Native electrophoresis and Western blot analysis (NEWeB): a method for characterization of different forms of potyvirus particles and similar nucleoprotein complexes in extracts of infected plant tissues.

A combination of native electrophoresis and immunodetection (Western blot) was used for the characterization of nucleoprotein particles of the potyvirus Plum pox virus (PPV). Virus particles were electrophoresed directly from plant extracts in agarose or mixed acrylamide-agarose gels under native conditions, blotted on nitrocellulose membranes, and characterized with the aid of a coat protein-specific antibody. Using this combined methodology, called NEWeB (native electrophoresis and Western blotting), we could show that a population of particles that differ in their electrophoretic mobility can be detected in extracts of Nicotiana benthamiana, that two different strains of PPV can be distinguished in double infections of the same plant and that virus particles from leaves contain detectable levels of helper component proteinase molecules. The potential of the NEWeB method for the study of structure and function of virus particles and similar nucleoprotein complexes in single and mixed infections is discussed.

Ultrastructural localization of nonstructural and coat proteins of 19 potyviruses using antisera to bacterially expressed proteins of plum pox potyvirus.

Antisera to the bacterially expressed nonstructural proteins (NSP) HC-Pro, CI, NIa, and NIb and the coat protein (CP) of plum pox potyvirus (PPV) were used for analysing the composition of virus-induced cytoplasmic and nuclear inclusions by electron microscopy. The antisera reacted with NSP and CP of PPV on immunogold-labelled ultrathin sections. Antiserum to CP reacted with virions of seven out of 18 other potyviruses. CP was distributed throughout the cytoplasm of infected cells. Antisera to PPV NSP specifically reacted with virus-specific cytoplasmic and/or nuclear inclusions induced by 17 different potyviruses. NSP were furthermore localized in confined cytoplasmic areas in between complex accumulations of virus-specific inclusions. Cylindrical inclusions induced by the potyviruses were proven to consist of CI protein. Most other cytoplasmic or nuclear inclusions were shown to be composed of two or more NSP. An unexpected composition of virus-induced inclusions was observed for the crystalline nuclear inclusions of tobacco etch virus. Here, in addition to the expected presence of NIa and NIb, HC-Pro could be demonstrated. Furthermore, amorphous cytoplasmic inclusions induced by papaya ringspot virus contained the expected HC-Pro but additionally NIa, NIb and CI. Beet mosaic virus-induced nuclear inclusions ('satellite bodies') contained in their electron-dense matrix NIa, NIb, Hc-Pro and CI and in their lacunae CP in bundles of virion-like filaments. The results indicate that all cytoplasmic or nuclear inclusions of potyviruses have to be regarded as deposition sites of excessively produced viral NSP.

Analysis of six DNA components of the faba bean necrotic yellows virus genome and their structural affinity to related plant virus genomes.

Faba bean necrotic yellows virus (FBNYV) has a multicomponent circular ssDNA genome. In addition to a previously described genome component (C1) coding for a replicase-associated protein (Rep), five further components (C2 to C6) have now been identified. Each of the six components is about 1 kb in size, contains one major open reading frame (ORF) in the virion sense with a TATA box and polyadenylation signal, and has a noncoding region containing a highly conserved sequence possibly forming a stem-loop structure. Similar to C1, C2 encodes another putative Rep of 33.1 kDa, which is closely related to the Rep of banana bunchy top virus (BBTV). Based on bacterial expression and immunoblot analysis, the ORF of C5 encodes the capsid protein (CP) with a deduced molecular mass of 19 kDa. The FBNYV CP shares the highest amino acid (aa) identity (56.2%) with that of subterranean clover stunt virus (SCSV). The ORF of C4 potentially codes for a hydrophobic protein which appears to be structurally and functionally similar to the BBTV-C4 and SCSV-C1 proteins. No protein sequence similarities were found in databases for the C3 and C6 ORFs of FBNYV. FBNYV is clearly distinct from any known virus but is taxonomically related to BBTV and SCSV.

Host-controlled cell-to-cell movement of a hybrid barley stripe mosaic virus expressing a dianthovirus movement protein.

The triple gene block (TGB) of barley stripe mosaic virus (BSMV), coding for viral movement proteins (MPs), was replaced by the single MP gene of red clover necrotic mosaic virus (RCNMV). Accumulation of the hybrid virus in barley plants (the selective host for BSMV) was reduced compared to BSMV. The hybrid virus induced small necrotic local lesions on Chenopodium amaranticolor leaves and did not infect Nicotiana clevelandii (the selective host for RCNMV). The hybrid virus accumulated in the inoculated leaves of Nicotiana benthamiana, but not in the upper noninoculated leaves. Thus the RCNMV MP gene substituted for the BSMV TGB in cell-to-cell movement, but not in systemic spread. Hybrid virus movement was efficient only in N. benthamiana, the common host for BSMV and RCNMV. These data point to the involvement of host-specific factors in the function of virus-coded transport determinants.

Sequence analysis of a faba bean necrotic yellows virus DNA component containing a putative replicase gene.

Faba bean necrotic yellows virus (FBNYV) has a circular ssDNA genome possibly consisting of several components of about 1 kb each. The complete nucleotide sequence of one component of FBNYV (FBNYV DNA 1) containing a putative replicase gene is presented. This component consists of 1002 nucleotides and, in the virion orientation, contains one large open reading frame (ORF1) potentially encoding a 32.3 kDa replicase with the NTP-binding motif GGEGKS. No obvious functions could be assigned to two smaller ORFs (7.4 and 9.3 kDa) occurring in the complementary orientation. Amino acid sequence comparisons of the putative replicase of FBNYV with that of other similar ssDNA viruses yielded higher homologies to subterranean clover stunt virus than to banana bunchy top and coconut foliar decay viruses. A potential stem-loop structure and a TATA box were identified within the noncoding region. Two oligonucleotides derived from FBNYV DNA 1 were used for direct sequencing of the virion ssDNA to determine its virion polarity and for amplifying part of this component by immunocapture PCR in extracts from from FBNYV-infected plants.

Serotype A and B strains of bean common mosaic virus are two distinct potyviruses.

The serological relationships among strains of bean common mosaic virus (BCMV) (genus Potyvirus, family Potyviridae) were investigated by testing 13 isolates of the 10 known BCMV pathotypes with two monoclonal antibodies and six antisera to BCMV strains. In addition, other properties of serologically distinct BCMV strains were compared. Two groups of BCMV strains were obtained by ELISA and Western blot serology: serotype A contained the BCMV strains NL3, NL5, and NL8 and serotype B contained the BCMV strains NL1, NL2, NL4, NL6, US4, NL7, NY15, and Fla. SDS polyacrylamide gel electrophoresis and Western blotting of freshly purified preparations, and of extracts from leaves infected with eleven BCMV strains showed that the apparent molecular mass of the capsid protein of the serotype A isolates NL3, NL5, and NL8 are lower (about M(r) 33,000) than those of the serotype B isolates (M(r) 34,500 to 35,000). The normal lengths of the particles of the serotype A isolates were shorter (810-818 nm) than those of most isolates (except NL6 and NY15) of serotype B (847-886 nm). All isolates studied induced cytoplasmic pinwheel and scroll inclusions. Cells infected with serotype A isolates contained a specific type of proliferated endoplasmic reticulum which was never found in cells infected with serotype B isolates. The capsid protein gene of a representative member of each serotype was cloned and sequenced. Molecular mass calculations based upon nucleotide sequence-derived amino acid sequences yielded M(r) of 29,662 and 32,489 for the capsid proteins of the serotype A isolate NL8 and the serotype B isolate NL4, respectively. Comparison of the coat-protein sequences showed considerable differences at the N-termini whereas the core regions and the C-termini appeared to be highly conserved. Marked differences were also observed within the 3' non-coding regions of cloned cDNAs of NL 4 and NL 8. The striking differences between the two serotypes of BCMV strongly suggest that they be classified as two distinct potyviruses which naturally infect Phaseolus beans.