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1.
Bortz E  Wang L  Jia Q  Wu TT  Whitelegge JP  Deng H  Zhou ZH  Sun R 《Journal of virology》2007,81(18):10137-10150
The tegument, a semiordered matrix of proteins overlying the nucleocapsid and underlying the virion envelope, in viruses in the gamma subfamily of Herpesviridae is poorly understood. Murine gammaherpesvirus 68 (MHV-68) is a robust model for studying gammaherpesvirus virion structure, assembly, and composition, as MHV-68 efficiently completes the lytic phase and productively infects cultured cells. We have found that MHV-68 ORF52 encodes an abundant tegument protein conserved among gammaherpesviruses. Detergent sensitivity experiments revealed that the MHV-68 ORF52 protein is more tightly bound to the virion nucleocapsid than the ORF45 tegument protein but could be dissociated from particles that retained the ORF65 small capsomer protein. ORF52, tagged with enhanced green fluorescent protein or FLAG epitope, localized to the cytoplasm. A recombinant MHV-68 bacterial artificial chromosome mutant with a nonsense mutation incorporated into ORF52 exhibited viral DNA replication, expression of late lytic genes, and capsid assembly and packaging at levels near those of the wild type. However, the MHV-68 ORF52-null virus was deficient in the assembly and release of infectious virion particles. Instead, partially tegumented capsids produced by the ORF52-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, the ORF64 and ORF67 tegument proteins, but virtually no ORF45 tegument protein. Thus, ORF52 is essential for the tegumentation and egress of infectious MHV-68 particles in the cytoplasm, suggesting an important conserved function in gammaherpesvirus virion morphogenesis.  相似文献   

2.
Tegument is the unique structure of a herpesvirion which occupies the space between nucleocapsid and envelope. Accumulating data have indicated that interactions among tegument proteins play a key role in virion morphogenesis. Morphogenesis of gammaherpesviruses including Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) is poorly understood due to the lack of efficient de novo lytic replication in cell culture. Murine gammaherpesvirus-68 (MHV-68) is genetically related to these two human herpesviruses and serves as an effective model to study the lytic replication of gammaherpesviruses. We previously showed that ORF33 of MHV-68 encodes a tegument protein and plays an essential role in virion maturation in the cytoplasm. However, the molecular mechanism of how ORF33 participates in virion morphogenesis has not been elucidated. In this study we demonstrated that ORF38 of MHV-68 is also a tegument protein and is localized to cytoplasmic compartments during both transient transfection and viral infection. Immuno-gold labeling assay showed that ORF38 is only present on virions that have entered the cytoplasmic vesicles, indicating that ORF38 is packaged into virions during secondary envelopment. We further showed that ORF38 co-localizes with ORF33 during viral infection; therefore, the interaction between ORF38 and ORF33 is conserved among herpesviruses. Notably, we found that although ORF33 by itself is distributed in both the nucleus and the cytoplasm, in the presence of ORF38, ORF33 is co-localized to trans-Golgi network (TGN), a site where secondary envelopment takes place.  相似文献   

3.
Chickenpox(varicella) is caused by primary infection with varicella zoster virus(VZV), which can establish long-term latency in the host ganglion. Once reactivated, the virus can cause shingles(zoster) in the host. VZV has a typical herpesvirus virion structure consisting of an inner DNA core, a capsid, a tegument, and an outer envelope. The tegument is an amorphous layer enclosed between the nucleocapsid and the envelope, which contains a variety of proteins. However, the types and functions of VZV tegument proteins have not yet been completely determined. In this review, we describe the current knowledge on the multiple roles played by VZV tegument proteins during viral infection. Moreover, we discuss the VZV tegument protein-protein interactions and their impact on viral tissue tropism in SCID-hu mice. This will help us develop a better understanding of how the tegument proteins aid viral DNA replication, evasion of host immune response, and pathogenesis.  相似文献   

4.
Human cytomegalovirus (HCMV) virions are composed of a DNA-containing nucleocapsid surrounded by a tegument layer and host-derived lipid envelope studded with virally encoded glycoproteins. These complex virions are estimated to be composed of more than 50 viral proteins. Assembly of HCMV virions is poorly understood, especially with respect to acquisition of the tegument; however, it is thought to involve the stepwise addition of virion components through protein-protein interactions. We sought to identify interactions among HCMV virion proteins using yeast two-hybrid analysis. Using 33 known capsid and tegument proteins, we tested 1,089 pairwise combinations for binary interaction in the two-hybrid assay. We identified 24 interactions among HCMV virion proteins, including 13 novel interactions among tegument proteins and one novel interaction between capsid proteins. Several of these novel interactions were confirmed by coimmunoprecipitation of protein complexes from transfected cells. In addition, we demonstrate three of these interactions in the context of HCMV infection. This study reveals several new protein-protein interactions among HCMV tegument proteins, some of which are likely important for HCMV replication and pathogenesis.  相似文献   

5.
Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, has a complex multilayered extracellular virion that is structurally conserved among other herpesviruses. PRV virions contain a double-stranded DNA genome within a proteinaceous capsid surrounded by the tegument, a layer of viral and cellular proteins. The envelope layer, which encloses the capsid and tegument, contains viral transmembrane proteins anchored in a phospholipid bilayer. The viral and host proteins contained within virions execute important functions during viral spread and pathogenesis, but a detailed understanding of the composition of PRV virions has been lacking. In this report, we present the first comprehensive proteomic characterization of purified PRV virions by mass spectrometry using two complementary approaches. To exclude proteins present in the extracellular medium that may nonspecifically associate with virions, we also analyzed virions treated with proteinase K and samples prepared from mock-infected cells. Overall, we identified 47 viral proteins associated with PRV virions, 40 of which were previously localized to the capsid, tegument, and envelope layers using traditional biochemical approaches. Additionally, we identified seven viral proteins that were previously undetected in virions, including pUL8, pUL20, pUL32, pUL40 (RR2), pUL42, pUL50 (dUTPase), and Rsp40/ICP22. Furthermore, although we did not enrich for posttranslational modifications, we detected phosphorylation of four virion proteins: pUL26, pUL36, pUL46, and pUL48. Finally, we identified 48 host proteins associated with PRV virions, many of which have known functions in important cellular pathways such as intracellular signaling, mRNA translation and processing, cytoskeletal dynamics, and membrane organization. This analysis extends previous work aimed at determining the composition of herpesvirus virions and provides novel insights critical for understanding the mechanisms underlying PRV entry, assembly, egress, spread, and pathogenesis.  相似文献   

6.
Wang L  Guo H  Reyes N  Lee S  Bortz E  Guo F  Sun R  Tong L  Deng H 《Journal of virology》2012,86(3):1348-1357
Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologically associated with several types of human malignancies. However, as these two human gammaherpesviruses do not replicate efficiently in cultured cells, the morphogenesis of gammaherpesvirus virions is poorly understood. Murine gammaherpesvirus 68 (MHV-68) provides a tractable model to define common, conserved features of gammaherpesvirus biology. ORF52 of MHV-68 is conserved among gammaherpesviruses. We have previously shown that this tegument protein is essential for the envelopment and egress of viral particles and solved the crystal structure of ORF52 dimers. To more closely examine its role in virion maturation, we performed immunoelectron microscopy of MHV-68-infected cells and found that ORF52 localized to both mature, extracellular virions and immature viral particles in the cytoplasm. ORF52 consists of three α-helices followed by one β-strand. To understand the structural requirements for ORF52 function, we constructed mutants of ORF52 and examined their ability to complement an ORF52-null MHV-68 virus. Mutations in conserved residues in the N-terminal α1-helix and C terminus, or deletion of the α2-helix, resulted in a loss-of-function phenotype. Furthermore, the α1-helix was crucial for the predominantly punctate cytoplasmic localization of ORF52, while the α2-helix was a key domain for ORF52 dimerization. Immunoprecipitation experiments demonstrated that ORF52 interacts with another MHV-68 tegument protein, ORF42; however, a single point mutation in R95 in the C terminus of ORF52 led to the loss of this interaction. Moreover, the homologues of MHV-68 ORF52 in Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus complement the defect in ORF52-null MHV-68 and interact with MHV-68 ORF52. Taken together, these data uncover the relationship between the α-helical structure and the molecular basis for ORF52 function. This is the first structure-based functional domain mapping study for an essential gammaherpesvirus tegument protein.  相似文献   

7.
Rozen R  Sathish N  Li Y  Yuan Y 《Journal of virology》2008,82(10):4742-4750
Herpesvirus virions are highly organized structures built through specific protein-protein interactions. Thus, revelation of the protein interactions among virion proteins will shed light on the processes and the mechanisms of virion formation. Recently, we identified 24 virion proteins of Kaposi's sarcoma-associated herpesvirus (KSHV), using a proteomic approach (F. X. Zhu et al., J. Virol. 79:800-811, 2005). In the current study, a comprehensive analysis of protein-protein interaction between KSHV virion proteins was carried out using yeast two-hybrid (Y2H) and coimmunoprecipitation (co-IP) approaches. Every pairwise combination between KSHV tegument and capsid proteins, between tegument and envelope proteins, and among tegument proteins was tested for possible binary interaction. Thirty-seven protein-protein interactions were identified by both Y2H and co-IP analyses. The results revealed interactions between tegument and capsid proteins such as that of open reading frame 64 (ORF64) with ORF25 (major capsid protein [MCP]), ORF62 (triplex-1 [TRI-1]), and ORF26 (TRI-2). Many interactions were detected among the tegument proteins. ORF64 was found to interact with several tegument proteins including ORF11, ORF21, ORF33, ORF45, ORF63, ORF75, and ORF64 itself, suggesting that ORF64 may serve as a hub protein and play a role in recruiting tegument proteins during tegumentation and virion assembly. Our investigation also revealed redundant interactions between tegument proteins and envelope glycoproteins. These interactions are believed to contribute to final envelopment in virion assembly. Overall, this study allows us to establish a virion-wide protein interaction map, which provides insight into the architecture of the KSHV virion and sets up a foundation for exploring the functions of these proteins in viral particle assembly.  相似文献   

8.
9.
To A  Bai Y  Shen A  Gong H  Umamoto S  Lu S  Liu F 《PloS one》2011,6(4):e17796
Human cytomegalovirus (HCMV) is the largest human herpesvirus and its virion contains many viral encoded proteins found in the capsid, tegument, and envelope. In this study, we carried out a yeast two-hybrid (YTH) analysis to study potential binary interactions among 56 HCMV-encoded virion proteins. We have tested more than 3,500 pairwise combinations for binary interactions in the YTH analysis, and identified 79 potential interactions that involve 37 proteins. Forty five of the 79 interactions were also identified in human cells expressing the viral proteins by co-immunoprecipitation (co-IP) experiments. To our knowledge, 58 of the 79 interactions revealed by YTH analysis, including those 24 that were also identified in co-IP experiments, have not been reported before. Novel potential interactions were found between viral capsid proteins and tegument proteins, between tegument proteins, between tegument proteins and envelope proteins, and between envelope proteins. Furthermore, both the YTH and co-IP experiments have identified 9, 7, and 5 interactions that were involved with UL25, UL24, and UL89, respectively, suggesting that these "hub" proteins may function as the organizing centers for connecting multiple virion proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly. Our study provides a framework to study potential interactions between HCMV proteins and investigate the roles of protein-protein interactions in HCMV virion formation or maturation process.  相似文献   

10.
Human cytomegalovirus (HCMV) is the most genetically and structurally complex human herpesvirus and is composed of an envelope, a tegument, and a dsDNA-containing capsid. HCMV tegument plays essential roles in HCMV infection and assembly. Using cryo electron tomography (cryoET), here we show that HCMV tegument compartment can be divided into two sub-compartments: an inner and an outer tegument. The inner tegument consists of densely-packed proteins surrounding the capsid. The outer tegument contains those components that are loosely packed in the space between the inner tegument and the pleomorphic glycoprotein-containing envelope. To systematically characterize the inner tegument proteins interacting with the capsid, we used chemical treatment to strip off the entire envelope and most tegument proteins to obtain a tegumented capsid with inner tegument proteins. SDS-polyacrylamide gel electrophoresis analyses show that only two tegument proteins, UL32-encoded pp150 and UL48-encoded high molecular weight protein (HMWP), remains unchanged in their abundance in the tegumented capsids as compared to their abundance in the intact particles. Three-dimensional reconstructions by single particle cryo electron microscopy (cryoEM) reveal that the net-like layer of icosahedrally-ordered tegument densities are also the same in the tegumented capsid and in the intact particles. CryoET reconstruction of the tegumented capsid labeled with an anti-pp150 antibody is consistent with the biochemical and cryoEM data in localizing pp150 within the ordered tegument. Taken together, these results suggest that pp150, a betaherpesvirus-specific tegument protein, is a constituent of the net-like layer of icosahedrally-ordered capsid-bound tegument densities, a structure lacking similarities in alpha and gammaherpesviruses.  相似文献   

11.
Meckes DG  Wills JW 《Journal of virology》2008,82(21):10429-10435
We have made the surprising discovery that the interactions of herpes simplex virus with its initial cell attachment receptor induce a rapid and highly efficient structural change in the tegument, the region of the virion situated between the membrane and the capsid. It has been known for nearly a decade that viruses can trigger host signaling pathways when they bind to receptors on the cell surface; however, until now there has been no evidence that a signal can be sent in reverse--from the "outside in"--across a viral membrane. Evidence for this signaling event was found during studies of UL16, a tegument protein that is conserved among all the herpesviruses. Previous work has demonstrated that UL16 is bound to capsids isolated from the cytoplasm of infected cells, but this interaction is destabilized during subsequent egress steps, leading to release of the extracellular virion. Pretreatment with N-ethylmaleimide, a small, membrane-permeating compound that covalently modifies free cysteines, restabilizes the interaction, thereby permitting the capsid-UL16 complex to be isolated following disruption of virions with NP-40. In the experiments described here, we found that the natural signal for release of UL16 from capsids is sent when virions merely bind to cells at 4 degrees C. The internal change was also observed upon binding to immobilized heparin in a manner that requires viral glycoprotein C. This represents the first example of signaling across a viral envelope following receptor binding.  相似文献   

12.
Murine gammaherpesvirus 68 (MHV-68) has been developed as a model for the human gammaherpesviruses Epstein-Barr virus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), which are associated with several types of human diseases. Open reading frame 45 (ORF45) is conserved among the members of the Gammaherpesvirinae subfamily and has been suggested to be a virion tegument protein. The repression of ORF45 expression by small interfering RNAs inhibits MHV-68 viral replication. However, the gene product of MHV-68 ORF45 and its function have not yet been well characterized. In this report, we show that MHV-68 ORF45 is a phosphorylated nuclear protein. We constructed an ORF45-null MHV-68 mutant virus (45STOP) by the insertion of translation termination codons into the portion of the gene encoding the N terminus of ORF45. We demonstrated that the ORF45 protein is essential for viral gene expression immediately after the viral genome enters the nucleus. These defects in viral replication were rescued by providing ORF45 in trans or in an ORF45-null revertant (45STOP.R) virus. Using a transcomplementation assay, we showed that the function of ORF45 in viral replication is conserved with that of its KSHV homologue. Finally, we found that the C-terminal 23 amino acids that are highly conserved among the Gammaherpesvirinae subfamily are critical for the function of ORF45 in viral replication.  相似文献   

13.
The tegument is a layer of proteins between the nucleocapsid and the envelope of herpesviruses. The functions of most tegument proteins are still poorly understood. In murine gammaherpesvirus 68, ORF52 is an abundant tegument protein of 135 residues that is required for the assembly and release of infectious virus particles. To help understand the molecular basis for the function of this protein, we have determined its crystal structure at 2.1 A resolution. The structure reveals a dimeric association of this protein. Interestingly, an N-terminal alpha-helix that assumes different conformation in the two monomers of the dimer mediates the formation of an asymmetrical tetramer and contains many highly conserved residues. Structural and sequence analyses suggest that this helix is more likely involved in interactions with other components of the tegument or nucleocapsid of the virus and that ORF52 functions as a symmetrical dimer. The asymmetrical tetramer of ORF52 may be a "latent" form of the protein, when it is not involved in virion assembly. The self-association of ORF52 has been confirmed by co-immunoprecipitation and fluorescence resonance energy transfer experiments. Deletion of the N-terminal alpha-helix, as well as mutation of the conserved Arg(95) residue, abolished the function of ORF52. The results of the functional studies are fully consistent with the structural observations and indicate that the N-terminal alpha-helix is a crucial site of interaction for ORF52.  相似文献   

14.
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a recently discovered DNA tumor virus that belongs to the γ-herpesvirus subfamily. Though numerous studies on KSHV and other herpesviruses, in general, have revealed much about their multilayered organization and capsid structure, the herpesvirus capsid assembly and maturation pathway remains poorly understood. Structural variability or irregularity of the capsid internal scaffolding core and the lack of adequate tools to study such structures have presented major hurdles to earlier investigations employing more traditional cryo-electron microscopy (cryoEM) single particle reconstruction. In this study, we used cryo-electron tomography (cryoET) to obtain 3D reconstructions of individual KSHV capsids, allowing direct visualization of the capsid internal structures and systematic comparison of the scaffolding cores for the first time. We show that B-capsids are not a structurally homogenous group; rather, they represent an ensemble of “B-capsid-like” particles whose inner scaffolding is highly variable, possibly representing different intermediates existing during the KSHV capsid assembly and maturation. This information, taken together with previous observations, has allowed us to propose a detailed pathway of herpesvirus capsid assembly and maturation.  相似文献   

15.
Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for Epstein-Barr virus and Kaposi''s sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in trans or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.Gammaherpesviruses are associated with tumorigenesis. Like other herpesviruses, they are characterized as having two distinct stages in their life cycle: lytic replication and latency (15, 16, 18, 21, 54). Latency provides the viruses with advantages to escape host immune surveillance and to establish lifelong persistent infection and contributes to transformation and development of malignancies. However, it is through lytic replication that viruses propagate and transmit among hosts to maintain viral reservoirs. Both viral latency and lytic replication play important roles in tumorigenesis. The gammaherpesvirus subfamily includes Epstein-Barr virus (EBV), Kaposi''s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 and murine gammaherpesvirus 68 (MHV-68), among others. EBV is associated with Burkitt''s lymphoma, nasopharyngeal carcinoma, Hodgkin''s disease, and lymphoproliferative diseases in immunodeficient patients (28). KSHV is etiologically linked with Kaposi''s sarcoma, primary effusion lymphoma, and multicentric Castleman''s disease (11-13, 22, 52). Neither in vivo nor in vitro studies of EBV and KSHV are convenient due to their propensity to establish latency in cell culture and their limited host ranges.MHV-68 is genetically related to these two human gammaherpesviruses, especially to KSHV, based on the alignment of their genomic sequences and other biological properties (55). As a natural pathogen of wild rodents, MHV-68 also infects laboratory mice (6, 40, 46) and replicates to a high titer in a variety of fibroblast and epithelial cell lines. These advantages make MHV-68 an excellent model for studying the lytic replication of gammaherpesviruses in vitro and certain aspects of virus-host interactions in vivo. In addition, the MHV-68 genome has been cloned as a bacterial artificial chromosome (BAC) that can propagate in Escherichia coli (1, 2, 36, 51), making it convenient to study the function of each open reading frame (ORF) by genetic methods. Exploring the functions of MHV-68 ORFs will likely shed light on the functions of their homologues in human gammaherpesviruses.Gammaherpesviral particles have a characteristic multilayered architecture. An infectious virion contains a double-stranded DNA genome, an icosahedral capsid shell, a thick, proteinaceous tegument compartment, and a lipid bilayer envelope spiked with glycoproteins (14, 30, 47, 49). As a unique structure of herpesviruses, the tegument plays important roles in multiple aspects of the viral life cycle, including virion assembly and egress (38, 48, 53), translocation of nucleocapsids into the nucleus, transactivation of viral immediate-early genes, and modulation of host cell gene expression, innate immunity, and signal transduction (9, 10, 23, 60). Some components of MHV-68 tegument have been identified by a mass spectrometric study (8), and the functions of some tegument proteins have been revealed, such as ORF45, ORF52, and ORF75c (7, 24, 29).MHV-68 ORF33 is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. Its homologues include human herpes simplex virus type 1 (HSV-1) UL16, human herpes simplex virus type 2 (HSV-2) UL16, human cytomegalovirus (HCMV) UL94, EBV BGLF2, KSHV ORF33, and rhesus monkey rhadinovirus (RRV) ORF33. HSV-1 UL16 has been identified as a tegument protein and may function in viral DNA packaging, virion assembly, budding, and egress (5, 32, 35, 41, 44). HCMV UL94 is a virion associated protein and might function in virion assembly and budding (31, 57). EBV BGLF2, KSHV ORF33, and RRV ORF33 are also virion-associated proteins, but their functions are not clear (26, 43, 59). The mass spectrometric study of MHV-68 did not identify ORF33 as a virion component (8), although ORF33 is found to be essential for viral lytic replication by transposon mutagenesis of the MHV-68 genome cloned as a BAC (51). However, insertion of the 1.2-kbp Mu transposon in that study may influence the expression of ORFs approximate to ORF33. Consequently, the role ORF33 plays in viral replication needs to be confirmed, preferably through site-directed mutagenesis. Whether ORF33 is a tegument protein and the exact viral replication stage in which it functions also need to be investigated.We determined that MHV-68 ORF33 encodes a tegument protein and is expressed with true late kinetics. To explore the function of ORF33 in viral lytic phase, we used site-directed mutagenesis and generated an ORF33-null mutant, taking advantage of the MHV-68 BAC system. We showed that the ORF33-null mutant is capable of viral DNA replication, early and late gene expression, capsid assembly, and DNA packaging, but incapable of virion release. The defect of ORF33-null mutant can be rescued in trans by an ORF33 expression plasmid.  相似文献   

16.
Tulane virus (TV) is a newly isolated cultivatable calicivirus that infects juvenile rhesus macaques. Here we report a 6.3 Å resolution cryo-electron microscopy structure of the TV virion. The TV virion is about 400 Å in diameter and consists of a T = 3 icosahedral protein capsid enclosing the RNA genome. 180 copies of the major capsid protein VP1 (∼57 KDa) are organized into two types of dimers A/B and C/C and form a thin, smooth shell studded with 90 dimeric protrusions. The overall capsid organization and the capsid protein fold of TV closely resemble that of other caliciviruses, especially of human Norwalk virus, the prototype human norovirus. These close structural similarities support TV as an attractive surrogate for the non-cultivatable human noroviruses. The most distinctive feature of TV is that its C/C dimers are in a highly flexible conformation with significantly reduced interactions between the shell (S) domain and the protruding (P) domain of VP1. A comparative structural analysis indicated that the P domains of TV C/C dimers were much more flexible than those of other caliciviruses. These observations, combined with previous studies on other caliciviruses, led us to hypothesize that the enhanced flexibility of C/C dimer P domains are likely required for efficient calicivirus-host cell interactions and the consequent uncoating and genome release. Residues in the S-P1 hinge between the S and P domain may play a critical role in the flexibility of P domains of C/C dimers.  相似文献   

17.
Loret S  Guay G  Lippé R 《Journal of virology》2008,82(17):8605-8618
The herpes simplex virus type 1 (HSV-1) genome is contained in a capsid wrapped by a complex tegument layer and an external envelope. The poorly defined tegument plays a critical role throughout the viral life cycle, including delivery of capsids to the nucleus, viral gene expression, capsid egress, and acquisition of the viral envelope. Current data suggest tegumentation is a dynamic and sequential process that starts in the nucleus and continues in the cytoplasm. Over two dozen proteins are assumed to be or are known to ultimately be added to virions as tegument, but its precise composition is currently unknown. Moreover, a comprehensive analysis of all proteins found in HSV-1 virions is still lacking. To better understand the implication of the tegument and host proteins incorporated into the virions, highly purified mature extracellular viruses were analyzed by mass spectrometry. The method proved accurate (95%) and sensitive and hinted at 8 different viral capsid proteins, 13 viral glycoproteins, and 23 potential viral teguments. Interestingly, four novel virion components were identified (UL7, UL23, UL50, and UL55), and two teguments were confirmed (ICP0 and ICP4). In contrast, UL4, UL24, the UL31/UL34 complex, and the viral UL15/UL28/UL33 terminase were undetected, as was most of the viral replication machinery, with the notable exception of UL23. Surprisingly, the viral glycoproteins gJ, gK, gN, and UL43 were absent. Analyses of virions produced by two unrelated cell lines suggest their protein compositions are largely cell type independent. Finally, but not least, up to 49 distinct host proteins were identified in the virions.  相似文献   

18.
Herpesviruses acquire their envelope by budding into the lumen of cytoplasmic membrane vesicles. This process is initiated by component(s) on viral particles, which recognize the budding site where the viral glycoproteins are present and recruit cellular cargo transport and sorting machinery to the site to complete the budding process. Proteins in the tegument layer, connecting capsid and envelope, are candidates for the recognition of budding sites on vesicle membrane and induction of budding and final envelopment. We examined several outer and matrix tegument proteins of Kaposi’s sarcoma-associated herpesvirus (KSHV) and found that ORF45 associates with lipid rafts (LRs) of cellular membrane. LRs are membrane micro-domains, which have been implicated as relay stations in intracellular signaling and transport including viral entry and virion assembly. The ability of ORF45 to target LR is dependent on the mono-ubiquitylation of ORF45 at Lys297 as the mutation at Lys297 (K297R) abolished LR-association of ORF45. The K297R mutation also impairs ORF45 and viral particle co-localization with trans-Golgi network and endosomes, but facilitates ORF45 and viral particles co-localizing with lysosomes. More importantly, the recombinant KSHV carrying ORF45 K297R mutant (BAC-K297R) was found severely defective in producing mature and infectious virion particles in comparison to wild type KSHV (BAC16). Taken together, our results reveal a new function of KSHV tegument protein ORF45 in targeting LR of host cell membrane, promoting viral particles co-localization with trans-Golgi and endosome vesicles and facilitating the maturation and release of virion particles, suggesting that ORF45 plays a role in bringing KSHV particles to the budding site on cytoplasmic vesicle membrane and triggering the viral budding process for final envelopment and virion maturation.  相似文献   

19.
Lee JI  Luxton GW  Smith GA 《Journal of virology》2006,80(24):12086-12094
The herpesvirus tegument is a layer of viral and cellular proteins located between the capsid and envelope of the virion. The VP1/2 tegument protein is critical for the propagation of all herpesviruses examined. Using an infectious clone of the alphaherpesvirus pseudorabies virus, we have made a collection of truncation and in-frame deletion mutations within the VP1/2 gene (UL36) and examined the resulting viruses for spread between cells. We found that the majority of the VP1/2 protein either was essential for virus propagation or did not tolerate large deletions. A recently described amino-terminal deubiquitinase-encoding domain was dispensable for alphaherpesvirus propagation, but the rate of propagation in an epithelial cell line and the frequency of transport in axons of primary sensory neurons were both reduced. We mapped one essential domain to a conserved sequence at the VP1/2 carboxy terminus and demonstrated that this domain sufficient to redirect the green fluorescent protein to capsid assemblons in nuclei of infected cells.  相似文献   

20.
Different tissue culture cell lines infected with a number of alphaherpesviruses produce, in addition to virions, light particles (L particles). L particles are composed of the envelope and tegument components of the virion but totally lack the proteins of the capsid and the virus genome; therefore, they are noninfectious. In this electron microscopy report, we show that L particles are produced during primary replication of the alphaherpesvirus pseudorabies virus (PRV) in the nasal mucosa of experimentally infected swine, its natural host. Although PRV infected different types of cells of the respiratory and olfactory mucosae, PRV L particles were found to be produced exclusively by epithelial cells and fibroblasts. We observed that formation of noninfectious particles occurred by budding of condensed tegument at the inner nuclear membrane and at membranes of cytoplasmic vesicles, resulting in intracisternal and intravesicular L particles, respectively. Both forms of capsidless particles were clearly distinguishable by the presence of prominent surface projections on the envelope and the higher electron density of the tegument, morphological features which were only observed in intravesicular L particles. Moreover, intravesicular but not intracisternal L particles were found to be released by exocytosis and were also identified extracellularly. Comparative analysis between PRV virion and L-particle morphogenesis indicates that both types of virus particles share a common intracellular pathway of assembly and egress but that they show different production patterns during the replication cycle of PRV.  相似文献   

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