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1.
Diverse animal and plant viruses are able to translocate their virions between neighboring cells via intercellular connections. In this work, we analyze the virion assembly and cell-to-cell movement of a plant closterovirus and reveal a strong correlation between these two processes. The filamentous virions of a closterovirus possess a long body formed by the major capsid protein (CP) and a short tail formed by the minor capsid protein (CPm). Genetic and biochemical analyses show that the functions of these virion components are distinct. A virion body is required primarily for genome protection, whereas a tail represents a specialized device for cell-to-cell movement. Furthermore, tail assembly is mediated by the viral Hsp70 homolog (Hsp70h) that becomes an integral part of the virion. Inactivation of the ATPase domain of Hsp70h results in assembly of tailless virions that are incapable of translocation. A dual role for the viral molecular chaperone Hsp70h in virion assembly and transport, combined with the previous finding of this protein in intercellular channels, allowed us to propose a model of closteroviral movement from cell to cell. 相似文献
2.
3.
Valentin V. Makarov Eugeny V. Skurat Pavel I. Semenyuk Dmitry A. Abashkin Natalya O. Kalinina Alexsandr M. Arutyunyan Andrey G. Solovyev Eugeny N. Dobrov 《PloS one》2013,8(4)
Virions of Barley stripe mosaic virus (BSMV) were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP) were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV), a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed. 相似文献
4.
Lucy R. Stewart Vicente Medina Tongyan Tian Massimo Turina Bryce W. Falk James C. K. Ng 《Journal of virology》2010,84(23):12165-12173
The Lettuce infectious yellows virus (LIYV) RNA 2 mutant p1-5b was previously isolated from Bemisia tabaci-transmitted virus maintained in Chenopodium murale plants. p1-5b RNA 2 contains a single-nucleotide deletion in the minor coat protein (CPm) open reading frame (ORF) that is predicted to result in a frameshift and premature termination of the protein. Using the recently developed agroinoculation system for LIYV, we tested RNA 2 containing the p1-5b CPm mutant genotype (agro-pR6-5b) in Nicotiana benthamiana plants. We showed that plant infection triggered by agro-pR6-5b spread systemically and resulted in the formation of virions similar to those produced in p1-5b-inoculated protoplasts. However, virions derived from these mutant CPm genotypes were not transmitted by whiteflies, even though virion concentrations were above the typical transmission thresholds. In contrast, and as demonstrated for the first time, an engineered restoration mutant (agro-pR6-5bM1) was capable of both systemic movement in plants and whitefly transmission. These results provide strong molecular evidence that the full-length LIYV-encoded CPm is dispensable for systemic plant movement but is required for whitefly transmission.Members of the genus Crinivirus are emerging plant viruses in many parts of the world. An important factor contributing to the increase in the incidence of these viruses is their association with and transmission by whitefly vectors that have increased in distribution in the last several decades. Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus (family Closteroviridae), is specifically transmitted by the sweet potato whitefly, Bemisia tabaci biotype A, in a semipersistent, noncirculative manner (6). The virus is confined to phloem cells within infected plants and is not transmissible to plants by leaf rub inoculation. The bipartite single-stranded positive-sense LIYV genome components, consisting of RNA 1 (approximately 8.1 kb) and RNA 2 (approximately 7.2 kb), are separately encapsidated in flexuous filamentous particles that are characteristic of the family Closteroviridae (8, 11). These virions are comprised of four protein components: the major coat protein (CP), the minor coat protein (CPm), an Hsp70 homolog (Hsp70h), and a 59-kDa protein (P59). Like other viruses in the family Closteroviridae, LIYV has bipolar virions with a “body” composed mainly of the CP and a “head” that is formed by the assembly of CPm subunits (2, 4, 7, 22, 28). Hsp70h and P59 are detected in LIYV virions (22), but their locations have not been identified, as they are not readily detected by immunogold labeling and transmission electron microscopy (IGL-TEM). For two members of the family Closteroviridae, Citrus tristeza virus (CTV) and Beet yellows virus (BYV), the combination of Hsp70h, P61 (the homolog of LIYV P59 in CTV) or P64 (the homolog of LIYV P59 in BYV), and CPm encapsidates the 5′ end (∼630 to 650 nucleotides [nt]) of the RNA genome, demonstrating the complex interactions that exist among the capsid proteins and the genomic RNA (15, 21).In our previous studies, we demonstrated the transmission of LIYV using an in vitro acquisition and whitefly transmission system (13, 22). Results from previous work implicated a role for LIYV CPm in whitefly transmission. Antibodies to CPm blocked the in vitro acquisition/transmission of LIYV virion preparations by B. tabaci biotype A, while antibodies to CP, Hsp70h, and P59 did not (22). The in vitro whitefly membrane-feeding system had also been used to demonstrate B. tabaci biotype A transmission of virions that were derived from cloned infectious cDNAs of LIYV RNA 1 and RNA 2 of several genotypes, including pR6 (the first cloned wild-type [WT] infectious cDNA of LIYV RNA 2 [10]), establishing for the first time that these cloned constructs contained all of the information necessary for protoplast infection, virion formation, whitefly transmission, and infection in plants (12). In that study, the mutant p1-5b was among the cloned LIYV RNA 2 cDNAs derived from B. tabaci biotype A-transmitted virus maintained in Chenopodium murale plants.p1-5b contains a single-adenine-residue deletion in the CPm open reading frame (ORF) at nucleotide 592, a deletion that is predicted to result in a frameshift, 14 new amino acids, and premature termination of the protein (12). The predicted p1-5b CPm has 211 amino acids, compared to 453 amino acids in the wild-type (pR6 genotype) protein. The p1-5b genotype also contains three other nucleotide changes in the CPm ORF relative to the pR6 infectious clone sequence (27), all of which result in amino acid changes. In contrast, the p1-5b CP, Hsp70h, and P59 sequences are identical to that of pR6 (12). Possible polymorphisms throughout the rest of the p1-5b clone were not characterized. In a prior study, B. tabaci biotype A transmission of p1-5b virions was not observed, even though the mutation did not affect its infectivity in protoplasts (as determined by virion yields) and apparent particle morphology (12). However, those studies were disadvantaged by the necessity of propagation in protoplasts to obtain specific genotypes from infectious cloned cDNAs. Protoplasts yield low quantities of virion relative to plants, and virion concentration is a critical parameter in whitefly transmission (13). Although virion concentrations in those experiments were above typical thresholds for whitefly transmission (12, 13), low concentrations may still be limiting for transmission, making negative transmission results difficult to interpret. Obtaining adequate virion concentrations of specific genotypes for whitefly transmission to plants has therefore been a significant hurdle to LIYV transmission studies.The recently developed agroinoculation method for LIYV (24) permits the study of systemic plant infection by distinct LIYV genotypes, including those that are whitefly transmission deficient, and the recovery of higher virion yields than were possible using protoplasts. The objective of this study was to further examine the function of the LIVY CPm by extending our observations of p1-5b. We constructed mutants with the CPm frameshift restored to determine if engineered mutations that either restored or disrupted the formation of an intact CPm also affected systemic plant infection, virion formation, and B. tabaci biotype A transmission. Our study revealed that a mutant engineered with the restored CPm ORF produced a WT infection profile characterized by systemic virus movement within agroinoculated plants and the generation of CPm-containing virions that were whitefly transmissible. Intriguingly, systemic virus movement was also observed for a mutant engineered to express the 1-5b CPm, but the virions lacked an identifiable CPm and were defective in whitefly transmission. These results represent a significant advance in addressing challenging questions and hypotheses about Crinivirus whitefly transmission properties not testable using earlier systems. 相似文献
5.
Fernández-Fernández MR Martínez-Torrecuadrada JL Roncal F Domínguez E García JA 《Journal of virology》2002,76(24):12646-12653
PEPSCAN analysis has been used to characterize the immunogenic regions of the capsid protein (CP) in virions of plum pox potyvirus (PPV). In addition to the well-known highly immunogenic N- and C-terminal domains of CP, regions within the core domain of the protein have also shown high immunogenicity. Moreover, the N terminus of CP is not homogeneously immunogenic, alternatively showing regions frequently recognized by antibodies and others that are not recognized at all. These results have helped us to design efficient antigen presentation vectors based on PPV. As predicted by PEPSCAN analysis, a small displacement of the insertion site in a previously constructed vector, PPV-gamma, turned the derived chimeras into efficient immunogens. Vectors expressing foreign peptides at different positions within a highly immunogenic region (amino acids 43 to 52) in the N-terminal domain of CP were the most effective at inducing specific antibody responses against the foreign sequence. 相似文献
6.
Murine monoclonal antibodies (mAbs) to lucerne transient streak sobemovirus (LTSV) were used as probes for examining the virion capsid organization. A panel of nine mAbs from approximately 100 hybridomas were chosen for this study. All of these mAbs interacted effectively with sites on intact capsid and isolated coat protein sub-units (i.e. metatopes) in ELISA and Western blots. Only one of these mAbs reacted with virions in gel diffusion test producing a visible precipitin band. Competitive binding assays showed that these mAbs recognized the same or very similar metatopes. None of the mAbs neutralized LTSV virion infectivity but rabbit polyclonal antibodies drastically reduced infectivity. Treatment of virions with EDTA (swollen LTSV). protein crosslinking reagents or sodium dodecyl sulphate caused no detectable alterations in their reactivities with these mAbs, The binding sites for monoclonal and polyclonal antibodies were located on denatured 5.5 kDa fragments resulting from partial trypsinization of LTSV coat protein and 8 kDa fragments formed with chymopapain proteolysis. These results indicate that these LTSV epitopes are of linear (or sequential) configuration and are located on the exposed, structurally stable domain of the virion capsid. 相似文献
7.
Satellite panicum mosaic virus (SPMV), an 824-nucleotide, positive-sense, single-stranded RNA virus, depends on Panicum mosaic virus (PMV) for replication and spread in host plants. Compared with PMV infection alone, symptoms are intensified and develop faster on millet plants infected with SPMV and PMV. SPMV encodes a 157 amino acid capsid protein (CP) (17.5 kDa) to encapsidate SPMV RNA and form T = 1 satellite virions. The present study identifies additional biological activities of the SPMV CP, including the induction of severe chlorosis on proso millet plants (Panicum miliaceum cv. Sunup or Red Turghai). Initial deletion mutagenesis experiments mapped the chlorosis-inducing domain to amino acids 50 to 157 on the C-terminal portion of the SPMV CP. More defined analyses revealed that amino acids 124 to 135 comprised a critical domain associated with chlorosis induction and virion formation, whereas the extreme C-terminal residues 148 to 157 were not strictly essential for either role. The results also demonstrated that the absence of SPMV CP tended to stimulate the accumulation of defective RNAs. This suggests that the SPMV CP plays a significant role in maintaining the structural integrity of the full-length satellite virus RNA and harbors multiple functions associated with pathogenesis in SPMV-infected host plants. 相似文献
8.
The proteins that compose a herpesvirus virion are thought to contain the functional information required for de novo infection, as well as virion assembly and egress. To investigate functional roles of Kaposi's sarcoma-associated herpesvirus (KSHV) virion proteins in viral productive replication and de novo infection, we attempted to identify virion proteins from purified KSHV by a proteomic approach. Extracellular KSHV virions were purified from phorbol-12-tetradecanoate-13-acetate-induced BCBL-1 cells through double-gradient ultracentrifugation, and their component proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirty prominent protein bands were excised and subjected to high-performance liquid chromatography ion trap mass spectrometric analysis. This study led to the identification of 24 virion-associated proteins. These include five capsid proteins, eight envelope glycoproteins, six tegument proteins, and five proteins whose locations in the virions have not yet been defined. Putative tegument proteins encoded by open reading frame 21 (ORF21), ORF33, and ORF45 were characterized and found to be resistant to protease digestion when purified virions were treated with trypsin, confirming that they are located within the virion particles. The ORF64-encoded large tegument protein was found to be associated with capsid but sensitive to protease treatment, suggesting its unique structure and array in KSHV virions. In addition, cellular beta-actin and class II myosin heavy chain type A were found inside KSHV virions and associated with tegument-capsid structure. Identification of KSHV virion proteins makes it possible to study the functional roles of these virion proteins in KSHV replication and pathogenicity. 相似文献
9.
Pederson DM Welsh LC Marvin DA Sampson M Perham RN Yu M Slater MR 《Journal of molecular biology》2001,309(2):401-421
The PH75 strain of filamentous bacteriophage (Inovirus) grows in the thermophilic bacterium Thermus thermophilus at 70 degrees C. We have characterized the viral DNA and determined the amino acid sequence of the major coat protein, p8. The p8 protein is synthesized without a leader sequence, like that of bacteriophage Pf3 but unlike that of bacteriophage Pf1, both of which grow in the mesophile Pseudomonas aeruginosa. X-ray diffraction patterns from ordered fibres of the PH75 virion are similar to those from bacteriophages Pf1 and Pf3, indicating that the protein capsid of the PH75 virion has the same helix symmetry and subunit shape, even though the primary structures of the major coat proteins are quite different and the virions assemble at very different temperatures. We have used this information to build a molecular model of the PH75 protein capsid based on that of Pf1, and refined the model by simulated annealing, using fibre diffraction data extending to 2.4 A resolution in the meridional direction and to 3.1 A resolution in the equatorial direction. The common design may reflect a fundamental motif of alpha-helix packing, although differences exist in the DNA packaging and in the means of insertion of the major coat protein of these filamentous bacteriophages into the membrane of the host bacterial cell. These may reflect differences in the assembly mechanisms of the virions. 相似文献
10.
Display of heterologous proteins on gp64null baculovirus virions and enhanced budding mediated by a vesicular stomatitis virus G-stem construct 总被引:2,自引:1,他引:1
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The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the gp64 gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a gp64null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV gp64null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the gp64null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on gp64null AcMNPV budded virions. The combined use of gp64null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production. 相似文献
11.
Dobrov EN Nemykh MA Lukashina EV Baratova LA Drachev VA Efimov AV 《Molekuliarnaia biologiia》2007,41(4):706-710
We propose the modified model of the structure of coat protein (CP) subunits in filamentous virions of potato virus X (PVX). The model is similar to the one proposed by us in 2001 for the CP of another helical plant virus (potato virus A) belonging to other (potyvirus) group. In this model the PVX CP molecule consist of two main domains--a bundle of four alpha-helices located close to the virion long axis and a so-called RNP-fold (or abCd-fold) located near the virion surface. Basing on this model we suggest possible mechanism of described by J.G. Atabekov and colleagues structural transition ("remodeling") of the PVX virions resulting from their interaction with virus-specific TGB-1 protein. 相似文献
12.
The 17-kDa capsid protein (CP) of satellite panicum mosaic virus (SPMV) contains a distinct N-terminal arginine-rich motif (N-ARM) which is required for SPMV virion assembly and the activity of SPMV CP to promote systemic accumulation of its cognate RNA. The present study indicates that SPMV CP also is involved in SPMV RNA accumulation in inoculated leaves and that this activity is also dependent on a functional N-ARM. In addition, deletions of a C-terminal region abolish virion assembly and impair SPMV RNA accumulation in both inoculated and systemic leaves. Unlike the N-ARM mutations, substantial deletions of the SPMV CP C-terminus do not affect SPMV RNA binding activity. Interestingly, SPMV CP also binds Panicum mosaic virus genomic RNA via N-ARM-mediated CP:RNA interactions. Mutations of the N-ARM and the C-terminal regions significantly reduce SPMV CP titers and result in symptom attenuation. In contrast, virions were not associated per se with symptom exacerbation or successful SPMV RNA accumulation. The results show the existence of a correlation between N- and C-termini-mediated contributions for CP accumulation, symptom induction, defective-interfering RNA accumulation, and temperature sensitivity of SPMV RNA maintenance. The data provide further evidence that SPMV CP has multiple roles during infection, which might involve the formation of nonvirion CP:RNA complexes whose stability is controlled in a biologically relevant manner by the N- and C-termini of the CP. 相似文献
13.
E. N. Dobrov M. A. Nemykh E. V. Lukashina L. A. Baratova V. A. Drachev A. V. Efimov 《Molecular Biology》2007,41(4):638-641
A modified model was proposed for the tertiary structure of the coat protein (CP) molecules in potato virus X (PVX) virions, similar to the original model of 2001 describing the structure of CP of potato virus A, a member of another group of filamentous viruses. According to the new model, CP comprises two main structural domains, namely, a bundle of α-helices, located near the long axis of the virion, and the socalled RNP fold (or abCd fold), located in the vicinity of its surface. The model made it possible to suggest a possible mechanism of the PVX virion structural rearrangement (remodeling) resulting from translational activation of virions by the TGB1 movement protein according to Atabekov and colleagues. 相似文献
14.
Rakitina DV Kantidze OL Leshchiner AD Solovyev AG Novikov VK Morozov SY Kalinina NO 《FEBS letters》2005,579(22):4955-4960
Coat proteins (CPs) of plant viruses are involved in different stages of the viral life cycle such as virion assembly, replication, movement, vector transmission, and regulation of host defense responses. Here, we report that the CPs of two filamentous RNA viruses, potato virus X (PVX, Potexvirus) and potato virus A (PVA, Potyvirus) exhibit an enzyme activity. The CP isolated from PVX virions possesses ATP-binding and ATPase activities. Recombinant PVX and PVA CPs produced in Escherichia coli show Mg2+-dependent ATPase and UTPase activities inhibited by antibodies against virus particles. Deletion of the C-terminal regions of these proteins diminishes their ATPase activity. 相似文献
15.
Consigli RA Haynes JI Chang D Grenz L Richter D 《Transactions of the Kansas Academy of Science. Kansas Academy of Science》1992,95(1-2):62-69
Polyoma virions have different attachment proteins which are responsible for hemagglutination of erythrocytes and attachment to cultured mouse kidney cells (MKC). Virion binding studies demonstrated that MKC possess specific (productive infection) and nonspecific (nonproductive) receptors. Empty polyoma capsids have hemagglutination activity and bind to non-specific MKC receptors, but they are not capable of competing for specific virion cell receptors or preventing productive infection. Isoelectric focusing of the virion major capsid protein, VP1, separated this protein into six species (A through F). These species had identical amino acid sequences, but differed in degree of modification (phosphorylation, acetylation, sulfation and hydroxylation). Evidence based upon precipitation with specific antisera supports the view that VP1 species E is required for specific adsorption and that D and F are required for hemagglutination. The virion attachment domain has been localized to an 18 kilodalton fragment of the C-terminal region of VP1. Monopinocytotic vesicles containing 125I-labeled polyoma virions were isolated from infected MKC. A crosslinker was used to bind the MKC cell receptor(s) covalently to VP1 attachment protein, and a new 120 kilodalton band was identified by SDS-PAGE. An anti-idiotype antibody prepared against a neutralizing polyoma monoclonal antiody was used to identify a putative 50 kilodalton receptor protein from a detergent extract of MKC, as well as from MKC membrane preparation. 相似文献
16.
Role of VP3 in human rotavirus internalization after target cell attachment via VP7. 总被引:27,自引:20,他引:7
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A cell lysate prepared from MA104 cells that had been infected with human rotavirus KUN strain (HRV-KUN) contained a 35-kilodalton protein capable of binding to MA104 cells. The binding of the 35-kilodalton protein was inhibited by a serotype 2-specific antiserum but not by antisera to other serotypes. Not only trypsin-treated, infectious HRV-KUN but also untreated, noninfectious virions effectively competed with the 35-kilodalton protein for the same cell surface binding sites. One monoclonal anti-VP7 (AH6) absorbed the 35-kilodalton protein from the HRV-KUN-infected cell lysate, whereas another monoclonal anti-VP7 (S2-2G10) inhibited the virions to compete with the 35-kilodalton protein for the cell surface binding sites. Both anti-VP7 (S2-2G10) and anti-VP3 (K-1532, K-376) monoclonal antibodies had the virus-neutralization activity, but only anti-VP7 inhibited virus adsorption. On the other hand, anti-VP3 monoclonal antibodies were capable of completely inhibiting the infection of preadsorbed HRV-KUN as long as virions were not yet internalized. Subsequent studies with [35S]methionine-labeled and purified HRV-KUN showed that not only trypsin-treated, infectious virions but also untreated, noninfectious virions were capable of efficient target cell binding and internalization. The internalization modes of these two HRV-KUN preparations were, however, quite different. Only the components of the inner capsid were internalized from trypsin-treated virions, whereas no such selective internalization was seen with untreated virions. Furthermore, anti-VP3 inhibited this selective internalization of the inner capsid from the infectious virions. From these results we conclude that VP7 is the HRV-KUN cell attachment protein and that adsorption of HRV-KUN via VP7 is independent of trypsin treatment, whereas the limited cleavage of VP3 by trypsin, which is essential for the development of HRV-KUN infectivity, is needed for the selective internalization of the inner capsid components, a process that is apparently essential for HRV-KUN infection. 相似文献
17.
Interaction between long-distance transport factor and Hsp70-related movement protein of Beet yellows virus 总被引:2,自引:0,他引:2
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Systemic spread of viruses in plants involves local movement from cell to cell and long-distance transport through the vascular system. The cell-to-cell movement of the Beet yellows virus (BYV) is mediated by a movement protein that is an Hsp70 homolog (Hsp70h). This protein is required for the assembly of movement-competent virions that incorporate Hsp70h. By using the yeast two-hybrid system, in vitro coimmunoprecipitation, and in planta coexpression approaches, we show here that the Hsp70h interacts with a 20-kDa BYV protein (p20). We further demonstrate that p20 is associated with the virions presumably via binding to Hsp70h. Genetic and immunochemical analyses indicate that p20 is dispensable for assembly and cell-to-cell movement of BYV but is required for the long-distance transport of virus through the phloem. These results reveal a novel activity for the Hsp70h that provides a molecular link between the local and systemic spread of a plant virus by docking a long-distance transport factor to virions. 相似文献
18.
In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of capsid assembly and provide insights into underlying mechanisms. CA multimerization was triggered by multivalent anions providing evidence that in vitro assembly is an electrostatically controlled process. In the case of RSV, in vitro assembly was a well-behaved nucleation-driven process that led to the formation of structures with morphologies similar to those found in virions. Isolated RSV dimers, when mixed with monomeric protein, acted as efficient seeds for assembly, eliminating the lag phase characteristic of a monomer-only reaction. This demonstrates for the first time the purification of an intermediate on the assembly pathway. Differences in the intrinsic tryptophan fluorescence of monomeric protein and the assembly-competent dimer fraction suggest the involvement of the NTD in the formation of the functional dimer. Furthermore, in vitro analysis of well-characterized CTD mutants provides evidence for assembly dependence on the second domain and suggests that the establishment of an NTD-CTD interface is a critical step in capsid assembly initiation. Overall, the data provide clear support for a model whereby capsid assembly within the maturing virion is dependent on the formation of a specific nucleating complex that involves a CA dimer and is directed by additional virion constituents. 相似文献
19.
Differences in the subpopulations of the structural proteins of polyoma virions and capsids: biological functions of the multiple VP1 species. 总被引:25,自引:22,他引:3
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The structural proteins of polyoma virions and capsids were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyoma virion VP1 was found to be composed of six distinct species which had pI's between pH 6.75 and 5.75. Polyoma capsid VP1 was found to contain four species with pI's between pH 6.60 and 5.75. The different forms of virion and capsid VP1 appeared to be generated by modifications (phosphorylation and acetylation) of the initial translation product. The most basic of the virion VP1 species (pI, pH 6.75) was absent in capsids and was found to be exclusively associated with the viral nucleoprotein complex. Three of the virion VP1 species and three of the capsid VP1 species were found in capsomere preparations enriched for hexon subunits. Two VP1 species were specifically immune precipitated from virions with hemagglutination-inhibiting antibodies. These two VP1 species were common to both virions and capsids. Polyoma virions, but not capsids, possessed a single VP1 species which was immune precipitated with neutralizing antibodies. Both virion and capsid VP2 were found to have pI's of approximately pH 5.50. Virion VP3 had a pI of approximately pH 7.00, whereas capsid VP3 had a pI of approximately pH 6.50. 相似文献
20.
The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication. 总被引:14,自引:9,他引:5
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In this study, we investigated the role of the conserved neuraminidase (NA) cytoplasmic tail residues in influenza virus replication. Mutants of influenza A virus (A/WSN/33 [H1N1]) with deletions of the NA cytoplasmic tail region were generated by reverse genetics. The resulting viruses, designated NOTAIL, contain only the initiating methionine of the conserved six amino-terminal residues. The mutant viruses grew much less readily and produced smaller plaques than did the wild-type virus. Despite similar levels of NA cell surface expression by the NOTAIL mutants and wild-type virus, incorporation of mutant NA molecules into virions was decreased by 86%. This reduction resulted in less NA activity per virion, leading to the formation of large aggregates of progeny mutant virions on the surface of infected cells. A NOTAIL virus containing an additional mutation (Ser-12 to Pro) in the transmembrane domain incorporated three times more NA molecules into virions than did the NOTAIL parent but approximately half of the amount incorporated by the wild-type virus. However, aggregation of the progeny virions still occurred at the cell surface. All NOTAIL viruses were attenuated in mice. We conclude that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus. In addition, the relative abundance of long filamentous particles formed by the NOTAIL mutants, compared with the largely spherical wild-type particles, indicates a role for the NA cytoplasmic tail in virion morphogenesis. 相似文献