Stabilization of cauliflower mosaic virus P3 tetramer by covalent linkage

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein (P3) that is indispensable for viral infectivity. Although P3 has been shown to be a prerequisite for CaMV aphid transmission, its role in viral replication remains unknown. We previously showed that P3 forms a tetramer in planta and that P3 tetramer co-sediments with viral coat protein on sucrose gradient centrifugation, suggesting that a tetramer may be the functional form of P3. We presumed that disulfide bonds were involved in tetramer formation because 1) the tetramer was detected by Western blotting after electrophoresis under non-reducing conditions, and 2) the cysteine-X-cysteine motif is well conserved in CaMV P3 and P3 homologues among Caulimoviruses. Therefore we mutated either or both of the cysteine residues of CaMV P3. The mutant viruses were infectious and accumulated to a similar extent as the wild-type. An analysis of mutant proteins confirmed that the wild-type P3 molecules in the tetramer are covalently bound with one another through disulfide bonds. It was also suggested that mutant proteins are less stable than wild-type protein in planta. Furthermore, sedimentation study suggested that the disulfide bonds are involved in stable association of P3 with CaMV virions or virion-like particles, or both. The mutant viruses could be transmitted by aphids. These results suggested that the covalent bonds in P3 tetramer are dispensable for biological activity of P3 under experimental situations and may have some biological significance in natural infection in the field.     

Virus Factories of Cauliflower Mosaic Virus Are Virion Reservoirs That Engage Actively in Vector Transmission

Cauliflower mosaic virus (CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.     

Structure of the mature P3-virus particle complex of cauliflower mosaic virus revealed by cryo-electron microscopy

The cauliflower mosaic virus (CaMV) has an icosahedral capsid composed of the viral protein P4. The viral product P3 is a multifunctional protein closely associated with the virus particle within host cells. The best-characterized function of P3 is its implication in CaMV plant-to-plant transmission by aphid vectors, involving a P3-virion complex. In this transmission process, the viral protein P2 attaches to virion-bound P3, and creates a molecular bridge between the virus and a putative receptor in the aphid's stylets. Recently, the virion-bound P3 has been suggested to participate in cell-to-cell or long-distance movement of CaMV within the host plant. Thus, as new data accumulate, the importance of the P3-virion complex during the virus life-cycle is becoming more and more evident. To provide a first insight into the knowledge of the transmission process of the virus, we determined the 3D structures of native and P3-decorated virions by cryo-electron microscopy and computer image processing. By difference mapping and biochemical analysis, we show that P3 forms a network around the capsomers and we propose a structural model for the binding of P3 to CaMV capsid in which its C terminus is anchored deeply in the inner shell of the virion, while the N-terminal extremity is facing out of the CaMV capsid, forming dimers by coiled-coil interactions. Our results combined with existing data reinforce the hypothesis that this coiled-coil N-terminal region of P3 could coordinate several functions during the virus life-cycle, such as cell-to-cell movement and aphid-transmission.     

Biomarker discovery from the top down: Protein biomarkers for efficient virus transmission by insects (Homoptera: Aphididae) discovered by coupling genetics and 2-D DIGE

Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are vectored by aphids. The identification of vector proteins mediating virus transmission is critical to develop sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Previously, we applied 2-D DIGE to an aphid filial generation 2 population to identify proteins correlated with the transmission phenotype that were stably inherited and expressed in the absence of the virus. In the present study, we examined the expression of the DIGE candidates in previously unstudied, field-collected aphid populations. We hypothesized that the expression of proteins involved in virus transmission could be clinically validated in unrelated, virus transmission-competent, field-collected aphid populations. All putative biomarkers were expressed in the field-collected biotypes, and the expression of nine of these aligned with the virus transmission-competent phenotype. The strong conservation of the expression of the biomarkers in multiple field-collected populations facilitates new and testable hypotheses concerning the genetics and biochemistry of virus transmission. Integration of these biomarkers into current aphid-scouting methodologies will enable rational strategies for vector control aimed at judicious use and development of precision pest control methods that reduce plant virus infection.     

Cauliflower mosaic virus protein P6-TAV plays a major role in alteration of aphid vector feeding behaviour but not performance on infected Arabidopsis

Emerging evidence suggests that viral infection modifies host plant traits that in turn alter behaviour and performance of vectors colonizing the plants in a way conducive for transmission of both nonpersistent and persistent viruses. Similar evidence for semipersistent viruses like cauliflower mosaic virus (CaMV) is scarce. Here we compared the effects of Arabidopsis infection with mild (CM) and severe (JI) CaMV isolates on the feeding behaviour (recorded by the electrical penetration graph technique) and fecundity of the aphid vector Myzus persicae. Compared to mock-inoculated plants, feeding behaviour was altered similarly on CM- and JI-infected plants, but only aphids on JI-infected plants had reduced fecundity. To evaluate the role of the multifunctional CaMV protein P6-TAV, aphid feeding behaviour and fecundity were tested on transgenic Arabidopsis plants expressing wild-type (wt) and mutant versions of P6-TAV. In contrast to viral infection, aphid fecundity was unchanged on all transgenic lines, suggesting that other viral factors compromise fecundity. Aphid feeding behaviour was modified on wt P6-CM-, but not on wt P6-JI-expressing plants. Analysis of plants expressing P6 mutants identified N-terminal P6 domains contributing to modification of feeding behaviour. Taken together, we show that CaMV infection can modify both aphid fecundity and feeding behaviour and that P6 is only involved in the latter.     

Genetics coupled to quantitative intact proteomics links heritable aphid and endosymbiont protein expression to circulative polerovirus transmission

Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid, while six proteins were predicted to be associated with the accessory salivary glands or hemolymph. Knowledge of the proteins that regulate virus transmission and their predicted locations will aid in understanding the biochemical mechanisms regulating circulative virus transmission in aphids, as well as in identifying new targets to block transmission.     

Interaction of chandipura virus N and P proteins: identification of two mutually exclusive domains of N involved in interaction with P

The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N(0)) form (N(0)-P complex). It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1-180 form the N(0)-P interacting region, whereas, C-terminal residues spanning aa 320-390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N(0).     

Effect of osmolytes and chaperone-like action of P-protein on folding of nucleocapsid protein of Chandipura virus.

Amino acid sequences of nucleocapsid proteins are mostly conserved among different rhabdoviruses. The protein plays a common functional role in different RNA viruses by enwrapping the viral genomic RNA in an RNase-resistant form. Upon expression of the nucleocapsid protein alone in COS cells and in bacteria, it forms large insoluble aggregates. In this work, we have reported for the first time the full-length cloning of the N gene of Chandipura virus and its expression in Escherichia coli in a soluble monomeric form and purification using nonionic detergents. The biological activity of the soluble recombinant protein has been tested, and it was found to possess efficient RNA-binding ability. The state of aggregation of the recombinant protein was monitored using light scattering. In the absence of nonionic detergents, it formed large aggregates. Aggregation was significantly reduced in the presence of osmolytes such as d-sorbitol. Aggregate formation was suppressed in the presence of another viral product, phosphoprotein P, in a chaperone-like manner. Both the osmolyte and phosphoprotein P also suppressed aggregation to a great extent during refolding from a guanidine hydrochloride-denatured form. The function of the phosphoprotein and osmolyte appears to be synergistic to keep the N-protein in a soluble biologically competent form in virus-infected cells.     

A single amino acid position in the helper component of cauliflower mosaic virus can change the spectrum of transmitting vector species

Viruses frequently use insect vectors to effect rapid spread through host populations. In plant viruses, vector transmission is the major mode of transmission, used by nearly 80% of species described to date. Despite the importance of this phenomenon in epidemiology, the specificity of the virus-vector relationship is poorly understood at both the molecular and the evolutionary level, and very limited data are available on the precise viral protein motifs that control specificity. Here, using the aphid-transmitted Cauliflower mosaic virus (CaMV) as a biological model, we confirm that the "noncirculative" mode of transmission dominant in plant viruses (designated "mechanical vector transmission" in animal viruses) involves extremely specific virus-vector recognition, and we identify an amino acid position in the "helper component" (HC) protein of CaMV involved in such recognition. Site-directed mutagenesis revealed that changing the residue at this position can differentially affect transmission rates obtained with various aphid species, thus modifying the spectrum of vector species for CaMV. Most interestingly, in a virus line transmitted by a single vector species, we observed the rapid appearance of a spontaneous mutant specifically losing its transmissibility by another aphid species. Hence, in addition to the first identification of an HC motif directly involved in specific vector recognition, we demonstrate that change of a virus to a different vector species requires only a single mutation and can occur rapidly and spontaneously.     

烟草在PVYN病毒与烟蚜作用下对高CO2浓度的响应

以CO2浓度为主处理因子,研究了加倍CO2浓度和对照大气CO2浓度条件下,烟蚜、马铃薯Y病毒N株(PVYN)以及二者共同作用下烟草各指标的响应。结果表明,在当前CO2浓度条件下,PVYN、烟蚜及两者联合作用对烟草生物量影响不显著;而在未来高CO2浓度条件下,PVYN、烟蚜及两者联合作用对烟草生物量影响很大。CO2浓度升高后,PVYN和蚜虫二者联合作用显著降低烟草产量,危害加重,高CO2的"肥料"作用被极大地削弱。在有烟蚜、PVYN以及两者共同作用时烟草的化学物质及主要的次生代谢物烟碱的含量对CO2浓度升高的响应也发生一定的变化,表现在:高CO2浓度条件下,蚜虫、蚜虫与PVYN共同作用显著增加了烟草的含氮量;显著减少了烟叶含糖量;PVYN及其与蚜虫共同作用显著升高叶片可溶性蛋白含量;当高CO2浓度下,各处理的烟草烟碱含量均显著下降,而且PVYN感染的烟叶烟碱含量无论在哪一种CO2浓度条件下,都比无毒无虫的对照烟叶烟碱含量升高。结果显示,烟蚜和马铃薯Y病毒N株(PVYN)对烟草的产量、营养物质及防御物质都有影响;CO2浓度升高对烟草的生长有促进作用,增加了烟草的产量,但蚜虫的危害和PVYN感染使烟草产量下降,在高CO2浓度条件下,烟蚜和PVYN共同作用相对于目前CO2浓度对烟草产量的危害加重。     

Localization of domains within the Drosophila Ref(2)P protein involved in the intracellular control of sigma rhabdovirus multiplication.

The ref(2)P gene of Drosophila melanogaster interferes with sigma rhabdovirus multiplication. This gene is highly variable, and the different alleles are considered permissive or restrictive according to their effects on virus replication. In all cases, the mechanisms involve intracellular interactions between the sigma virus and Ref(2)P proteins. We showed that the N-terminal domain of the Ref(2)P protein was required for its activity in vivo. The protein was inactive in the null p(od)2 mutant when its first 82 amino acids were deleted. The p delta n gene was constructed so that the first 91 amino acids coded for by the restrictive alleles could be expressed in vivo. It was active in a transformed line. This sequence was sufficient to impart a restrictive phenotype to an adult D. melanogaster fly after it was injected with the virus. However, the truncated protein expressed by p delta n did not have an effect on the hereditary transmission of the sigma virus to the offspring of the infected flies, even though it contained the restriction site. The native Ref(2)P protein has been previously shown to have conformation-dependent epitopes common with some of those of the viral N protein. We demonstrated the following. (i) These epitopes were found in a domain of the Ref(2)P protein distinct from the site involved in restriction. (ii) They were modified in the N protein of the haP7 sigma virus mutant selected as being adapted to the restrictive alleles of the ref(2)P gene; only one mutation in the N gene, leading to an amino acid substitution, distinguished the haP7 mutant from the original virus. (iii) The virus strains partially or totally adapted to the effects of the full restrictive protein expressed by pp were always found to multiply to a lesser extent in the presence of the protein expressed by p delta n. These data suggest that two distinct domains of the Ref(2)P protein are involved in the control of sigma virus multiplication.     

HIV-1 integrase crosslinked oligomers are active in vitro

The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors.     

Aphid transmission of cauliflower mosaic virus: The role of the host plant

Transmission of plant viruses is the result of interactions between a given virus, the host plant and the vector. Most research has focused on molecular and cellular virus-vector interactions, and the host has only been regarded as a reservoir from which the virus is acquired by the vector more or less accidentally. However, a growing body of evidence suggests that the host can play a crucial role in transmission. Indeed, at least one virus, Cauliflower mosaic virus, exploits the host''s cellular pathways to form specialized intracellular structures that optimize virus uptake by the vector and hence transmission.Key words: virus, vector, host plant, transmission, interactionsTransmission is a step in a virus''s life cycle that is often neglected. Nonetheless, it is obvious that also this step is obligatory for a virus, as it could not maintain itself without dispersing to other hosts and infecting them. Most plant viruses are transmitted by insects, using two different strategies: “circulant transmission” where the virus, once taken up by the vector during feeding on an infected plant, passes from the intestine via the body lumen to the salivary glands and is finally inoculated with the saliva into a new host plant; the second strategy is “non-circulant transmission” where transmissible virus particles attach only to the exterior mouthpieces of the insect from which they are released into a new host. Whereas the first strategy obviously requires highly specific interactions between the virus and the vector to allow for passage of the virus through the vector, non-circulant transmission was initially thought of as a more or less accidental event, where virus sticks non-specifically to the mouthpieces. However, it becomes more and more evident that also non-circulant transmission is the result of sophisticated interactions between a given virus, a host and a vector. The vectors are most often aphids that, due to their non-destructive feeding behavior, are ideally suited as virus vectors. In fact, once landed on a plant, aphids first probe the prospective food source by short, only seconds lasting intracellular punctures in epidermis and mesophyll cells that do not even kill the punctured cells.¹ After these exploratory punctures and when they judge the plant as suited, the aphids insert their proboscis-like mouthpieces (stylets) into the phloem and feed from its sap for time spans that may exceed several hours. Depending on the tissues they infect, plant viruses can be acquired by aphids during either or only one of the two puncture phases. For example, Luteoviruses are only acquired from the vascular tissues,² whereas Cauliflower mosaic virus is acquired from both tissues.³Cauliflower mosaic virus (CaMV) is one of the best studied viruses on what concerns non-circulant transmission, the most often used transmission mode employed by plant viruses. For its transmission, a transmissible complex must form that attaches to a protein receptor located in the stylets of the aphid.⁴ This complex is not only, as for some viruses, composed of the virus particle, but also, as for many non-circulantly transmitted plant viruses, of a viral helper protein that with one domain interacts with the virus particle and with another with the stylet receptor⁵ (Fig. 1A). The helper protein of CaMV, P2, seems to have no other function but to assist in transmission as CaMV mutants deleted of P2 are perfectly infectious but not transmissible.⁶ A puzzling fact is that P2 may be acquired independently of the virus particle, meaning that it alone can bind to the stylet receptor and that virus particles either attach concomitantly with P2 onto the stylets or later attach to pre-bound P2. This has consequences for the composition of the transmitted viral population as it can be compiled of virus particles originating from the same cell from which P2 was acquired, but also from other cells and even sieve tubes that themselves do not contain P2.³ In fact, this potentially sequential acquisition mode of CaMV by the vector is controlled by the intracellular⁷ and tissue-specific localization of P2 that is only found in epidermis and parenchyma cells.³ In these cells, P2 localizes exclusively in a single viral inclusion, the transmission body, that has been proposed and recently been shown to be specialized for transmission:^8–10 if this structure does not form, CaMV can not be taken up by the aphid, even if functional P2 is present in the infected cell.Open in a separate windowFigure 1(A) The different strategies of non-circulant transmission: Viruses (V) using the capsid strategy (CS) attach directly to a receptor (R) in the tip of the a proboscis forming aphid stylets (blue), whereas in the helper strategy (HS) this interaction is mediated by the viral helper protein (H) that binds the virus particle to the receptor. Note that the helper protein can bind independently of the virus to the stylets. Whether the same receptor is used by different viruses as presented in the schema, is not known. (B) A turnip protoplast transfected with CaMV was double-labelled late in infection for CaMV helper protein P2 (red) and the marker protein for the virus factories P6 (green). It is visible that P2 localizes in a single, large transmission body, whereas the numerous virus factories are devoid of P2 (Colocalization would be revealed by yellowish color (M) in this superposition). (C and D) Turnip protoplasts were cotransfected with CaMV and TBK5-GFP and immunolabelled for P2 (red) and TBK5 was detected by GFP fluorescence. (C) shows a cell early in infection, where P2 and TBK5-GFP colocalize on a network that we identified as the microtubule cytoskeleton (unpublished data). (D) shows a cell later in infection where P2 and TBK5-GFP colocalize, as indicated by the yellowish color, in a transmission body. Note that TBK5-GFP also strongly labels the nucleus (N).This posed the interesting question how the transmission body forms during infection because elucidating this mechanism would show that CaMV hijacks cellular pathways for the sole purpose to ensure its transmission. It was known that besides the single transmission body a second type of viral inclusion bodies is found in infected cells: the numerous “electron-dense inclusions” that are assumed to be the virus factories (Fig. 1B) where all viral synthesis occurs¹¹ and where most virus particles accumulate. However, P2 was never described in the factories, presenting the paradox: if it is translated in the factories why is it not found there? Of different possible scenarios we chose to test the hypothesis that P2 is produced in the factories and then exported. Protoplasts were transfected with CaMV particles and kinetics of P2 accumulation followed by immunofluorescence. The results showed that P2 is indeed translated in the viral factories but then associates temporally with microtubules before finally condensing into a single transmission body. Also the other known components of the transmission body, the viral protein P3 and to a lesser degree, some virus particles, followed the same route from viral factories to the transmission body.Experiments with cytoskeleton drugs confirmed that transient localization of transmission body components with microtubules, but not with actin filaments, is necessary for transmission body formation. The results also indicated that both microtubules and actin filaments are apparently not required for other steps of the intracellular infection cycle because formation of viral factories was only slightly inhibited by the drugs.The results show that CaMV specifically uses the microtubule cytoskeleton to form the transmission body and thus enable vector transmission. Consequently, non-circulant transmission of at least this virus is not a random event where the vector takes up some transmissible complexes by chance. It is rather the result of highly specific interactions, where the virus “intentionally” (ab)uses cellular pathways to optimize acquisition by the vector, and this long before arrival of the latter on an infected plant.A lot of questions remain open, though. Are P2 and the other components of the transmission body actively transported on microtubules, or is their transient colocalization with microtubules part of an alternative transport mode? We started to more closely examine interaction between P2 and microtubules and privileged the hypothesis that the protein might be transported by a motor activity on microtubules. As preliminary data indicated that P2 does not possess an innate translocating activity, we looked for a cellular motor protein and tested as a candidate the kinesin TBK5.¹² This transport protein is, when overexpressed, able to bundle microtubules into a single focus, just as transmission bodies are singular structures in the cell. When healthy protoplasts were cotransfected with TBK5 and CaMV, TBK5 localized transiently with P2 on microtubules and in transmission bodies (Fig. 1C and D). This might be taken as the first evidence that a kinesin might be involved in formation of transmission bodies, but more experimentation is needed to confirm this hypothesis.A by far more important question is: Have also other viruses, whether from the plant or the animal kingdoms, that are noncirculantly (or mechanically, as animal virologists call this mode of transmission) transmitted, developed similar strategies that fine-tune interactions between the host and the virus to prepare and perfect transmission?     

The Interactions of Allium sativum leaf agglutinin with a chaperonin group of unique receptor protein isolated from a bacterial endosymbiont of the mustard aphid

The homopteran sucking insect, Lipaphis erysimi (mustard aphid) causes severe damage to various crops. This pest not only affects plants by sucking on the phloem, but it also transmits single-stranded RNA luteoviruses while feeding, which cause disease and damage in the crop. The mannose-binding Allium sativum (garlic) leaf lectin has been found to be a potent control agent of L. erysimi. The lectin receptor protein isolated from brush border membrane vesicle of insect gut was purified to determine the mechanism of lectin binding to the gut. Purified receptor was identified as an endosymbiotic chaperonin, symbionin, using liquid chromatography-tandem mass spectrometry. Symbionin from endosymbionts of other aphid species have been reported to play a significant role in virus transmission by binding to the read-through domain of the viral coat protein. To understand the molecular interactions of the said lectin and this unique symbionin molecule, the model structures of both molecules were generated using the Modeller program. The interaction was confirmed through docking of the two molecules forming a complex. A surface accessibility test of these molecules demonstrated a significant reduction in the accessibility of the complex molecule compared with that of the free symbionin molecule. This reduction in surface accessibility may have an effect on other molecular interactive processes, including "symbionin virion recognition", which is essential for such symbionin-mediated virus transmission. Thus, garlic leaf lectin provides an important component of a crop management program by controlling, on one hand, aphid attack and on the other hand, symbionin-mediated luteovirus transmission.     

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.     

Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate the virus mutation rate and for replication in macrophages

Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.     

A Trio of Viral Proteins Tunes Aphid-Plant Interactions in Arabidopsis thaliana

Background

Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae (common names: ‘peach-potato aphid’, ‘green peach aphid’).

Methodology/Principal Findings

Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0.

Conclusions/Significance

Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between the effects of Fny-CMV on this plant and those seen previously in tobacco (inhibition of resistance to aphids) may have important epidemiological consequences.