The Open Reading Frame III Product of Cauliflower Mosaic Virus Forms a Tetramer through a N-terminal Coiled-coil

Leclerc, Denis; Burri, Lena; Kajava, Andrey V.; Mougeot, Jean-Luc; Heß, Daniel; Lustig, Ariel; Kleemann, Gerd R.; Höhn, Thomas

doi:10.1074/jbc.273.44.29015

Cited by 44 publications

(47 citation statements)

References 33 publications

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“…Sequence-based secondary-structure predictions indicate that P3 is composed of two amphipathic ␣-helices, with residues 3 to 32 and 38 to 57, respectively, having high and moderate propensities to form coiled-coil structures-a widespread protein-protein interaction and oligomerization motif (1)-thought to be assembled as a parallel tetramer (22,42). The domain organization of the P3 protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to the two-stranded antiparallel conformation when anchored to virions (37), the N-terminal ␣-helical domain of P3 has been demonstrated to form a distinct coiled-coil structure as a parallel tetramer when free in solution (22). Consistently, a tetrameric form of P3 has been extracted from infected plants (45,46) and also shown to exist in other species of the family Caulimoviridae (42), suggesting a different biological role for this conformation.…”

mentioning

confidence: 98%

“…However, sequence analyses revealed the presence of coiled-coil motifs, subsequently confirmed by circular-dichroism measurements of the purified protein (data not shown). The parallel tetrameric organization was also proposed from molecular-modeling studies (22). Initial phases of the hexagonal crystal form were determined by exploratory molecular replacement using different canonical coiled-coil models (5 ).…”

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confidence: 99%

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Structural Insights into the Molecular Mechanisms of Cauliflower Mosaic Virus Transmission by Its Insect Vector

Hoh

Uzest

Drucker

et al. 2010

J Virol

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Cauliflower mosaic virus (CaMV) is transmitted from plant to plant through a seemingly simple interaction with insect vectors. This process involves an aphid receptor and two viral proteins, P2 and P3. P2 binds to both the aphid receptor and P3, itself tightly associated with the virus particle, with the ensemble forming a transmissible viral complex. Here, we describe the conformations of both unliganded CaMV P3 protein and its virion-associated form. X-ray crystallography revealed that the N-terminal domain of unliganded P3 is a tetrameric parallel coiled coil with a unique organization showing two successive four-stranded subdomains with opposite supercoiling handedness stabilized by a ring of interchain disulfide bridges. A structural model of virus-liganded P3 proteins, folding as an antiparallel coiled-coil network coating the virus surface, was derived from molecular modeling. Our results highlight the structural and biological versatility of this coiled-coil structure and provide new insights into the molecular mechanisms involved in CaMV acquisition and transmission by the insect vector.Cauliflower mosaic virus (CaMV) is the type member of the plant virus family Caulimoviridae. This family is grouped together with hepadnaviruses into the pararetrovirus group due to its mode of replication via reverse transcription of a pregenomic RNA intermediate (17). The CaMV genome is a double-stranded circular DNA of approximately 8,000 bp, comprising seven major open reading frames (ORFs), only six of which have clearly identified biological functions. The product of ORF VI (P6) is expressed early in infection from the monocistronic 19S RNA. In addition to its recently reported role in the suppression of RNA silencing (14), P6 rapidly self-aggregates into the so-called electron-dense inclusion bodies, demonstrated to be the viral factory (17). Within these structures, P6 then trans-activates the translation of ORFs I to V from the polycistronic pregenomic 35S RNA according to a complex reinitiation process (39). ORFs I to V encode the major capsid protein (P4, encoded by ORF IV), reverse transcriptase (P5, encoded by ORF V), and three auxiliary proteins: P1 (ORF I) is involved in cell-to-cell and long-distance within-plant movement, P2 (ORF II) is involved in aphid transmission, and P3 (ORF III) is tightly associated with the virus particles and has a complex regulatory function in the virus infection cycle, particularly during plant-to-plant vector transmission.The CaMV viral particle is roughly spherical, 520 Å in diameter, with an icosahedral T7 symmetry. It is constituted of three concentric shells, built from 420 capsid protein subunits, surrounding a large inner cavity with a diameter of approximately 250 Å (4, 37). The physical association between P3 and viral particles has been consistently demonstrated in mature CaMV virions by copurification, immunolabeling experiments, and in vitro interactions (7,(23)(24)(25). Recently, cryoelectron microscopy (cryo-EM) image reconstruction further showed that P3 molecu...

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Section: Resultsmentioning

confidence: 99%

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confidence: 98%

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confidence: 99%

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Structural Insights into the Molecular Mechanisms of Cauliflower Mosaic Virus Transmission by Its Insect Vector

Hoh

Uzest

Drucker

et al. 2010

J Virol

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“…Interestingly, a coiled-coil domain promotes the interaction between the PIII and PII proteins of Cauliflower mosaic virus during transmission by vector aphids (Leclerc et al, 1998 ;Leh et al, 1999). Two mutations of the PEBV TpA56 2b protein are known to severely affect nematode transmission of the virus.…”

Section: Tobraviruses Which Include Pea Early-browning Virus (Pebv)mentioning

confidence: 99%

Substitution of a single amino acid in the 2b protein of Pea early-browning virus affects nematode transmission

Vellios

Brown

MacFarlane

2002

Journal of General Virology

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The 2b protein of Pea early-browning virus (PEBV) is required for transmission of the virus by nematodes. Comparison of the 2b proteins of highly transmissible (TpA56) and poorly transmissible (SP5) isolates of PEBV identified two amino acid substitutions (G90S and G177R) that might be responsible for the poor transmission of isolate SP5. Hybrid viruses were created in which the TpA56 2b protein carried SP5-specific substitutions at residue 90 or 177, and in which the SP5 2b protein carried TpA56-specific substitutions at these positions. Transmission tests showed that the G177R substitution is sufficient to prevent nematode transmission of the virus. Examination of the 2b proteins from PEBV and other tobraviruses predicted the presence of a coiled-coil domain in the central region of the protein. This structural element is important for the association of interacting proteins and, thus, might mediate interaction of the 2b protein with the virus coat protein or with the vector nematode.Tobraviruses, which include Pea early-browning virus (PEBV), Tobacco rattle virus (TRV) and Pepper ringspot virus (PepRSV) are transmitted between plants by soil-inhabiting nematodes (Taylor & Brown, 1997). Glasshouse tests have demonstrated that particular tobravirus isolates can be transmitted only by certain nematode species. For example, TRV isolate PpK20 can be transmitted by Paratrichodorus pachydermus but not by P. anemones or Trichodorus primitivus (Herna! ndez et al., 1997). In contrast, TRV isolate PaY4 can be transmitted by both P. pachydermus and P. anemones (Vassilakos, 2000 ; Vassilakos et al., 2001), whereas PEBV isolate TpA56 is transmitted by T. primitivus but not by P. pachydermus (MacFarlane & Brown, 1995).Tobraviruses have two positive-stranded genomic RNAs, each of which is encapsidated separately in a rod-shaped particle. The larger RNA (RNA1) encodes proteins involved in Author for correspondence : Stuart MacFarlane.Fax j44 1382 562426. e-mail s.macfarlane!scri.sari.ac.uk the replication and spread of the virus in plants, and can infect plants systemically in the complete absence of the second, smaller genomic RNA (RNA2) (Harrison & Robinson, 1986). RNA2 was shown to carry the determinants for nematode transmission by the analysis of pseudorecombinant isolates of TRV in which the genomic RNAs from the nematodetransmissible PpK20 isolate and the non-transmissible PLB isolate were re-assorted (Ploeg et al., 1993). These experiments showed that inclusion of RNA2 from TRV PpK20 led to the transmission of TRV PLB RNA1, and the replacement of PpK20 RNA2 with PLB RNA2 resulted in failure to transmit PpK20 RNA1. Thus, RNA2 of TRV PpK20 encoded a transmission determinant(s) that was lacking in RNA2 of TRV PLB.RNA2 has been sequenced from many tobravirus isolates, revealing a great variation in the size and gene content (MacFarlane, 1999). All isolates encode a coat protein (CP), although deleted forms of RNA2 lacking the CP gene can be generated in infected plants (Herna! ndez et al., 1996). Many isolates include at ...

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“…However, studies have suggested that CaMV virions do not appear to directly interact with the MP. Instead, the MP interacts with the CaMV P3 protein (also known as the virion-associated protein [VAP]), which forms a trimeric structure that is anchored into the virions (Leclerc et al, 1998;Leclerc et al, 2001). Electron microscopy studies have indicated that MP and VAP colocalize with virions only at the entrance to or within the plasmodesmata, and it has been suggested that the VAP/virion complex travels to the plasmodesmata independently from the MP (Stavolone et al, 2005).…”

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Association of the P6 Protein of Cauliflower mosaic virus with Plasmodesmata and Plasmodesmal Proteins

et al. 2014

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The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.Through the years, numerous studies have focused on the characterization of viral replication sites within the cell, as well as how plant virus movement proteins (MPs) modify the plasmodesmata to facilitate cell-tocell movement (for review, see Benitez-Alfonso et al.,

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The Open Reading Frame III Product of Cauliflower Mosaic Virus Forms a Tetramer through a N-terminal Coiled-coil

Cited by 44 publications

References 33 publications

Structural Insights into the Molecular Mechanisms of Cauliflower Mosaic Virus Transmission by Its Insect Vector

Structural Insights into the Molecular Mechanisms of Cauliflower Mosaic Virus Transmission by Its Insect Vector

Substitution of a single amino acid in the 2b protein of Pea early-browning virus affects nematode transmission

Association of the P6 Protein of Cauliflower mosaic virus with Plasmodesmata and Plasmodesmal Proteins

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