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During egress from the nucleus, HSV capsids that contain DNA (termed C capsids) are preferentially enveloped at the inner nuclear membrane over capsid types lacking DNA. Using coimmu-noprecipitation and biochemical analyses of wil...
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During egress from the nucleus, HSV capsids that contain DNA (termed C capsids) are preferentially enveloped at the inner nuclear membrane over capsid types lacking DNA. Using coimmu-noprecipitation and biochemical analyses of wild-type and mutant capsids, we identify an interaction between a complex of pU_L17/ pU_L25, termed the C capsid-specific complex (CCSC), and pU_L31, a component of the nuclear egress complex (NEC). We also show that the interactions between these components are dependent on expression of all three proteins but occur independently of the pU_L31 interacting protein and NEC component pU_L34, as well as a kinase encoded by U_s3 that phosphorylates both pU_L31 and pU_L34. The interaction between the CCSC and pU_L31 in the NEC suggests a mechanism to conserve viral resources by promoting assembly of only those viral particles with the potential to become infectious.
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White spot syndrome virus (WSSV) is one of the most virulent pathogens affecting penaeid shrimp, causing high mortality in infected populations. Interactions between virus structural proteins are likely to be important for virus a...
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White spot syndrome virus (WSSV) is one of the most virulent pathogens affecting penaeid shrimp, causing high mortality in infected populations. Interactions between virus structural proteins are likely to be important for virus assembly. Many steps of the WSSV assembly and maturation pathway remain unclear. In the present study, the interaction between VP37 and envelope or nucleocapsid proteins was characterized. VP37 was expressed in Escherichia coli and confirmed by Western blotting. Pure WSSV virions were subjected to Triton X-100 treatment to separate the envelope and nucleocapsid fractions. Overlay assays showed that VP37 interacted with VP28 and VP26. The interaction of VP37 with VP28 and VP26 was confirmed further by His pull-down and matrix-assisted laser desorption ionization (MALDI) mass spectrographic assays. The binding assay of VP37 with VP28 by ELISA confirmed that the 2 proteins had direct interaction in vivo. This discovery will help elucidate the molecular mechanisms of virion morphogenesis,
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Influenza A virus vRNA segments contain specific packaging signals at their termini that overlap the coding regions. To further characterise segment 5 packaging signals, we introduced synonymous mutations into the terminal coding ...
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Influenza A virus vRNA segments contain specific packaging signals at their termini that overlap the coding regions. To further characterise segment 5 packaging signals, we introduced synonymous mutations into the terminal coding regions of the vRNA and characterised the replicative fitness of the resulting viruses. Most mutations tested were well-tolerated, but a virus with alterations to NP codons 464-466, near the 5'-end of the vRNA, produced small plaques and replicated to around one-tenth of the level of wild type virus. The mutant virus supported normal levels of NP and segment 5 vRNA synthesis but packaged reduced levels of both segment 5 and segment 3 into virus particles. This Suggests an interaction between segments 3 and 5 during influenza A virus assembly.
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Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system....
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Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system. Despite extensive genetic and biophysical characterization of many of the lambda proteins and the assembly pathways in which they are implicated, there is a relative dearth of structural information on many of the most critical proteins involved in lambda assembly and maturation, including that of the lambda major capsid protein. Toward this end, we have utilized a combination of chemical cross-linking/mass spectrometry and computational modeling to construct a pseudo-atomic model of the lambda major capsid protein as a monomer, as well as in the context of the assembled procapsid shell. The approach described here is generalizable and can be used to provide structural models for any biological complex of interest. The procapsid structural model is in good agreement with published biochemical data indicating that procapsid expansion exposes hydrophobic surface area and that this serves to nucleate assembly of capsid decoration protein, gpD. The model further implicates additional molecular interactions that may be critical to the assembly of the capsid shell and for the stabilization of the structure by the gpD decoration protein.
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The Archaea—and their viruses—remain the most enigmatic of life's three domains. Once thought to inhabit only extreme environments, archaea are now known to inhabit diverse environments. Even though the first archaeal virus was ...
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The Archaea—and their viruses—remain the most enigmatic of life's three domains. Once thought to inhabit only extreme environments, archaea are now known to inhabit diverse environments. Even though the first archaeal virus was described over 40 years ago, only 117 archaeal viruses have been discovered to date. Despite this small number, these viruses have painted a portrait of enormous morphological and genetic diversity. For example, research centered around the various steps of the archaeal virus life cycle has led to the discovery of unique mechanisms employed by archaeal viruses during replication, maturation, and virion release. In many instances, archaeal virus proteins display very low levels of sequence homology to other proteins listed in the public database, and therefore, structural characterization of these proteins has played an integral role in functional assignment. These structural studies have not only provided insights into structure-function relationships but have also identified links between viruses across all three domains of life.
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The Archaea—and their viruses—remain the most enigmatic of life's three domains. Once thought to inhabit only extreme environments, archaea are now known to inhabit diverse environments. Even though the first archaeal virus was ...
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The Archaea—and their viruses—remain the most enigmatic of life's three domains. Once thought to inhabit only extreme environments, archaea are now known to inhabit diverse environments. Even though the first archaeal virus was described over 40 years ago, only 117 archaeal viruses have been discovered to date. Despite this small number, these viruses have painted a portrait of enormous morphological and genetic diversity. For example, research centered around the various steps of the archaeal virus life cycle has led to the discovery of unique mechanisms employed by archaeal viruses during replication, maturation, and virion release. In many instances, archaeal virus proteins display very low levels of sequence homology to other proteins listed in the public database, and therefore, structural characterization of these proteins has played an integral role in functional assignment. These structural studies have not only provided insights into structure-function relationships but have also identified links between viruses across all three domains of life.
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The assembly and egress of herpes simplex virus (HSV) is a complicated multistage process that involves several different cellular compartments and the activity of many viral and cellular proteins. The process begins in the nucleu...
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The assembly and egress of herpes simplex virus (HSV) is a complicated multistage process that involves several different cellular compartments and the activity of many viral and cellular proteins. The process begins in the nucleus, with capsid assembly followed by genome packaging into the preformed capsids. The DNA-filled capsids (nucleocapsids) then exit the nucleus by a process of envelopment at the inner nuclear membrane followed by fusion with the outer nuclear membrane. In the cytoplasm nucleocapsids associate with tegument proteins, which form a complicated protein network that links the nucleocapsid to the cytoplasmic domains of viral envelope proteins. Nucleocapsids and associated tegument then undergo secondary envelopment at intracellular membranes originating from late secretory pathway and endosomal compartments. This leads to assembled virions in the lumen of large cytoplasmic vesicles, which are then transported to the cell periphery to fuse with the plasma membrane and release virus particles from the cell. The details of this multifaceted process are described in this chapter.
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Paramyxoviruses replicate in the cytoplasm of infected cells and newly synthesized viral nucleocapsids (vRNPs) are transported to the plasma membrane to be incorporated into progeny virions. In this study, we analyzed the impact o...
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Paramyxoviruses replicate in the cytoplasm of infected cells and newly synthesized viral nucleocapsids (vRNPs) are transported to the plasma membrane to be incorporated into progeny virions. In this study, we analyzed the impact of the Rab11-mediated recycling pathway in Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) vRNP transport. We found that suppression of Rab11 expression caused vRNP aggregation in the cytoplasm and reduced progeny virion formation. Overexpression of constitutively active Rab11Q70L, but not dominant negative Rab11S25N co-localized with vRNP, showing that vRNP specifically recognizes the GTP-bound active form of Rab11. Moreover, Rab11Q70L co-localized with the dominant negative tails of all three subtypes of myosins, Va, Vb, and Vc, while SeV and hPIV1 vRNPs co-localized with only myosin Vb and Vc. These results highlight the critical role of Rabll in vRNP trafficking, and suggest a specificity in the recycling endosomes parainfluenza viruses utilize for virus assembly. (C) 2015 Elsevier Inc. All rights reserved.
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Cocksfoot mottle virus is a plant virus that belongs to the genus Sobemovirus. The structure of the virus has been determined at 2.7A resolution. The icosahedral capsid has T = 3 quasisymmetry and 180 copies of the coat protein. E...
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Cocksfoot mottle virus is a plant virus that belongs to the genus Sobemovirus. The structure of the virus has been determined at 2.7A resolution. The icosahedral capsid has T = 3 quasisymmetry and 180 copies of the coat protein. Except for a couple of stacked bases, the viral RNA is not visible in the electron density map. The coat protein has a jelly-roll β-sandwich fold and its conformation is very similar to that of other sobemoviruses and tobacco necrosis virus. The N-terminal arm of one of the three quasiequivalent subunits is partly ordered and follows the same path in the capsid as the arm in rice yellow mottle virus, another sobemovirus. In other sobemoviruses, the ordered arm follows a different path, but in both cases the arms from three subunits meet and form a similar structure at a threefold axis. A comparison of the structures and sequences of viruses in this family shows that the only conserved parts of the protein-protein interfaces are those that form binding sites for calcium ions. Still, the relative orientations and position of the subunits are mantained.
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The X-ray crystal structure of Cowpea chlorotic mottle bromovirus (CCMV) revealed a unique tubular structure formed by the interaction of the N-termini from six coat protein subunits at each three-fold axis of the assembled virion...
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The X-ray crystal structure of Cowpea chlorotic mottle bromovirus (CCMV) revealed a unique tubular structure formed by the interaction of the N-termini from six coat protein subunits at each three-fold axis of the assembled virion. This structure, termed the β-hexamer, consists of six short β-strands. The β-hexamer was postulated to play a critical role in the assembly and stability of the virion by stabilizing hexameric capsomers (Speir et al., 1995). Mutational analyses of the β-hexamer structure, utilizing both in vitro and in vivo assembly assays, demonstrate that this structure is not required for virion formation devoid of nucleic acids in vitro or for RNA-containing virions in vivo. However, the β-hexamer structure does contribute to virion stability in vitro and modulates disease expression in vivo. These results support a model for CCMV assembly through pentamer intermediates.
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