Characterization of proteinase cleavage sites in the N-terminal region of the RNA1-encoded polyprotein from Arabis mosaic virus (subgroup A nepovirus)

Arabis mosaic virus is a subgroup A nepovirus. The RNA1-encoded polyprotein (P1) contains the domains for the NTP-binding protein (NTB), VPg, proteinase (Pro) and polymerase at its C-terminus. Putative cleavage sites delineating these domains have been proposed. However, the number and location of cleavage sites upstream of the NTB domain are not known. Using in vitro processing assays, we have confirmed proteolytic cleavage at the NTB-VPg and VPg-Pro sites. In addition, we have identified two cleavage sites in the N-terminal region of P1. Site-directed mutagenesis and immunoprecipitation experiments using inserted peptide tags confirmed that the position of these cleavage sites corresponds to that of cleavage sites delineating the X1 and X2 domains in Tomato ringspot virus (subgroup C nepovirus). Amino acid alignments implied the presence of similar cleavage sites in the P1 polyprotein of other nepoviruses. Our results suggest that the presence of two protein domains upstream of NTB is a common feature of nepoviruses.     

Molecular characterization of the interaction between the N-terminal region of Potato virus X (PVX) coat protein (CP) and Nicotiana benthamiana PVX CP-interacting protein, NbPCIP1

Using yeast two-hybrid assays and a Nicotiana benthamiana cDNA library, we previously identified an N. benthamiana protein, NbPCIP1, that interacts with Potato virus X (PVX) coat protein (CP). We also previously determined that NbPCIP1 enhances PVX replication in plants. To determine the domains and/or amino acid residues required for PVX CP and NbPCIP1 interaction, here we used yeast two-hybrid and β-galactosidase filter assays to test the effects of deletion and site-directed mutations on the interaction. Truncation analysis revealed that the N-terminal region of PVX CP interacts with NbPCIP1. To identify which N-terminal region PVX CP amino acid(s) interact with NbPCIP1, we substituted the 12 charged amino acids on the PVX CP N-terminal region to alanine. Yeast two-hybrid, β-galactosidase filter, and bimolecular fluorescence complementation (BiFC) assays confirmed that ten of the 12 alanine-substituted mutations blocked the interaction with NbPCIP1. The results suggest that the N-terminal region of PVX CP including its helical structure is important for interaction with NbPCIP1.     

Effects of monoparacoumarylputrescinium chloride on the hypersensitive reaction of Gomphrena globosa leaves to tomato bushy stunt virus

Monoparacoumarylputrescinium chloride (pCPH), supplied to detached leaves of Gomphrena globosa via the petiole, induced interference with tomato bushy stunt virus infection by reducing the number and size of the necrotic local lesions. The phenolic compound neither inactivated directly the virus in vitro, nor induced interference when supplied just after virus inoculation, all this indicating an effect on cell metabolism. The interference was positively correlated to both pCPH concentration and time of induction (viz., the intervals between pCPH supply and inoculation). Coumaric acid did not, whereas putrescine, but not spermidine or spermine, did induce interference. Disc electrophoresis in polyacrylamide gels revealed no changes in the soluble protein constitution between pCPH-treated and control leaves.     

Inhibition of long-distance movement of RNA silencing signals in Nicotiana benthamiana by Apple chlorotic leaf spot virus 50 kDa movement protein

Apple chlorotic leaf spot virus 50 kDa movement protein (P50) acts as a suppressor of systemic silencing in Nicotiana benthamiana. Here, we investigate the mode of action of P50 suppressor. An agroinfiltration assay in GFP-expressing N. benthamiana line16c (GFP-plant) showed that P50 could not prevent the short-distance spread of silencing. In grafting experiments, the systemic silencing was inhibited in GFP-plants (scion) grafted on P50-expressing N. benthamiana (P50-plant; rootstock) when GFP silencing was induced in rootstock. In double-grafted plants, GFP-plant (scion)/P50-plant (interstock)/GFP-plant (rootstock), the systemic silencing in scion was inhibited when GFP silencing was induced in rootstock. Analysis of P50 deletion mutants indicated that the N-terminal region (amino acids 1-284) is important for its suppressor activity. In gel mobility shift assay, P50 lacks binding ability with siRNAs. These results indicated that P50 has a unique suppressor activity that specifically inhibits the long-distance movement of silencing signals.     

Phosphorylation of the TGBp1 movement protein of Potato virus X by a Nicotiana tabacum CK2-like activity

The movement protein (MP) TGBp1 of the potexvirus Potato virus X (PVX) is a multifunctional protein required for cell-to-cell movement within the host plant. Recent work on other plant viruses has indicated that MP phosphorylation by host kinases can regulate MP function. In this study, we demonstrate that recombinant and native TGBp1 are phosphorylated by Nicotiana tabacum extracts from both PVX-infected and non-infected leaves. The phosphorylation activity present in plant extracts has distinctive characteristics of casein kinase 2 (CK2): it is inhibited by heparin, stimulated by polylysine, and uses either ATP or GTP as phosphoryl donors. We also demonstrate that TGBp1 is efficiently phosphorylated by recombinant tobacco CK2 alpha subunit and by partially purified tobacco CK2. Phosphopeptide mass mapping reveals that TGBp1 is phosphorylated in Ser-165, which is localized within a CK2 consensus sequence. Our results strongly suggest that a N. tabacum kinase of the CK2 family is involved in TBGp1 phosphorylation during the course of viral infection.     

Live-attenuated recombinant equine herpesvirus type 1 (EHV-1) induces a neutralizing antibody response against West Nile virus (WNV)

The immunogenicity in horses of a recombinant equine herpesvirus type 1 (EHV-1) vaccine expressing West Nile virus (WNV) prM and E proteins was studied. To construct the recombinant EHV-1, two-step en passant mutagenesis was employed for manipulation of a bacterial artificial chromosome (BAC) of vaccine strain RacH. Recombinant EHV-1 stably expressed the WNV prM and E proteins as demonstrated by indirect immunofluorescence and Western blotting. In addition, growth properties in vitro of the EHV-1/WNV recombinant were found to not be significantly different from those of the parental virus. To determine if vaccination of horses induces an antibody response, 10 horses were allocated in two groups. Group A consisted of six horses that were vaccinated three times with the recombinant EHV-1/WNV virus in 28- to 31-day intervals. Group B consisted of four horses that were sham-vaccinated using the same regimen. Serum was collected on days 0, 31, 45 and 66. Plaque reduction neutralization test and IgG(T)- and IgGb-specific WNV E antibody-capture ELISAs were used. After a single vaccination (day 31), at least four of the six horses from group A had detectable levels of serum neutralizing antibodies against WNV, and three horses retained SN titers until the end of the study. None of the horses in the control group B sero-converted. On days 31 and 45, five of the six horses in group A had a marked increase of WNV-specific IgG(T), and at least four exhibited modestly elevated WNV-specific IgGb titers. From the results, we concluded that the EHV-1 vectored virus is able to express the WNV structural proteins and that vaccination of horses results in the induction of WNV E-protein-specific IgG(T), IgGb, and neutralizing antibodies.     

DNA-A of a highly pathogenic Indian cassava mosaic virus isolated from Jatropha curcas causes symptoms in Nicotiana benthamiana

Jatropha curcas mosaic disease (JcMD) is a newly emerging disease that has been reported in Africa and India. Here, we report the complete nucleotide sequence of a new Indian cassava mosaic virus isolate (ICMV-SG) from Singapore. Infection of ICMV-SG showed more severe JcMD in Jatropha curcas and Nicotiana benthamiana than the other ICMV isolates reported previously, though ICMV-SG shares high sequence identity with the other ICMV isolates. Agroinfectious DNA-A alone sufficiently induced systemic symptoms in N. benthamiana, but not in J. curcas. Results from agroinfection assays showed that systemic infection of ICMV-SG in J. curcas required both DNA-A and DNA-B components.     

Complete nucleotide sequence and analysis of the putative polyprotein of maize dwarf mosaic virus genomic RNA (Bulgarian isolate)

Summary.  The complete nucleotide sequence of maize dwarf mosaic virus Bulgarian isolate (MDMV-Bg) was determined. The viral genome was 9515 nt and contained an open reading frame encoding 3042 amino acids, flanked by 3′- and 5′-UTRs of 139 and 250 nucleotides, respectively. MDMV-Bg was more conserved in the coding region (52.9%) than in the UTRs (45.8%) when compared to the 15 other potyviruses. Of ten putative gene products of MDMV-Bg, the P1 was the most variable protein (24.9%) while the NIb was the most conserved protein (67.3%). Several sequence variations were observed between MDMV-Bg and Johnson grass mosaic virus (JGMV), and more between MDMV-Bg and the dicot potyviruses. Phylogenetic analysis suggested that MDMV-Bg was the most closely related to JGMV. Received January 15, 1998 Accepted April 29, 1998     

Differential diagnosis between type-specific duck hepatitis virus type 1 (DHV-1) and recent Korean DHV-1-like isolates using a multiplex polymerase chain reaction

Duck hepatitis can be caused by four types of viruses: duck hepatitis virus (DHV) type 1 (DHV-1), DHV-1a (a variant strain of DHV-1), DHV-2 and DHV-3. In Korea, duck hepatitis has been associated with two types of DHV-1, original DHV-1 type-specific strain (DHV-1s) and the recently reported DHV-1 variant strains (DHV-1v). The pathogenicity and pathological findings of ducklings infected with the recent DHV-1v isolates, AP-04114 and AP-04203, were almost identical to those infected with members of the DHV-1s, DHV-HS and the type-specific strain DRL-62. To be able to monitor the epidemiological patterns exhibited by the two Korean types, a specific gene-based differential diagnostic method based on multiplex polymerase chain reaction was developed. The primers selected were designed to bind to and amplify conserved regions within the RNA-dependent RNA polymerase (3D) gene, the complete capsid (P1) region or the 5′-untranslated region to distinguish between the DHV-1s and DHV-1v groups. The described multiplex polymerase chain reaction method was able to selectively recognize ducklings infected with either of the two groups of Korean isolates. The method was also able to distinguish between DHVs and other avian-originated RNA viruses. The detection limit of the diagnostic method was determined to correspond to 10³ copies viral RNA and 100 pg used as starting template. As a result, the use of this test allows rapid and early diagnosis of two different virus types affecting the commercial duckling industry.     

Modeling-based characterization of the elicitor function of amino acid 461 of Cucumber mosaic virus 1a protein in the hypersensitive response

The Ns strain of Cucumber mosaic virus (CMV) induces hypersensitive response (HR) on Nicotiana tabacum cv. Xanthi-nc and on Nicotiana glutinosa. The genetic determinant of the HR induction was localized earlier to amino acid 461 of the 1a protein. The 3D structure of the 1a protein is still unknown and building a homology model is impossible. Nevertheless, on the basis of secondary structure predictions we have created partial protein models for the region surrounding residue 461 which can account structurally for the effect of aa 461 on elicitor function. Seven different amino acid mutations were designed and introduced to the position 461 of the 1a protein in RNA 1. Three of the mutations (proline, glutamic acid, asparagine) inhibited virus replication. Two of the mutants caused systemic symptom development (lysine and arginine). Two mutants (alanine and serine) resulted in localization of the virus, but strong necrosis similar to the original Ns-CMV strain was not observed. Inoculation of purified Ns-CMV virions at extremely high concentration provoked systemic symptoms.     

Enhancement of the immune response by a catabolic product of normal rabbit IgG. Effect of F(ab')2-like fragment.

The serum of partially hepatectomized rabbit taken 4 h after operation possessed the proerty of enhancing the immune response to sheep red blood cells (SRBC) in homologous recipients. An exhaustive absorption of the serum with SRBC stromata did not affect its adjuvant-like activity indicating that an active factor augmented the immune response non-specifically. It was shown that the serum of partially hepatectomized rabbits contained a 5-2S IgG fragment antigenically related to peptic F(ab')2 fragment. The i.v. injection of this fragment together with SRBC into rabbits resulted in enhancement of PFC response and haemagglutinin production. Similar enhancing effect was produced by peptic F(ab')2 fragment obtained from rabbit IgG devoid of anti-SRBC antibodies. Sera of partially hepatectomized rabbits did not contain F(ab')2-like fragments and failed to enhance the immune response if the animals were treated with proteinase inhibitor ('Trasylol'), indicating that the fragment was a cetabolic product of IgG. The results are interpreted in terms of regulation of immunoglobulin synthesis by a split product of autologous IgG.     

The RNA of the human syncytium-forming (foamy) virus

Summary Human syncytium-forming (foamy) virus was labeled with³H-uridine and banded isopycnically in sucrose gradients (buoyant density = 1.16 to 1.18 g/cm³). Viral RNA extracted from the banded virus was analyzed either by rate zonal separation in sucrose gradients or by polyacrylamide-agarose gel electrophoresis. The results indicated that purified HSFV contains a 60S RNA component plus several smaller molecular weight RNA components. On dissociation with heat, smaller RNA structures were released from the 60S component. These results indicate that the genome of HSFV, like the other members of theRetroviridae family, is composed of an aggregate of several RNA species.With 3 Figures     

The 28 S genomic RNA of avian sarcoma virus PRCII codes for the transformation-specific polyprotein P105

We have characterized the genomic RNA of the defective avian sarcoma virus PRCII. Replicating virus which consists of transforming PRCII and a nontransforming but replication-competent helper, PRCII-AV, contains two RNA species. One is identical in size to the 35 S genome of avian leukosis helper viruses. The second component migrates slightly faster than 28 S ribosomal RNA in polyacrylamide gel electrophoresis but co-sediments with this RNA in sucrose gradients. In vitro translation across a gradient of velocity-sedimented poly(A)-containing PRCII virion RNA yielded three major proteins. The virion protein precursors Pr76^gag and Pr180^gag-pol were translated from 35 S RNA, while the transformation-specific polyprotein P105 was translated from 28 S RNA. P105 may be the only protein coded for by the PRCII genome, although this product would not exhaust the coding capacity of 28 S RNA. Whether translated in vitro or immunoprecipitated from transformed cells, P105 was essentially identical as demonstrated by comparative peptide maps.     

Nucleotide sequence of RNA 3 of the British type isolate (Blencowe strain) of tomato aspermy virus

The complete sequence of RNA 3 of the Blencowe, British (B) isolate of tomato aspermy virus (TAV) is presented. The RNA 3 of TAV-B is dicistronic, encoding the putative movement protein 3a and the capsid protein.