Potato virus Y HCPro localization at distinct, dynamically related and environment-influenced structures in the cell cytoplasm.

Potyvirus HCPro is a multifunctional protein that, among other functions, interferes with antiviral defenses in plants and mediates viral transmission by aphid vectors. We have visualized in vivo the subcellular distribution and dynamics of HCPro from Potato virus Y and its homodimers, using green, yellow, and red fluorescent protein tags or their split parts, while assessing their biological activities. Confocal microscopy revealed a pattern of even distribution of fluorescence throughout the cytoplasm, common to all these modified HCPros, when transiently expressed in Nicotiana benthamiana epidermal cells in virus-free systems. However, in some cells, distinct additional patterns, specific to some constructs and influenced by environmental conditions, were observed: i) a small number of large, amorphous cytoplasm inclusions that contained α-tubulin; ii) a pattern of numerous small, similarly sized, dot-like inclusions distributing regularly throughout the cytoplasm and associated or anchored to the cortical endoplasmic reticulum and the microtubule (MT) cytoskeleton; and iii) a pattern that smoothly coated the MT. Furthermore, mixed and intermediate forms from the last two patterns were observed, suggesting dynamic transports between them. HCPro did not colocalize with actin filaments or the Golgi apparatus. Despite its association with MT, this network integrity was required neither for HCPro suppression of silencing in agropatch assays nor for its mediation of virus transmission by aphids.

Molecular characterization of a Chinese isolate of potato virus A (PVA) and evidence of a genome recombination event between PVA variants at the 3'-proximal end of the genome.

Potato plants that exhibited mosaic symptoms were collected in Xiangxi, Hunan province, China. Multiplex RT-PCR screening for common viruses revealed the presence of potato virus A (PVA) in these samples. ELISA with virus-specific antibodies confirmed infection by PVA in the plants. Rod-shaped virions of ~750 nm in length and ~13 nm in width were observed by transmission electron microscopy. One virus isolate (designated PVA-Hunan) was subjected to molecular characterization. The viral genome consisted of 9,567 nucleotides, excluding the poly(A) tail, and encoded a polyprotein of 3,059 amino acids. A second characteristic potyvirus open reading frame (ORF), pretty interesting Potyviridae ORF (pipo), was located at nucleotides 2,834-3,139. The isolate shared 84% to 98% and 93% to 99% sequence identity with other PVA isolates at the nucleotide and amino acid level, respectively. Phylogenetic analysis demonstrated that, within the PVA group, PVA-Hunan clustered most closely with the Finnish isolate Her, then with isolates 143, U, Ali, M and B11. The isolate TamMV stood alone at a separate branch. However, scanning of complete genome sequences using SimPlot revealed 99%-sequence identity between PVA-Hunan and TamMV in the 3'-proximal end of the genome (~nt 9,160 to the 3'end) and a 50%-94% (average~83%) identity upstream of nt 9,160. In contrast, 98% identity between PVA-Hunan and isolates M and B11 was detected for nucleotides 1 to ~9,160, but only ~94% for the 3'-proximal region, suggesting a genome recombination event (RE) at nt 9,133. The recombination breakpoint also was identified by the Recombination Detection Program (RDP). The RE was further confirmed by analysis of the CP gene, where the apparent RE was located.

[Properties of potato virus M and potato virus Y isolates in Ukraine].

Monitoring of viruses of tomato plants (Lycopersicon esculentum Mill.) was carried out. Twenty-seven varieties of tomatoes from different regions of Ukraine were tested for the virus presence. New symptoms, which had not been described before, were revealed. It was found out that the diseases were caused by Potato virus M and Potato virus Y. This is the first report about the infection of tomato plants with such viruses in Ukraine. Some biological, physical and chemical properties of the pathogens are studied. Differences between PVM, PVY and the known isolates were found in morphology and molecular weight of structural protein.

A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia.

A suspected virus disease was identified from an arborescent Brugmansia x candida Pers. (syn. Datura candida Pers.) tree. The causal agent was aphid transmissible at low rates. Viral particles were purified from infected tobacco tissue, analyzed, and purified virions were inoculated into healthy tobacco plants to recreate the symptoms. The virions had a mean length of 720-729 nm, and infected cells contained inclusion bodies typical of potyvirus infections. Analysis of infected tissues and purified virions with a panel of potyvirus-specific antibodies confirmed identification as a potyvirus. Viral host range, dilution end point, thermal tolerance and aphid transmission characteristics were examined. The viral genome (9761 nt) is typical of potyviruses, with the closest related potyvirus being pepper mottle virus, at 72 % nt sequence identity. Based on conventions for naming novel potyviruses, the virus was determined to be a member of a previously undescribed species, tentatively named "Brugmansia mosaic virus" (BruMV).

Top 10 plant viruses in molecular plant pathology.

Many scientists, if not all, feel that their particular plant virus should appear in any list of the most important plant viruses. However, to our knowledge, no such list exists. The aim of this review was to survey all plant virologists with an association with Molecular Plant Pathology and ask them to nominate which plant viruses they would place in a 'Top 10' based on scientific/economic importance. The survey generated more than 250 votes from the international community, and allowed the generation of a Top 10 plant virus list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Brome mosaic virus and (10) Potato virus X, with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato bushy stunt virus. This review article presents a short review on each virus of the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant virology community, as well as laying down a benchmark, as it will be interesting to see in future years how perceptions change and which viruses enter and leave the Top 10.

Incidencia de potyvirus y caracterización molecular de PVY en regiones productoras de papa (Solanum tuberosum L) de Colombia / Incidence of potyvirus and molecular characterization of PVY in potato (Solanum tuberosum L) growing regions of Colombia

Los problemas virales reducen los rendimientos y la calidad del tubérculo semilla en cultivos de papa de todo el mundo. Esta investigación se planteó con el fin de evaluar los niveles de incidencia de potyvirus en diez de las principales regiones cultivadoras de papa de los departamentos de Antioquia, Boyacá, Cundinamarca y Nariño (Colombia), y las características genotípicas del virus Y de la papa (Potato virus Y, PVY), seleccionado por ser el potyvirus más limitante de este cultivo. Para la evaluación de la incidencia se utilizaron pruebas de Elisa con anticuerpos que reconocen epítopes comunes a los potyvirus, mientras que las pruebas moleculares incluyeron el análisis filogenético de secuencias parciales del gen de la cápside viral de 33 aislamientos, así como la secuenciación de una porción de los extremos 5´ y 3´del genoma de dos cepas colombianas de este virus. Los resultados confirmaron la presencia de potyvirus en los cultivos de los cuatro departamentos evaluados, con una incidencia promedio del 72%, siendo este nivel superior al 56% en todas las zonas evaluadas. Los análisis moleculares del PVY, permitieron asociar las cepas colombianas estudiadas con las razas PVYN y la variante PVYNTN, esta última responsable de la enfermedad conocida en el mundo como PTNRD (Potato tuber necrotic ringspot disease).

Potato viruses are responsible for significant reductions in seed quality and crop yields around the world. In this study, we evaluate the levels of incidence of potyvirus in ten potato growing regions of Colombia from the provinces of Antioquia, Boyacá, Cundinamarca and Nariño. As PVY is the most limiting potyvirus in potato farming, a molecular characterization of Colombian PVY strains was also performed. Incidence was evaluated by ELISA using general potyvirus antibodies. Phylogenetic analysis were made on the partial sequence of the capsid gene from 33 isolates. A portion of the 5´ and 3' genome ends was obtained from two Colombian strains. Results confirmed the presence of potyvirus in the four provinces with an average incidence of 72%. The lowest incidence value was 56%. Molecular analysis clustered all Colombian isolates with strains PVYN and PVYNTN, the latter responsible for the disease known as PTNRD (Potato tuber necrotic ringspot disease).

Rapid diagnosis of plant virus diseases by transmission electron microscopy.

A clear and rapid diagnosis of plant virus diseases is of great importance for agriculture and scientific experiments in plant phytopathology. Even though negative staining and transmission electron microscopy (TEM) are often used for detection and identification of viral particles and provide rapid and reliable results, it is necessary to examine ultrastructural changes induced by viruses for clear identification of the disease. With conventional sample preparation for TEM it can take several days to obtain ultrastructural results and it is therefore not suitable for rapid diagnosis of virus diseases of plants. The use of microwave irradiation can reduce the time for sample preparation for TEM investigations. Two model virus-plant systems [Nicotiana tabacum plants infected with Tobacco mosaic virus (TMV), Cucurbita pepo plants infected with Zucchini yellow mosaic virus (ZYMV)] demonstrate that it is possible to diagnose ultrastructural alterations induced by viruses in less than half a day by using microwave irradiation for preparation of samples. Negative staining of the sap of plants infected with TMV and ZYMV and the examination of ultrastructure and size were also carried out during sample preparation thus permitting diagnosis of the viral agent by TEM in a few hours. These methods will contribute towards a rapid and clear identification of virus diseases of plants and will be useful for diagnostic purposes in agriculture and in plant phytopathology.

Turnip mosaic virus RNA replication complex vesicles are mobile, align with microfilaments, and are each derived from a single viral genome.

Nicotiana benthamiana plants were agroinoculated with an infectious cDNA clone of Turnip mosaic virus (TuMV) that was engineered to express a fluorescent protein (green fluorescent protein [GFP] or mCherry) fused to the viral 6K2 protein known to induce vesicle formation. Cytoplasmic fluorescent discrete protein structures were observed in infected cells, corresponding to the vesicles containing the viral RNA replication complex. The vesicles were motile and aligned with microfilaments. Intracellular movement of the vesicles was inhibited when cells were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. It was also observed that viral accumulation in the presence of this drug was reduced. These data indicate that microfilaments are used for vesicle movement and are necessary for virus production. Biogenesis of the vesicles was further investigated by infecting cells with two recombinant TuMV strains: one expressed 6K2GFP and the other expressed 6K2mCherry. Green- and red-only vesicles were observed within the same cell, suggesting that each vesicle originated from a single viral genome. There were also vesicles that exhibited sectors of green, red, or yellow fluorescence, an indication that fusion among individual vesicles is possible. Protoplasts derived from TuMV-infected N. benthamiana leaves were isolated. Using immunofluorescence staining and confocal microscopy, viral RNA synthesis sites were visualized as punctate structures distributed throughout the cytoplasm. The viral proteins VPg-Pro, RNA-dependent RNA polymerase, and cytoplasmic inclusion protein (helicase) and host translation factors were found to be associated with these structures. A single-genome origin and presence of protein synthetic machinery components suggest that translation of viral RNA is taking place within the vesicle.

Three heterologous proteins simultaneously expressed from a chimeric potyvirus: infectivity, stability and the correlation of genome and virion lengths.

Three heterologous proteins were simultaneously expressed from a chimeric potyvirus Potato virus A (PVA) in Nicotiana benthamiana. The genes for green fluorescent protein of Aequoria victoriae ("G"; 714 nucleotides, nt), luciferase of Renilla reniformis ("L", 933 nt) and beta-glucuronidase of Escherichia coli ("U", 1806 nt) were inserted into the engineered cloning sites at the N-terminus of the P1 domain, the junction of P1 and helper component protein (HC-Pro), and the junction of the viral replicase (NIb) and coat protein (CP), respectively, in an infectious PVA cDNA. The proteins were expressed as part of the viral polyprotein and subsequently released by cleavage at the flanking proteolytic cleavage sites by P1 (one site) or the NIa-Pro proteinase (other sites). The engineered viral genome (pGLU, 13311 nt) was 39.2% larger than wild-type PVA (9565 nt) and infected plants of N. benthamiana systemically. pGLU was stable and expressed all three heterologous proteins, also following the second infection cycle initiated by sap-inoculation of new plants with the progeny viruses. The gene for GUS showed some inherent instabilities, as also reported in other studies. Accumulation of pGLU in infected leaves was lower by a magnitude as compared to the vector viruses pG0U and p0LU used to express two heterologous proteins. Hence, pGLU may have reached the maximum genome size that can still function and complete the PVA infection cycle. Examination of virions by electron microscopy indicated that the virion lengths of PVA chimera with various numbers of inserts were directly proportional to their genome lengths.

A new potyvirus from butterfly flower (Iris japonica Thunb.) in Zhejiang, China.

A filamentous virus isolated from Iris japonica with mosaic symptoms was pathogenic to I. japonica and I. bulleyana but not to 12 virus indicator species. Sequencing of the 3'-terminus of the genome showed that it was a potyvirus, most closely related to sunflower mosaic virus (63.2% identical), and phylogenetic analysis also showed a more distant grouping with tobacco etch and Colombian datura viruses. The virus was detected by RT-PCR in symptomatic I. japonica plants from several regions of China. The virus appears to be an isolate of a new species in the genus Potyvirus, tentatively named butterfly flower mosaic virus.

Localization of sweet potato chlorotic stunt virus (SPCSV) in synergic infection with potyviruses in sweet potato

Among diseases reported worldwidely for sweet potato (Ipomoea batatas (L) Lam) crop, one of the most frequent is the Sweet potato virus disease (SPVD), caused by sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) co-infection. In Argentina, there exists the sweet potato chlorotic dwarf (SPCD), a sweet potato disease caused by triple co-infection with SPCSV, SPFMV and sweet potato mild speckling virus (SPMSV). Both diseases cause a synergism between the potyviruses (SPFMV and SPMSV) and the crinivirus (SPCSV). Up to date, studies carried out on the interaction among these three viruses have not described their localization in the infected tissues. In single infections, virions of the crinivirus genus are limited to the phloem while potyviral virions are found in most tissues of the infected plant. The purpose of this work was to localize the heat shock protein 70 homolog (HSP70h), a movement protein for genus crinivirus, of an Argentinean SPCSV isolate in its single infection and in its double and triple co-infection with SPFMV and SPMSV. The localization was made by in situ hybridization (ISH) for electron microscopy (EM) on ultrathin sections of sweet potato cv. Morada INTA infected tissues. The results demonstrated that viral RNA coding HSP70h is restricted to phloem cells during crinivirus single infection, while it was detected outside the phloem in infections combined with the potyviruses involved in chlorotic dwarf disease.

Occurrence of Colombian datura virus in Brugmansia hybrids, Physalis peruviana L. and Solanum muricatum Ait. in Hungary.

Colombian datura virus (CDV) has been found to infect angel trumpets (Brugmansia spp.) frequently and cape gooseberry (Physalis peruviana) and pepino (Solanum muricatum) sporadically in Hungary. A CDV BRG/H isolate was characterized. It had flexuous thread-like virions of about 750 x 12 nm in size. Host range and symptomathological studies revealed its great similarity to authentic CDV isolates. Nicotiana tabacum cultivars and lines resistant to Potato virus Y (PVYN) either genically or transgenically proved highly susceptible to the BRG/H isolate. Tomato (L. esculentum cvs.) was systemically susceptible to this isolate, but some lines of Lycopersicon hirsutum and L. peruvianum turned out to be resistant. Browallia demissa, Ipomoea purpurea, N. megalosiphon and S. scabrum were demonstrated as new experimental hosts of CDV. The BRG/ H isolate proved to be transmissible by the aphid Myzus persicae Sulz. in a non-persistent manner. Potyvirus-specific coat protein (CP) gene sequences of about 1700 bp from angel trumpet, cape gooseberry and pepino plants were amplified by RT-PCR. The cloned BRG/H CP gene showed a 99.12-99.31% identity with other CDV isolates. CDV has been found for the first time to infect naturally cape gooseberry and pepino. Since the botanical genus name of original hosts of CDV has changed from Datura to Brugmansia, we propose to change the virus name from CDV to Angel trumpet mosaic virus (ATMV).

Further characterization of 'sweet potato virus 2': a distinct species of the genus Potyvirus.

An incompletely described potyvirus isolate from sweet potato in Taiwan, referred to as 'sweet potato virus 2' (SPV2), was further characterised. Electron microscopy revealed that SPV2 has filamentous particles of 850 nm in length and induces cytoplasmic cylindrical inclusions consisting of pinwheels and scrolls. The virus was mechanically transmitted to several species of the genera Chenopodium, Datura, Nicotiana, and Ipomoea. Two biotypes of Myzus persicae transmitted SPV2 in a non-persistent manner. Decoration titer experiments revealed a distant serological relationship between SPV2 and other potyviruses infecting sweet potato. The 3'-terminal 2006 nucleotides of the viral RNA were determined and shown to be a potyviral genome fragment comprising the coding region for the C-terminal half of the NIb protein, the entire coat protein cistron, and the 3' untranslated region (UTR). Comparison of the capsid protein and 3' UTR sequences of SPV2 with those of other potyviruses demonstrated that it is a distinct member of the genus Potyvirus (family Potyviridae). We propose that SPV2 is named Sweet potato virus Y.

Effect of pH, ions, bovine serum albumin and heterologous antisera on the stability of immunosorbed flexuous potato viruses.

Electron microscopic studies on the stability of immunosorbed (trapped) virions of potato viruses X, S and Y0 (PVX, PVS and PVY0) revealed disintegration and dislodging of PVY0 virions upon incubation with (1) antisera to PVX, PVS, or both diluted in saline, (2) 0.86% NaCl (saline) or 0.1 mol/l CaCl2 but not with 0.1 mol/l CaSO4 or 0.1 mol/l MgSO4. PVX virions, on the other hand, showed partial dislodging upon incubation with an antiserum to PVS diluted in saline, but complete disintegration and dislodging with saline. 0.1 mol/l CaCl2 caused partial dislodging while MgCl2, CaSO4 or MgSO4 (all 0.1 mol/l) had no apparent adverse effect. PVS virions were not affected by saline, CaCl2, MgCl2, CaSO4 or MgSO4 (all 0.1 mol/l) and were only partially dislodged by antisera to PVX or PVY0. Disintegration and/or dislodging of the PVX and PVY0 virions was prevented when (1) they were fixed with glutaraldehyde prior to incubation or (2) the virus extract contained bovine serum albumin (BSA) or (3) heterologous antisera were diluted in 0.1 mol/l phosphate buffer (PB) before use except the PVS antiserum which still caused disintegration and dislodging of PVY0 virions. Prior fixation of virions prevented their disruption and dislodging by saline only in the case of PVY0 but not PVX. On the other hand, BSA reverted the adverse effect of saline but not that of the PVS antiserum on PVY0 virions. The results presented here suggest (1) a disruptive effect of Cl' on PVX and PVY0 virions particularly when it was associated with Na+ and (2) an interaction between the immunosorbed virions of PVX or PVY0 and the antiserum to PVS.

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.

Encapsidation of potyviral RNA in various forms of transgene coat protein is not correlated with resistance in transgenic plants.

Transgenic plants expressing either bean yellow mosaic potyvirus or chimeric potyvirus coat protein (CP) were inoculated with various potyviruses. Antigen-coated plate, indirect enzyme-linked immunosorbent assay and immunoelectron microscopy of virus purified from transgenic plants showed that progeny virions contained from < 1% to as much as 25% transgenic CP. Different levels of transcapsidation may reflect the extent of compatibility between transgene CP and the viral CP.

Immunocytology shows the presence of tobacco etch virus P3 protein in nuclear inclusions.

Intracellular localization studies of various potyvirus proteins have been made in hope of finding clues to their function(s). Immunocytological studies localized many of the tobacco etch virus (TEV)-encoded proteins in infected cells. We used antiserum against the nonstructural P3 protein of TEV to determine the subcellular location of the P3 protein in ultrathin sections of virus-infected cells. Immunogold labeling with the antiserum showed labels associated with nucleoli, nuclei, or NIs, Absorption of antiserum with purified NIs or P3 protein resulted in no labeling. TEV NIs are known to contain a bifunctional genome-linked protein-viral proteinase (NIa-VPg) and RNA-dependent RNA polymerase (NIb). It appeared that the TEV P3 protein was a third nonstructural viral protein of NIs of TEV if the NIa-VPg is considered one protein. The presence of P3 in NIs was also supported by Western blot assays. P3 protein in the nucleolus and nucleus could indicate that it, too, is involved in early stages of viral replication.

Loss of potyvirus transmissibility and helper-component activity correlate with non-retention of virions in aphid stylets.

The hypothesis that loss of aphid transmissibility of potyvirus mutants is due to non-retention of virions in the mouthparts was tested by feeding aphids through membranes on purified virions of aphid transmissible (AT or HAT) and non-aphid-transmissible (NAT) tobacco vein mottling virus (TVMV) or tobacco etch virus (TEV), in the presence of functional [potato virus Y (PVY) HC or TVMV HC] or non-functional (PVC HC) helper component (HC). TVMV virions were detected, by electron microscopic examination of immunogold-labelled thin sections, in the food canal or cibarium of 57% of 28 aphids fed on the transmissible combination of TVMV-AT and functional HC, while no virions were found in these structures in 25 aphids fed on the non-transmissible combinations: TVMV-NAT and PVY HC, or TVMV-AT and PVC HC. Autoradiography of intact stylets allowed the examination of much larger numbers of aphids, fed on 125I-labelled TEV; 48% of 523 aphids fed on the TEV-HAT and PVY HC combination retained label in the stylets: this correlated well with the percentage transmission in bioassays. In contrast, in non-transmissible combinations, label was found in the stylets of 0.77% of 389 aphids fed on TEV-NAT and PVY HC, and 1.35% of 223 aphids fed on TEV-HAT and PVC HC. No differences were found in the overall amount of label in the bodies of aphids fed on the transmissible and non-transmissible combinations. There was a strong tendency for virions to be retained in the distal third of the stylets; 56% of aphids positive for TVMV, and 82% of those positive for TEV, had label in this area. These data support the concept that virions retained within the stylets are those that are primarily involved in potyvirus transmission.

The tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication.

The tobacco etch potyvirus (TEV) genome encodes a polyprotein that is processed by three virus-encoded proteinases. Although replication of TEV likely occurs in the cytoplasm, two replication-associated proteins, VPg-proteinase (nuclear inclusion protein a) (NIa) and RNA-dependent RNA polymerase (nuclear inclusion protein b) (NIb), accumulate in the nucleus of infected cells. The 6-kDa protein is located adjacent to the N terminus of NIa in the TEV polyprotein, and, in the context of a 6-kDa protein/NIa (6/NIa) polyprotein, impedes nuclear translocation of NIa (M. A. Restrepo-Hartwig and J. C. Carrington, J. Virol. 66:5662-5666, 1992). The 6-kDa protein and three polyproteins containing the 6-kDa protein were identified by affinity chromatography of extracts from infected plants. Two of the polyproteins contained NIa or the N-terminal VPg domain of NIa linked to the 6-kDa protein. To investigate the role of the 6-kDa protein in vivo, insertion and substitution mutagenesis was targeted to sequences coding for the 6-kDa protein and its N- and C-terminal cleavage sites. These mutations were introduced into a TEV genome engineered to express the reporter protein beta-glucuronidase (GUS), allowing quantitation of virus amplification by a fluorometric assay. Three-amino-acid insertions at each of three positions in the 6-kDa protein resulted in viruses that were nonviable in tobacco protoplasts. Disruption of the N-terminal cleavage site resulted in a virus that was approximately 10% as active as the parent, while disruption of the C-terminal processing site eliminated virus viability. The subcellular localization properties of the 6-kDa protein were investigated by fractionation and immunolocalization of 6-kDa protein/GUS (6/GUS) fusion proteins in transgenic plants. Nonfused GUS was associated with the cytosolic fraction (30,000 x g centrifugation supernatant), while 6/GUS and GUS/6 fusion proteins sedimented with the crude membrane fraction (30,000 x g centrifugation pellet). The GUS/6 fusion protein was localized to apparent membranous proliferations associated with the periphery of the nucleus. These data suggest that the 6-kDa protein is membrane associated and is necessary for virus replication.

Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants.

Tobacco etch potyvirus engineered to express the reporter protein beta-glucuronidase (TEV-GUS) was used for direct observation and quantitation of virus translocation in plants. Four TEV-GUS mutants were generated containing capsid proteins (CPs) with single amino acid substitutions (R154D and D198R), a double substitution (DR), or a deletion of part of the N-terminal domain (delta N). Each modified virus replicated as well as the parental virus in protoplasts, but was defective in cell-to-cell movement through inoculated leaves. The R154D, D198R and DR mutants were restricted essentially to single, initially infected cells. The delta N variant exhibited slow cell-to-cell movement in inoculated leaves, but was unable to move systemically due to a lack of entry into or replication in vascular-associated cells. Both cell-to-cell and systemic movement defects of each mutant were rescued in transgenic plants expressing wild-type TEV CP. Cell-to-cell movement, but not systemic movement, of the DR mutant was rescued partially in transgenic plants expressing TEV CP lacking the C-terminal domain, and in plants expressing CP from the heterologous potyvirus, potato virus Y. Despite comparable levels of accumulation of parental virus and each mutant in symptomatic tissue of TEV CP-expressing transgenic plants, virions were detected only in parental virus- and delta N mutant-infected plants, as revealed using three independent assays. These data suggest that the potyvirus CP possesses distinct, separable activities required for virion assembly, cell-to-cell movement and long-distance transport.