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

Manoussopoulos, Yiannis; Maiß, E.; Tsagris, Mina

doi:10.1099/0022-1317-81-9-2295

Cited by 28 publications

(15 citation statements)

References 11 publications

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“…4). In agreement with this assumption, binding between HCPro and CP from non-aphidtransmissible viruses was previously observed (14,67), which suggests that the interaction between these two viral factors might have a biological role besides aphid-mediated plant-to-plant transmission, such as the one proposed here.…”

Section: Discussionsupporting

confidence: 71%

“…Besides the essential role of CP in viral particle assembly, there is no information about other viral or host factors required for this event. In addition to the expected detection of VPg (55), HCPro was found associated with viral particles (14) in a protrusion located at one of the ends (15). Further experiments also detected the presence of CI in these protruding tips (56).…”

Section: Discussionmentioning

confidence: 84%

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A Novel Role of the Potyviral Helper Component Proteinase Contributes To Enhance the Yield of Viral Particles

Vallí

Gallo

Calvo

et al. 2014

J Virol

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The helper component proteinase (HCPro) is an indispensable, multifunctional protein of members of the genus Potyvirus and other viruses of the family Potyviridae. This viral factor is directly involved in diverse steps of viral infection, such as aphid transmission, polyprotein processing, and suppression of host antiviral RNA silencing. In this paper, we show that although a chimeric virus based on the potyvirus Plum pox virus lacking HCPro, which was replaced by a heterologous silencing suppressor, caused an efficient infection in Nicotiana benthamiana plants, its viral progeny had very reduced infectivity. Making use of different approaches, here, we provide direct evidence of a previously unknown function of HCPro in which the viral factor enhances the stability of its cognate capsid protein (CP), positively affecting the yield of virions and consequently improving the infectivity of the viral progeny. Site-directed mutagenesis revealed that the ability of HCPro to stabilize CP and enhance the yield of infectious viral particles is not linked to any of its previously known activities and helped us to delimit the region of HCPro involved in this function in the central region of the protein. Moreover, the function is highly specific and cannot be fulfilled by the HCPro of a heterologous potyvirus. The importance of this novel requirement in regulating the sorting of the viral genome to be subjected to replication, translation, and encapsidation, thus contributing to the synchronization of these viral processes, is discussed. IMPORTANCEPotyviruses form one of the most numerous groups of plant viruses and are a major cause of crop loss worldwide. It is well known that these pathogens make use of virus-derived multitasking proteins, as well as dedicated host factors, to successfully infect their hosts. Here, we describe a novel requirement for the proper yield and infectivity of potyviral progeny. In this case, such a function is performed by the extensively studied viral factor HCPro, which seems to use an unknown mechanism that is not linked to its previously described activities. To our knowledge, this is the first time that a factor different from capsid protein (CP) has been shown to be directly involved in the yield of potyviral particles. Based on the data presented here, we hypothesize that this capacity of HCPro might be involved in the coordination of mutually exclusive activities of the viral genome by controlling correct assembly of CP in stable virions.

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Section: Discussionsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 84%

A Novel Role of the Potyviral Helper Component Proteinase Contributes To Enhance the Yield of Viral Particles

Vallí

Gallo

Calvo

et al. 2014

J Virol

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“…42 The HC-Pro interacts also with the N terminus of the CP potyviral CP. 18,31,32,43 Such an interaction is thought to be essential for virus transmission. 12,18,43 The N terminus is exposed in the assembled particle and the interaction with HC-Pro may take place during formation of the tip with the CP domain(s) at one (concave) extremity containing the 5 0 end of RNA, rather than along the entire length of the particles.…”

Section: Molecular Architecture Of Potyvirus Particlesmentioning

confidence: 99%

“…Approximately 10% of PVA particles (24/253 particles) imaged in the air or liquid environment contained such a tip structure at one end ( Figure 1 Previous work has demonstrated an interaction between potyviral HC-Pro and virus particles or CP. 18,31,32 To investigate whether HC-Pro was associated with PVY particles, ELISA experiments were done following the method of Roudet-Tavert et al, 32 where virus particles were trapped from infected leaf extracts in ELISA plates coated with a monoclonal antibody (mAb) against the CP (mAb SCR 39). HC-Pro or virus CP were detected by anti-HC-Pro or anti-CP antiserum (rabbit) followed by anti-rabbit alkaline phosphatase conjugate.…”

mentioning

confidence: 99%

An Unusual Structure at One End of Potato Potyvirus Particles

Torrance¹,

Андреев

Gabrenaite-Verhovskaya

et al. 2006

Journal of Molecular Biology

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“…Later, it was exploited for the separation of soluble protein complexes of mitochondrial matrix [4], plasma membrane protein complexes [5][6][7], or whole cell lysates [8]. Moreover, an adaptation of the technique to agarose gel matrices with a higher separation limit was utilized for separation of very large protein complexes such as pyruvate dehydrogenase complex [4] or virus particles [9]. The procedure has several advantages that make the BN-PAGE suitable also for separation of nuclear protein complexes.…”

Section: Introductionmentioning

confidence: 99%

Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis

et al. 2006

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The nucleus is a highly structured organelle with distinct compartmentalization of specific functions. To understand the functions of these nuclear compartments, detailed description of protein complexes which form these structures is of crucial importance. We explored therefore the potential of blue native PAGE (BN-PAGE) method for the separation of nuclear protein complexes. We focused on (i) solubility and stability of nuclear complexes under conditions prerequisite for the separation by BN-PAGE, (ii) improved separation of native nuclear protein complexes using 2-D colorless native/blue native PAGE (CN-/BN-PAGE), and (iii) mass spectrometric analysis of protein complexes which were isolated directly from native 1-D or from 2-D CN/BN-PAGE gels. The suitability of BN-PAGE for nuclear proteomic research is demonstrated by the successful separation of polymerase I and polymerase II complexes, and by mass spectrometric determination of U1 small nuclear ribonucleoprotein particle composition. Moreover, practical advice for sample preparation is provided.

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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

Cited by 28 publications

References 11 publications

A Novel Role of the Potyviral Helper Component Proteinase Contributes To Enhance the Yield of Viral Particles

A Novel Role of the Potyviral Helper Component Proteinase Contributes To Enhance the Yield of Viral Particles

An Unusual Structure at One End of Potato Potyvirus Particles

Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis

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