Open Reading Frame 1a Research Articles

Citrus tristeza virus: a pathogen that changed the course of the citrus industry.

Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) is the causal agent of devastating epidemics that changed the course of the citrus industry. Adapted to replicate in phloem cells of a few species within the family Rutaceae and to transmission by a few aphid species, CTV and citrus probably coevolved for centuries at the site of origin of citrus plants. CTV dispersal to other regions and its interaction with new scion varieties and rootstock combinations resulted in three distinct syndromes named tristeza, stem pitting and seedling yellows. The first, inciting decline of varieties propagated on sour orange, has forced the rebuilding of many citrus industries using tristeza-tolerant rootstocks. The second, inducing stunting, stem pitting and low bearing of some varieties, causes economic losses in an increasing number of countries. The third is usually observed by biological indexing, but rarely in the field. CTV polar virions are composed of two capsid proteins and a single-stranded, positive-sense genomic RNA (gRNA) of approximately 20 kb, containing 12 open reading frames (ORFs) and two untranslated regions (UTRs). ORFs 1a and 1b, encoding proteins of the replicase complex, are directly translated from the gRNA, and together with the 5' and 3'UTRs are the only regions required for RNA replication. The remaining ORFs, expressed via 3'-coterminal subgenomic RNAs, encode proteins required for virion assembly and movement (p6, p65, p61, p27 and p25), asymmetrical accumulation of positive and negative strands during RNA replication (p23), or suppression of post-transcriptional gene silencing (p25, p20 and p23), with the role of proteins p33, p18 and p13 as yet unknown. Analysis of genetic variation in CTV isolates revealed (1) conservation of genomes in distant geographical regions, with a limited repertoire of genotypes, (2) uneven distribution of variation along the gRNA, (3) frequent recombination events and (4) different selection pressures shaping CTV populations. Measures to control CTV damage include quarantine and budwood certification programmes, elimination of infected trees, use of tristeza-tolerant rootstocks, or cross protection with mild isolates, depending on CTV incidence and on the virus strains and host varieties predominant in each region. Incorporating resistance genes into commercial varieties by conventional breeding is presently unfeasible, whereas incorporation of pathogen-derived resistance by plant transformation has yielded variable results, indicating that the CTV-citrus interaction may be more specific and complex than initially thought. A deep understanding of the interactions between viral proteins and host and vector factors will be necessary to develop reliable and sound control measures.

Molecular characterization and detection of plum bark necrosis stem pitting-associated virus

The complete RNA genome of plum bark necrosis stem pitting-associated virus (PBNSPaV) was cloned and sequenced and was determined to be 14, 214 nts long. The genome structure revealed seven major open reading frames (ORFs), and nontranslated regions at the 5' and 3' ends. PBNSPaV represents the simplest genome organization in the genus Ampelovirus, family Closteroviridae. The ORFs 1a and 1b encode, respectively, a large polyprotein with a molecular mass (Mr) of 259.6 kDa containing conserved domains characteristic of a papain-like protease, methyltransferase and helicase (ORF1a) and a 64.1-kDa protein of eight conserved motifs characteristic of viral RNA-dependent RNA polymerase (RdRp) (ORF1b). ORF1b is presumably expressed via a +1 ribosomal frameshift mechanism. ORF2 encodes a small 6.3-kDa hydrophobic protein of unknown function. ORF3 encodes a 57.4-kDa protein, a homologue of the HSP70 family of heat shock proteins. ORF4 encodes a 61.6-kDa protein with unknown function. ORF5 encodes a 35.9-kDa capsid protein (CP). Lastly, ORF6 encodes a 25.2-kDa minor capsid protein (CPm). Phylogenetic analyses performed on sequences of the HSP70h RdRp and CP support classification of the virus in the genus Ampelovirus. A real-time TaqMan RT-PCR assay and a one-step RT-PCR were developed for PBNSPaV detection and compared using three different sample preparation methods.

Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 Replication

Coronaviruses express open reading frame 1a (ORF1a) and ORF1b polyproteins from which 16 nonstructural proteins (nsp) are derived. The highly conserved region at the carboxy terminus of ORF1a is processed by the nsp5 proteinase (Mpro) into mature products, including nsp7, nsp8, nsp9, and nsp10, proteins with predicted or identified activities involved in RNA synthesis. Although continuous translation and proteolytic processing of ORF1ab by Mpro is required for replication, it is unknown whether specific cleavage events within the polyprotein are dispensable. We determined the requirement for the nsp7 to nsp10 proteins and their processing during murine hepatitis virus (MHV) replication. Through use of an MHV reverse genetics system, in-frame deletions of the coding sequences for nsp7 to nsp10, or ablation of their flanking Mpro cleavage sites, were made and the effects upon replication were determined. Viable viruses were characterized by analysis of Mpro processing, RNA transcription, and growth fitness. Deletion of any of the regions encoding nsp7 to nsp10 was lethal. Disruption of the cleavage sites was lethal with the exception of that of the nsp9-nsp10 site, which resulted in a mutant virus with attenuated replication. Passage of the attenuated nsp9-nsp10 cleavage mutant increased fitness to near-wild-type kinetics without reversion to a virus capable of processing nsp9-nsp10. We also confirmed the presence of a second cleavage site between nsp7 and nsp8. In order to determine whether a distinct function could be attributed to preprocessed forms of the polyprotein, including nsp7 to nsp10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus was not viable, suggesting that the uncleaved protein may be essential for replication or proteolytic processing.

The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein–protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8–ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.

Neither the RNA nor the Proteins of Open Reading Frames 3a and 3b of the Coronavirus Infectious Bronchitis Virus Are Essential for Replication

Gene 3 of infectious bronchitis virus is tricistronic; open reading frames (ORFs) 3a and 3b encode two small nonstructural (ns) proteins, 3a and 3b, of unknown function, and a third, structural protein E, is encoded by ORF 3c. To determine if either the 3a or the 3b protein is required for replication, we first modified their translation initiation codons to prevent translation of the 3a and 3b proteins from recombinant infectious bronchitis viruses (rIBVs). Replication in primary chick kidney (CK) cells and in chicken embryos was not affected. In chicken tracheal organ cultures (TOCs), the recombinant rIBVs reached titers similar to those of the wild-type virus, but in the case of viruses lacking the 3a protein, the titer declined reproducibly earlier. Translation of the IBV E protein is believed to be initiated by internal entry of ribosomes at a structure formed by the sequences corresponding to ORFs 3a and 3b. To assess the necessity of this mechanism, we deleted most of the sequence representing 3a and 3b to produce a gene in which ORF 3c (E) was adjacent to the gene 3 transcription-associated sequence. Western blot analysis revealed that the recombinant IBV produced fivefold less E protein. Nevertheless, titers produced in CK cells, embryos, and TOCs were similar to those of the wild-type virus, although they declined earlier in TOCs, probably due to the absence of the 3a protein. Thus, neither the tricistronic arrangement of gene 3, the internal initiation of translation of E protein, nor the 3a and 3b proteins are essential for replication per se, suggesting that these proteins are accessory proteins that may have roles in vivo.

Genome Prediction of Putative Genome-Linked Viral Protein (VPg) of Astroviruses

Positive-sense single-stranded RNA (+ssRNA) viruses replicate by uncoating the RNA genome for translation to provide viral proteins essential for genome replication and the production of new viral particles. The viral proteins are synthesized from a polyprotein precursor, which is cleaved nascently. The synthesized proteins include viral RNA-dependent RNA polymerase (RdRP), viral genome-linked protein (VPg), and a helicase. VPg is covalently attached to the genomic form of +ssRNA viruses. Helicases and NTPase unwind the RNA before replication. VPg and helicases have been identified in +ssRNA families, however, the presence of VPg and helicase in the Astroviridae, another +ssRNA family, has not been fully elucidated. Computational tools were utilized to provide sequence analysis evidence for the presence and genomic location of astrovirus VPg and helicase. HMMER program v2.1.1 was used to build Hidden Markov Model (HMM) profile for calicivirus VPg to search for conserved motifs in the astrovirus genome. We performed phylogenetic analysis of two genomic regions of astroviruses and caliciviruses (encoding the RdRP and VPg). We identified a putative VPg coding region in astrovirus. This region was located in open reading frame 1a (ORF1 a) and included sites with high sequence similarity to the VPg coding regions of Caliciviridae, Piconaviridae, and Potyviridae. A region encoding a putative astrovirus helicase identified conserved motifs only with pestivirus helicase sequences. Sequence analysis and comparison to other +ssRNA viruses supports the presence of VPg in the Astroviridae. Further laboratory analysis will be necessary to confirm these findings.

Infectious Bronchitis Virus 3a Protein Localizes to a Novel Domain of the Smooth Endoplasmic Reticulum

All coronaviruses possess small open reading frames (ORFs) between structural genes that have been hypothesized to play important roles in pathogenesis. Infectious bronchitis virus (IBV) ORF 3a is one such gene. It is highly conserved among group 3 coronaviruses, suggesting that it has an important function in infection. IBV 3a protein is expressed in infected cells but is not detected in virions. Sequence analysis predicted that IBV 3a was a membrane protein; however, only a fraction behaved like an integral membrane protein. Microscopy and immunoprecipitation studies demonstrated that IBV 3a localized to the cytoplasm in a diffuse pattern as well as in sharp puncta in both infected and transfected cells. These puncta did not overlap cellular organelles or other punctate structures. Confocal microscopy demonstrated that IBV 3a puncta lined up along smooth endoplasmic reticulum (ER) tubules and, in a significant number of instances, were partially surrounded by these tubules. Our results suggest that IBV 3a is partially targeted to a novel domain of the smooth ER.

Identification of differentially expressed genes in human pineal parenchymal tumors by microarray analysis

Human pineal parenchymal tumors (PPTs) are rare tumors, and little is known about their molecular pathogenesis. We used Atlas plastic human 8 K microarray analysis to identify the genes expressed in four human PPTs of different grades, in normal brain tissue and in a normal fetal pineal gland. We selected the most highly expressed genes in PPT (n=39) and compared their expression to that both in normal brain and fetal pineal gland. Nine genes were expressed more than twice as strongly and 3 at about half the level in PPT. Furthermore, real-time reverse transcription-PCR was performed to compare mRNA levels in the four PPTs, in four medulloblastomas (MBs) (the most common type of similar embryonal neoplasm in the cerebellum), and in normal brain, for 9 of the 39 genes. Among genes showing an expression similar to that obtained with microarray, puromycin-sensitive aminopeptidase and teratocarcinoma-derived growth factor 3 were up-regulated in PPT and in MB, and adenomatous polyposis coli like was down-regulated only in PPT. Up-regulated expression of chromosome 17 open reading frame 1A was seen in high-grade PPT and in MB, but not in lower grade PPT. In conclusion, our results identified a number of genes that are differentially expressed in PPT and MB, and some of them may serve as prognostic markers and can be used to define mechanisms of tumorigenesis.

Nucleotide sequence, genome organization and phylogenetic analysis of Strawberry pallidosis associated virus, a new member of the genus Crinivirus

The complete nucleotide sequence of Strawberry pallidosis associated virus (SPaV), a newly identified member of the genus Crinivirus, family Closteroviridae has been determined. RNA 1 is 8067 nucleotides long and encodes at least three open reading frames (ORFs). The first ORF (ORF 1a) specifies a multifunctional protein that has papain-like proteinase, methyltransferase and RNA helicase domains. The RNA-dependent-RNA polymerase is encoded in ORF 1b and is probably expressed by a +1 ribosomal frameshift. The 3' ORF of RNA 1 encodes a small protein with two potential transmembrane helices. RNA 2 is 7979 nucleotides long and encodes 8 ORFs, similar in amino acid sequence and arrangement with those of other criniviruses. SPaV encodes the largest structural protein of closteroviruses sequenced to date as the minor coat protein of the virus has molecular mass of approximately 80 kDa. The 3' non-translated regions share nucleotide sequence identities of about 56% and the predicted folding of the non-translated regions is similar. Phylogenetic analyses reveal that SPaV is related most closely to Abutilon yellows virus and Beet pseudo-yellows virus, another virus that has been identified recently to cause identical symptoms on strawberry indicator plants as SPaV.

Characterization of emerging European-like porcine reproductive and respiratory syndrome virus isolates in the United States.

European-like field isolates of porcine reproductive and respiratory syndrome virus (PRRSV) have recently emerged in North America. The full-length genomic sequence of an index isolate characterized in 1999, strain EuroPRRSV, served as the reference strain for further studies of the evolution and epidemiology of European-like isolates (type 1) in the United States. Strain EuroPRRSV shared 90.1 to 100% amino acid identity with the prototype European strain, Lelystad, within the structural and nonstructural open reading frames (ORFs) and 95.3% overall nucleotide identity. The 5' untranslated region and two nonstructural regions within ORF 1 were closely examined due to significant divergence from strain Lelystad. A 51-bp deletion in a region within ORF 1a, coding for nonstructural protein 2 (NSP2), was observed. Sequence analysis of the structural ORFs 2 to 7 of additional European-like isolates indicated that these isolates share 93% nucleotide identity with one another and 95 to 96% identity with the Lelystad strain but only 70% identity with the North American reference strain VR-2332. Phylogenetic analysis with published PRRSV ORF 3, 5, and 7 nucleotide sequences indicated that these newly emerging isolates form a clade with the Lelystad and United Kingdom PRRSV isolates. Detailed analysis of four of these isolates with a panel of 60 monoclonal antibodies directed against the structural proteins confirmed a recognition pattern that was more consistent with strain Lelystad than with other North American isolates.

Cis-acting elements required for efficient packaging of brome mosaic virus RNA3 in barley protoplasts.

Brome mosaic virus (BMV) is a positive-sense RNA plant virus, the tripartite genomic RNAs of which are separately packaged into virions. RNA3 is copackaged with subgenomic RNA4. In barley protoplasts coinoculated with RNA1 and RNA2, an RNA3 mutant with a 69-nucleotide (nt) deletion in the 3'-proximal region of the 3a open reading frame (ORF) was very poorly packaged compared with other RNA3 mutants and wild-type RNA3, despite their comparable accumulation in the absence of coat protein. Computer analysis of RNA secondary structure predicted two stem-loop (SL) structures (i.e., SL-I and SL-II) in the 69-nt region. Disruption of SL-II, but not of SL-I, significantly reduced RNA3 packaging. A chimeric BMV RNA3 (B3Cmp), with the BMV 3a ORF replacing that of cucumber mosaic virus (CMV), was packaged negligibly, whereas RNA4 was packaged efficiently. Replacement of the 3'-proximal region of the CMV 3a ORF in B3Cmp with the 3'-proximal region of the BMV 3a ORF significantly improved packaging efficiency, and the disruption of SL-II in the substituted BMV 3a ORF region greatly reduced packaging efficiency. These results suggest that the 3'-proximal region of the BMV 3a ORF, especially SL-II predicted between nt 904 and 933, plays an important role in the packaging of BMV RNA3 in vivo. Furthermore, the efficient packaging of RNA4 without RNA3 in B3Cmp-infected cells implies the presence of an element in the 3a ORF of BMV RNA3 that regulates the copackaging of RNA3 and RNA4.

Defective RNAs of Citrus tristeza virus analogous to Crinivirus genomic RNAs

The family Closteroviridae includes the genera Closterovirus and Ampelovirus with monopartite genomes and the genus Crinivirus with bipartite genomes. Plants infected with the Closterovirus, Citrus tristeza virus (CTV), often contain one or more populations of defective RNAs (dRNAs). Although most dRNAs are comparatively small (2–5 kb) consisting of the genomic RNA termini with large internal deletions, we recently characterized large dRNAs of ∼12 kb that retained the open reading frames (ORFs) 1a plus 1b. These were self-replicating RNAs and appeared to be analogous to the genomic RNA 1 of the bipartite criniviruses. The present report describes the finding of an additional group of large dRNAs (LdRNAs) that retained all or most of the 10 3′ ORFs and appeared to be analogous to genomic RNA 2 of criniviruses. Isolates associated with LdRNAs were found associated with double-recombinant dRNAs (DR-dRNAs) of various sizes (1.7 to 5.1 kb) that comprised the two termini and a noncontiguous internal sequence from ORF2. The genetic and epidemiological implications of the architectural identities of LdRNAs and DR dRNAs and their apparent analogy with the genomic RNA 2 of criniviruses are discussed.

Avian astroviruses

As poultry becomes more important in the world economy, it is increasingly important to fully understand the mechanisms of disease and poor production that affect the industry. To more accurately and reasonably treat these diseases, a more sophisticated understanding of interrelatedness is required. This review focuses on avian astroviruses (AAstVs), in particular the recent advances in our understanding of AAstV molecular biology, and also history, diagnosis, treatment and control. The known AastVs comprise duck astrovirus 1, turkey astrovirus 1 and 2, and avian nephritis virus of chickens. Nucleotide and amino acid identities between the avian and mammalian (human, ovine, bovine) astroviruses is very low (e.g. 20 to 25% and 12 to 15%, respectively) in open reading frame (ORF) 1a. There is also variation among the avian astroviruses, including between the two known types of turkey astrovirus. The ORF 1b sequence contains a number of conserved amino acid motifs; these could be the basis of degnerate oligonucleotide primers. A nomenclature for astroviruses is also proposed, based on: host species-astrovirus-type number/country(state)/reference number/year of isolation. For example, turkey astrovirus 2/North Carolina/034/1999.

RNA replication of mouse hepatitis virus takes place at double-membrane vesicles.

The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5'-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.

Positional Effect of Deletions on Viability, Especially on Encapsidation, of Brome mosaic virus D-RNA in Barley Protoplasts

Brome mosaic virus (BMV), a tripartite RNA plant virus, accumulates RNA3-derived defective RNAs (D-RNAs) in which 477–500 nucleotides (nt) are deleted in the central region of the 3a protein open reading frame (ORF), after prolonged infection in barley. In the present study, six artificial D-RNAs (AD-RNAs), having deletions of the same size as the naturally occurring D-RNA but at different positions in the 3a ORF, were constructed and tested for their amplification and encapsidation in barley protoplasts by coinoculation with BMV RNA1 and 2, or RNA1, 2, and 3. Northern blot analysis of RNA accumulation in total and virion fractions showed that deletions of 492 nt in the 3′-proximal and the 5′-proximal regions of the 3a ORF decreased encapsidation efficiency of the AD-RNAs compared with that of RNA3, whereas deletions in the central region enhanced encapsidation efficiency. The present results also show that deletion positions affect competition with RNA3 in the amplification and encapsidation of AD-RNAs.

Full-length sequence of a Canadian porcine reproductive and respiratory syndrome virus (PRRSV) isolate.

Summary. Presently, one of the most economically important pathogens affecting swine is the porcine reproductive and respiratory syndrome virus (PRRSV). This virus is prevalent in herds throughout the world and continues to pose a significant threat as newer and more virulent disease phenotypes emerge. In this report we describe the full-length nucleotide sequence of a Canadian PRRSV isolate, designated PA8. A consecutive sequence of 15,411 nucleotides was obtained from a set of overlapping cDNA clones. In order to determine the extent of genetic variation among isolates recovered from swine in Canada and the US, as well as to understand the molecular mechanisms governing the evolution of PRRSV, the full-length sequence of PA8 was compared with that of two US isolates, VR2332 and 16244B. The genomic sequence of PA8 shared 98.2% and 99.2% identity with 16244B and VR2332, respectively. The untranslated regions (UTR) at the 5′ and 3′ ends of the genome were very well conserved. Notable exceptions include an eight nucleotide difference at the 5′ end of the 5′ UTR of VR2332 relative to PA8 and 16244B and a two nucleotide difference in the 3′ UTR of PA8 relative to VR2332 and 16244B. In contrast to PA8 and VR2332, 16244B possessed two nucleotide differences within the RNA pseudoknot structure of the ribosomal frameshift region between open reading frame (ORF)1a and ORF1b. Amino acid differences were distributed throughout the genome, however they appeared to be most extensive in Nsp1β and ORF5 of the nonstructural and structural coding regions, respectively, suggesting that the evolutionary pressure to conserve these viral genes is somewhat lower.

Avian nephritis virus (ANV) as a new member of the family Astroviridae and construction of infectious ANV cDNA.

The complete RNA genome of the avian nephritis virus (ANV) associated with acute nephritis in chickens has been molecularly cloned and sequenced. Excluding the poly(A) tail, the genome comprises 6,927 nucleotides and contains three sequential open reading frames (ORFs). The first ORF (ORF 1a) contains a sequence encoding a serine protease motif, and the second ORF (ORF 1b) has a sequence encoding an RNA-dependent RNA polymerase. ORF 1a may be linked to the second ORF by a ribosomal frameshifting mechanism. The third ORF (ORF 2) may encode the virion structural proteins as a polyprotein precursor. Two RNAs, probably genonic and subgenonic RNA (7.5 and 3.0 kb), were detected in the cytoplasm of ANV-infected cells. ANV and human astroviruses have the same genonic organization, and both are characterized by the presence of two RNA bands. The amino acid homologies of the products of ORF 1a, 1b, and 2 were 20.3, 41.9, and 25.8% to products of the corresponding ORFs of human astrovirus serotype 1 (A/88 Newcastle strain). We have constructed a genonic-length cDNA clone of ANV to test whether the in vitro transcript is infectious. When a chicken kidney cell culture was transfected with RNA transcribed in vitro and the cDNA clone, infectious virus was produced with cytopathic effects in the absence of trypsin. These observations suggested that the ANV (G-4260 strain) is a new genus of the family Astroviridae.

Four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly.

The replicase gene (gene 1) of the coronavirus mouse hepatitis virus (MHV) encodes two co-amino-terminal polyproteins presumed to incorporate all the virus-encoded proteins necessary for viral RNA synthesis. The polyproteins are cotranslationally processed by viral proteinases into at least 15 mature proteins, including four predicted cleavage products of less than 25 kDa that together would comprise the final 59 kDa of protein translated from open reading frame 1a. Monospecific antibodies directed against the four distinct domains detected proteins of 10, 12, and 15 kDa (p1a-10, p1a-12, and p1a-15) in MHV-A59-infected DBT cells, in addition to a previously identified 22-kDa protein (p1a-22). When infected cells were probed by immunofluorescence laser confocal microscopy, p1a-10, -22, -12, and -15 were detected in discrete foci that were prominent in the perinuclear region but were widely distributed throughout the cytoplasm as well. Dual-labeling experiments demonstrated colocalization of the majority of p1a-22 in replication complexes with the helicase, nucleocapsid, and 3C-like proteinase, as well as with p1a-10, -12, and -15. p1a-22 was also detected in separate foci adjacent to the replication complexes. The majority of complexes containing the gene 1 proteins were distinct from sites of accumulation of the M assembly protein. However, in perinuclear regions the gene 1 proteins and nucleocapsid were intercalated with sites of M protein localization. These results demonstrate that the complexes known to be involved in RNA synthesis contain multiple gene 1 proteins and are closely associated with structural proteins at presumed sites of virion assembly.

Nucleotide sequence and organization of ten open reading frames in the genome of grapevine leafroll-associated virus 1 and identification of three subgenomic RNAs.

The genome of Grapevine leafroll-associated virus 1 (GLRaV-1) was cloned and the sequence of 12394 nts determined. It contains 10 major open reading frames (ORFs) and a 3'-non-coding region lacking a poly(A) tract. The first ORF (ORF 1a) encodes a putative RNA helicase at the C-terminal portion of an apparently larger protein. The downstream ORF, 1b, overlaps ORF 1a and lacks an initiation codon. This ORF encodes an RNA-dependent RNA polymerase of M(r) 59276. ORF 2 encodes a small hydrophobic protein of M(r) 6736, and ORF 3 encodes a homologue of the HSP70 family of heat shock proteins and has an M(r) of 59500. ORF 4 encodes a protein with an M(r) of 54648 that shows similarity to the corresponding proteins of other closteroviruses. ORF 5 encodes the viral coat protein (CP) with an M(r) of 35416. The identity of this ORF as the CP gene was confirmed by expression in Escherichia coli and testing with the viral antibody. ORFs 6 and 7 code for two CP-related products with M(r) of 55805 and 50164, respectively. ORFs 8 and 9 encode proteins of M(r) 21558 and 23771 with unknown functions. Using DNA probes to different regions of the GLRaV-1 sequence, three major 3'-coterminal subgenomic RNA species were identified and mapped on the GLRaV-1 genome. Phylogenetic analyses of the individual genes of GLRaV-1 demonstrated a closer relationship between GLRaV-1 and GLRaV-3 than with other closteroviruses.

Identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus Avian infectious bronchitis virus and characterization of the cleavage products.

The coronavirus Avian infectious bronchitis virus (IBV) employs polyprotein processing as a strategy to express its gene products. Previously we identified the first cleavage event as proteolysis at the Gly(673)-Gly(674) dipeptide bond mediated by the first papain-like proteinase domain (PLPD-1) to release an 87-kDa mature protein. In this report, we demonstrate a novel cleavage activity of PLPD-1. Expression, deletion, and mutagenesis studies showed that the product encoded between nucleotides 2548 and 8865 was further cleaved by PLPD-1 at the Gly(2265)-Gly(2266) dipeptide bond to release an N-terminal 195-kDa and a C-terminal 41-kDa cleavage product. Characterization of the cleavage activity revealed that the proteinase is active on this scissile bond when expressed in vitro in rabbit reticulocyte lysates and can act on the same substrate in trans when expressed in intact cells. Both the N- and C-terminal cleavage products were detected in virus-infected cells and were found to be physically associated. Glycosidase digestion and site-directed mutagenesis studies of the 41-kDa protein demonstrated that it is modified by N-linked glycosylation at the Asn(2313) residue encoded by nucleotides 7465 to 7467. By using a region-specific antiserum raised against the IBV sequence encoded by nucleotides 8865 to 9786, we also demonstrated that a 33-kDa protein, representing the 3C-like proteinase (3CLP), was specifically immunoprecipitated from the virus-infected cells. Site-directed mutagenesis and expression studies showed that a previously predicted cleavage site (Q(2583)-G(2584)) located within the 41-kDa protein-encoding region was not utilized by 3CLP, supporting the conclusion that the 41-kDa protein is a mature viral product.