马铃薯Y病毒属病毒P3和P3-PiPo蛋白功能研究进展

Potyvirus属于马铃薯Y病毒科Potyviridae,是最大的植物病毒属,给农业生产造成严重的经济损失。P3是Potyvirus属病毒中变异很大、功能较复杂的编码蛋白,涉及到病毒复制、侵染、抗性及细胞间运动;P3-PiPo是P3编码框内新近发现的Potyvirus重要编码蛋白,已证实它在病毒的细胞间运动中起着决定性的作用。对病毒蛋白功能的研究为该属病毒的研究发展提供重要的理论基础,对Potyvirus侵染机制及抗病机理研究具有指导价值。     

Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.     

Mutational analysis of interaction between coat protein and helper component-proteinase of Soybean mosaic virus involved in aphid transmission

Soybean mosaic virus (SMV), a member of the genus Potyvirus , is transmitted by aphids in a non-persistent manner. It has been well documented that the helper component-proteinase (HC-Pro) plays a role as a 'bridge' between virion particles and aphid stylets in the aphid transmission of potyviruses. Several motifs, including the KITC and PTK motifs on HC-Pro and the DAG motif on the coat protein (CP), have been found to be involved in aphid transmission. Previously, we have shown strong interaction between SMV CP and HC-Pro in a yeast two-hybrid system (YTHS). In this report, we further analysed this CP–HC-Pro interaction based on YTHS and an in vivo binding assay to identify crucial amino acid residues for this interaction. Through this genetic approach, we identified two additional amino acid residues (H256 on CP and R455 on HC-Pro), as well as G12 on the DAG motif, crucial for the CP–HC-Pro interaction. We introduced mutations into the identified residues using an SMV infectious clone and showed that these mutations affected the efficiency of aphid transmission of SMV. We also investigated the involvement of the PTK and DAG motifs in the CP–HC-Pro interaction and aphid transmission of SMV. Our results support the concept that physical interaction between CP and HC-Pro is important for potyviral aphid transmission. Based on the combination of our current results with previous findings, the possibility that aphid transmission may be regulated by more complex molecular interactions than the simple involvement of HC-Pro as a bridge is discussed.     

Uridylylation of the potyvirus VPg by viral replicase NIb correlates with the nucleotide binding capacity of VPg

Poty- and picornaviruses share similar genome organizations and polyprotein processing strategies. By analogy to picornaviruses it has been proposed that the genome-linked protein VPg may serve as a primer for genome replication of potyviruses. The multifunctional VPg of potato virus A (PVA; genus Potyvirus) was found to be uridylylated by NIb, the RNA polymerase of PVA. The nucleotidylation activity of NIb is more efficient in the presence of Mn(2+) than Mg(2+) and does not require an RNA template. Our results suggest that the nucleotidylation reaction exhibits weak preference for UTP over the other NTPs. An NTP-binding experiment with oxidized [alpha-(32)P]UTP revealed that PVA VPg contains an NTP-binding site. Deletion of a 7-amino acid-long putative NTP-binding site from VPg reduced nucleotide-binding capacity and debilitated uridylylation reaction. These results provide evidence that VPg may play a similar role in RNA synthesis of potyviruses as it does in the case of picornaviruses.     

Complete Protein Linkage Map of Poliovirus P3 Proteins: Interaction of Polymerase 3Dpol with VPg and with Genetic Variants of 3AB

Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3D^pol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3D^pol interaction. The viral proteinase 3C^pro was not found to interact with other 3C^pro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CD^pro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.     

马铃薯Y病毒属病毒HC-Pro蛋白功能研究进展

马铃薯Ｙ病毒属病毒基因组编码的ＨＣ－Ｐｒｏ 蛋白（蚜传辅助因子）具有多种功能,在病毒生活史各个环节中起重要作用。ＨＣ－Ｐｒｏ 蛋白具有蛋白酶活性,作为蚜传辅助因子参与病毒蚜传过程,调节病毒在宿主体内的转移,并在病毒复制、宿主症状表达及增强异源病毒复制等方面发挥作用。本文对ＨＣ－Ｐｒｏ 蛋白的既定、预测功能作一综述。深入了解ＨＣ－Ｐｒｏ 蛋白的功能不仅在理论上有助于明确病毒生活周期,而且在实践上也可根据其特性设计抗病毒的新策略。     

Plant virus HC-Pro is a determinant of eriophyid mite transmission

The eriophyid mite transmitted Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) shares a common genome organization with aphid transmitted species of the genus Potyvirus. Although both tritimoviruses and potyviruses encode helper component-proteinase (HC-Pro) homologues (required for nonpersistent aphid transmission of potyviruses), sequence conservation is low (amino acid identity, approximately 16%), and a role for HC-Pro in semipersistent transmission of WSMV by the wheat curl mite (Aceria tosichella [Keifer]) has not been investigated. Wheat curl mite transmissibility was abolished by replacement of WSMV HC-Pro with homologues of an aphid transmitted potyvirus (Turnip mosaic virus), a rymovirus (Agropyron mosaic virus) vectored by a different eriophyid mite, or a closely related tritimovirus (Oat necrotic mottle virus; ONMV) with no known vector. In contrast, both WSMV-Sidney 81 and a chimeric WSMV genome bearing HC-Pro of a divergent strain (WSMV-El Batán 3; 86% amino acid sequence identity) were efficiently transmitted by A. tosichella. Replacing portions of WSMV-Sidney 81 HC-Pro with the corresponding regions from ONMV showed that determinants of wheat curl mite transmission map to the 5'-proximal half of HC-Pro. WSMV genomes bearing HC-Pro of heterologous species retained the ability to form virions, indicating that loss of vector transmissibility was not a result of failure to encapsidate. Although titer in systemically infected leaves was reduced for all chimeric genomes relative to WSMV-Sidney 81, titer was not correlated with loss of vector transmissibility. Collectively, these results demonstrate for the first time that HC-Pro is required for virus transmission by a vector other than aphids.     

Viral genome-linked protein (VPg) controls accumulation and phloem-loading of a potyvirus in inoculated potato leaves

The viral protein covalently linked to the 5' end of the plus-sense, single-stranded RNA genome of potyviruses (genus Potyvirus) can be an avirulence determinant in incompatible potyvirus-host combinations in which the resistance prevents systemic virus infection. The mechanism is not well known. This study shows that virus strain-specific resistance to systemic infection with Potato virus A (PVA) in Solanum commersonii is overcome by a single amino acid (aa) substitution, His118Tyr, in the viral genome-linked protein (VPg). Virus localization and other experiments revealed that Tyr118, controls phloem loading of PVA. The critical boundary may be constituted in phloem parenchyma, companion cells, or both. Tyr118 also controls the cellular level of virus accumulation in infected leaves, including phloem cells. Amino acid substitutions at three additional positions of the central part (aa 116) and C terminus (aa 185) of the VPg and of the N terminus of the 6K2 protein (aa 5) affect virus accumulation and rate of systemic infection but are not sufficient for phloem loading of PVA. These data, together with previous studies, indicate that the PVA VPg aa residues crucial for systemic infection are host specific. Also, our data and previous studies on other potyvirus-host species combinations indicate that the central part of the VPg is a domain with universal importance to virus-host interactions required for systemic invasion of plants with potyviruses.     

The interaction of soybean reticulon homology domain protein (GmRHP) with Soybean mosaic virus encoded P3 contributes to the viral infection

Soybean mosaic virus (SMV), a member of the Potyvirus genus, is a prevalent and devastating viral pathogen in soybean-growing regions worldwide. Potyvirus replication occurs in the 6K2-induced viral replication complex at endoplasmic reticulum exit sites. Potyvirus-encoded P3 is also associated with the endoplasmic reticulum and is as an essential component of the viral replication complex, playing a key role in viral replication. This study provides evidence that the soybean (Glycine max) reticulon homology domain protein (designated as GmRHP) interacts with SMV-P3 by using a two-hybrid yeast system to screen a soybean cDNA library. A bimolecular fluorescence complementation assay further confirmed the interaction, which occurred on the cytomembrane, endoplasmic reticulum and cytoskeleton in Nicotiana benthamiana cells. The transient expression of GmRHP can promote the coupling of Turnip mosaic virus replication and cell-to-cell movement in N. benthamiana. The interaction between the membrane protein SMV-P3 and GmRHP may contribute to the potyvirus infection, and GmRHP may be an essential host factor for P3's involvement in potyvirus replication.     

Interaction of Sesbania mosaic virus movement protein with VPg and P10: implication to specificity of genome recognition

Sesbania mosaic virus (SeMV) is a single strand positive-sense RNA plant virus that belongs to the genus Sobemovirus. The mechanism of cell-to-cell movement in sobemoviruses has not been well studied. With a view to identify the viral encoded ancillary proteins of SeMV that may assist in cell-to-cell movement of the virus, all the proteins encoded by SeMV genome were cloned into yeast Matchmaker system 3 and interaction studies were performed. Two proteins namely, viral protein genome linked (VPg) and a 10-kDa protein (P10) c v gft encoded by OFR 2a, were identified as possible interacting partners in addition to the viral coat protein (CP). Further characterization of these interactions revealed that the movement protein (MP) recognizes cognate RNA through interaction with VPg, which is covalently linked to the 5' end of the RNA. Analysis of the deletion mutants delineated the domains of MP involved in the interaction with VPg and P10. This study implicates for the first time that VPg might play an important role in specific recognition of viral genome by MP in SeMV and shed light on the possible role of P10 in the viral movement.     

Two patches of amino acids on the E2 DNA binding domain define the surface for interaction with E1

The E1 and E2 proteins from bovine papillomavirus bind cooperatively to the viral origin of DNA replication (ori), forming a complex which is essential for initiation of DNA replication. Cooperative binding has two components, in which (i) the DNA binding domains (DBDs) of the two proteins interact with each other and (ii) the E2 transactivation domain interacts with the helicase domain of E1. By generating specific point mutations in the DBD of E2, we have defined two patches of amino acids that are involved in the interaction with the E1 DBD. These same mutations, when introduced into the viral genome, result in severely reduced replication of the viral genome, as well as failure to transform mouse cells in tissue culture. Thus, the interaction between the E1 and E2 DBDs is important for the establishment of the viral genome as an episome and most likely contributes to the formation of a preinitiation complex on the viral ori.     

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors.     

Degradation of cellular proteins during poliovirus infection: studies by two-dimensional gel electrophoresis.

Picornaviruses encode for their own proteinases, which are responsible for the proteolytic processing of the polyprotein encoded in the viral genome to produce the mature viral polypeptides. The two poliovirus proteinases, known as proteins 2A and 3C, use the poliovirus-encoded polyprotein as a substrate. The possibility that these poliovirus proteinases also degrade cellular proteins remains largely unexplored. High-resolution two-dimensional gel electrophoresis indicates that a few cellular proteins disappear after poliovirus infection. Thus, at least nine acidic and five basic cellular proteins, ranging in Mr from 120 to 30 kilodaltons, are clearly degraded during poliovirus infection of HeLa cells. The degradation of these cellular polypeptides is very specific because it does not occur upon infection of HeLa cells with encephalomyocarditis virus or Semliki Forest virus. Moreover, inhibitors of poliovirus replication, such as cycloheximide or 3-methylquercetin, block the disappearance of these polypeptides. These results suggest that the input virions are not responsible for this degradation and that active poliovirus replication is required for the proteolysis to occur. Analysis of the time course of the disappearance of these polypeptides indicates that it does not occur during the first 2 h of infection, clearly suggesting that this phenomenon is not linked to the poliovirus-induced shutoff of host protein synthesis. This conclusion is strengthened by the finding that 3-methylquercetin blocks proteolysis without preventing shutoff of host translation.     

Two potato proteins,including a novel RING finger protein (HIP1), interact with the potyviral multifunctional protein HCpro

Potyviral helper-component proteinase (HCpro) is a multifunctional protein exerting its cellular functions in interaction with putative host proteins. In this study, cellular protein partners of the HCpro encoded by Potato virus A (PVA) (genus Potyvirus) were screened in a potato leaf cDNA library using a yeast two-hybrid system. Two cellular proteins were obtained that interact specifically with PVA HCpro in yeast and in the two in vitro binding assays used. Both proteins are encoded by single-copy genes in the potato genome. Analysis of the deduced amino acid sequences revealed that one (HIP1) of the two HCpro interactors is a novel RING finger protein. The sequence of the other protein (HIP2) showed no resemblance to the protein sequences available from databanks and has known biological functions.     

The Mre11/Rad50/Nbs1 complex limits adeno-associated virus transduction and replication

Adeno-associated virus (AAV) is a parvovirus with a small single-stranded DNA genome that relies on cellular replication machinery together with functions supplied by coinfecting helper viruses. The impact of host factors on AAV infection is not well understood. We explored the connection between AAV helper functions supplied by adenovirus and cellular DNA repair proteins. The adenoviral E1b55K/E4orf6 proteins induce degradation of the cellular Mre11 repair complex (MRN) to promote productive adenovirus infection. These viral proteins also augment recombinant AAV transduction and provide crucial helper functions for wild-type AAV replication. Here, we show that MRN poses a barrier to AAV and that the helper function provided by E1b55K/E4orf6 involves MRN degradation. Using a fluorescent method to visualize the viral genome, we show an effect at the viral DNA level. MRN components accumulate at AAV replication centers and recognize the viral inverted terminal repeats. Together, our data suggest that AAV is targeted by MRN and has evolved to exploit adenoviral proteins that degrade these cellular factors.     

Polyprotein processing in picornavirus replication

The primary translation product of the picornavirus genome is a single large protein which is processed to the mature viral polypeptides by progressive, co- and post-translational cleavages. Replication of the picornaviruses is thus entirely dependent upon the proteolysis of viral precursor proteins. In poliovirus, two virus-encoded proteinases have been identified that catalyze all but the final cleavage of the viral polyprotein. The final processing event, maturation of the virion polypeptide VPO, appears to occur by an unusual autocatalytic serine proteinase-like mechanism. Proteolytic processing of viral precursor proteins is basically similar in all picornaviruses, but recently it has become clear that there are also important differences between these viruses. Understanding of the processing events in picornavirus replication may ultimately lead to the discovery of specific inhibitors of the viral enzymes that could prove clinically useful as anti-viral agents.     

Transgenic Brassica rapa plants over‐expressing eIF(iso)4E variants show broad‐spectrum Turnip mosaic virus (TuMV) resistance

The protein–protein interaction between VPg (viral protein genome‐linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad‐spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge‐based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap‐binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap‐binding pockets, and mutated. Yeast two‐hybrid assay and co‐immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E‐knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild‐type were over‐expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T₁ and T₂ transformants demonstrated that the over‐expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge‐based approaches for the engineering of broad‐spectrum resistance in Chinese cabbage.