capping of alphavirus mRNAs the guanine is methylated before linkage to the mRNA molecule.

Deletion and Duplication Mutations in the C-Terminal Nonconserved Region of Sindbis Virus nsP3: Effects on Phosphorylation and on Virus Replication in Vertebrate and Invertebrate Cells

Little is known concerning the function of the C-terminal nonconserved region of Sindbis virus nsP3. In this report, we created a number of in-frame deletions and duplications (from 12 to 159 residues) in this region and examined their effects on Sindbis virus replication. Sindbis RNA transcripts containing these mutations were infectious and gave rise to viable virus after transfection of chicken embryo fibroblasts (CEF). In CEF, the rate of virus release and accumulation of viral RNAs were similar for the mutants and the parental virus, although larger deletions resulted in decreased total viral RNA synthesis and lower virus yields early in infection. For some mutants, dramatic differences in nsP3 phosphorylation were noted, both in the level of phosphorylation and in the pattern of electrophoretically distinct forms. The two largest deletions resulted in only trace levels of nsP3 phosphorylation, which indicates that highly phosphorylated nsP3 is not necessary for efficient SIN replication in CEF. In the C7-10 mosquito cell line, the largest deletion mutants were defective at initiating a productive infection, generating plaques at only 1-2% the efficiency of the parental virus. However, once infection was established normal virus yields were produced. Thus, although nonessential, the nsP3 nonconserved region may be important for optimal virus replication in diverse host cells. The ability to engineer viable in-frame insertion mutations in this region of the genome provides yet another strategy for expression of heterologous polypeptides and RNAs using alphavirus vectors.

Nuclear targeting of Semliki Forest virus nsP2.

The Semliki Forest virus-specific nonstructural protein nsP2 is transported into the nuclei of both infected and transfected BHK cells. The pentapeptide sequence P 648R RRV is an essential part of the nuclear localization signal (NLS) of nsP2, the middle arginine being the most critical residue for nuclear targeting. Host DNA and RNA syntheses are rapidly inhibited in virus-infected cells, and nsP2 could be involved in these processes. It has been postulated that the inhibition of cellular replication could be due to viral NTPase activity. We have expressed and purified nsP2 in E. coli using the highly efficient T7 based expression system. Purified nsP2 was shown to have ATPase and GTPase activities, and these specific activities were increased in the presence of single-stranded RNA, a typical feature of RNA helicases. The role of nsP2 in the nucleus was studied by creating a mutant virus SFV-RDR, which contained an altered NLS (PRDRV). The mutation affected neither the processing nor the stability of nsP2, but it did render nsP2 completely cytoplasmic. SFV-RDR was shown to be fully infectious, and no difference could be seen in the expression of viral proteins. In addition, the inhibition of host DNA synthesis was almost equally efficient in both wild-type and mutant-infected cells. The pathogenic properties of the mutant will be further studied.

Expression of sindbis virus nsP1 and methyltransferase activity in Escherichia coli

We have constructed two plasmids, pSR5–42 and pSR5-Toto, which under lac control expressed the SV LM21 and the SV Toto forms, respectively, of the Sindbis virus nonstructural protein, nsP1. The induced protein, which was the major protein made following induction with IPTG, had an apparent molecular weight of 60, 000 and an amino terminal sequence in agreement with that expected for nsP1. Following induction with IPTG, cells carrying pSR5–42 (which contains the SV LM21 gene sequence) generated much higher RNA methyltransferase activity than cells carrying pSR5Toto (which contains the SV Toto gene sequence). This result is in agreement with what is observed when methyltransferase is measured in cells infected with SV LM21 and SV STD (or SV Toto), respectively. These results provide strong evidence that nsP1 has methyltransferase activity in the absence of any other viral nonstructural proteins.

Deletion analysis of brome mosaic virus 2a protein: effects on RNA replication and systemic spread

Brome mosaic virus (BMV) genomic RNA2 encodes the 94-kDa 2a protein, which is one of two BMV nonstructural proteins required for RNA replication and subgenomic mRNA transcription. 2a contains a central polymeraselike region, which has extensive sequence similarity with the Sindbis virus nsP4 and tobacco mosaic virus (TMV) 183-kDa replication proteins, and also contains N- and C-terminal flanking segments without counterparts in the Sindbis virus and TMV nonstructural proteins. To further investigate the roles of the central and flanking segments in 2a, we have constructed a series of deletion and frameshift mutants in a biologically active BMV RNA2 cDNA clone and tested their ability to support viral RNA replication in barley protoplasts and systemic infection in whole barley plants. The entire 125-amino-acid C-terminal segment following the polymeraselike region was dispensable for RNA replication and transcription. Within the 200-amino-acid N-terminal flanking segment, deletion of the first 50 residues dramatically reduced genomic and subgenomic RNA accumulation, and deletion of 100 or more residues abolished detectable RNA synthesis. All mutations removing residues from the central polymeraselike domain also blocked RNA replication in trans. Sequences required in cis for RNA2 replication or stability were found to occur within the first 300 nucleotides of the 2a coding region. In whole barley plants, systemic infection was inhibited even by 2a deletions that supported strong RNA replication in protoplasts. Some replication-competent 2a variants failed to spread to uninoculated leaves, while other showed 10- to 500-fold-reduced virus yield in both inoculated and uninoculated leaves. These reductions were not due to any defects in RNA2 encapsidation.