Herpes Simplex Virus Tegument Protein VP16 Is a Component of Primary Enveloped Virions (original) (raw)
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Journal of Virology, 2012
Herpes simplex virus 1 (HSV-1) viral glycoproteins gD (carboxyl terminus), gE, gK, and gM, the membrane protein UL20, and membrane-associated protein UL11 play important roles in cytoplasmic virion envelopment and egress from infected cells. We showed previously that a recombinant virus carrying a deletion of the carboxyl-terminal 29 amino acids of gD (gDΔct) and the entire gE gene (ΔgE) did not exhibit substantial defects in cytoplasmic virion envelopment and egress (H. C. Lee et al., J. Virol. 83:6115–6124, 2009). The recombinant virus ΔgM2, engineered not to express gM, produced a 3- to 4-fold decrease in viral titers and a 50% reduction in average plaque sizes in comparison to the HSV-1(F) parental virus. The recombinant virus containing all three mutations, gDΔct-ΔgM2-ΔgE, replicated approximately 1 log unit less efficiently than the HSV-1(F) parental virus and produced viral plaques which were on average one-third the size of those of HSV-1(F). The recombinant virus ΔUL11-ΔgM2...
Nuclear Localizations of the Herpes Simplex Virus Type 1 Tegument Proteins
Journal of Virology, 2005
Herpes simplex virus type 1 (HSV-1) induces microtubule reorganization beginning at approximately 9 h postinfection (hpi), and this correlates with the nuclear localization of the tegument protein VP22. Thus, the active retention of this major virion component by cytoskeletal structures may function to regulate its subcellular localization (A.
Journal of General Virology, 1987
Spikes of different kinds, distinct in size and appearance were detected on the surfaces of herpes simplex virions by electron microscopy of negatively stained preparations. Use of monoclonal antibodies coupled to colloidal gold permitted identification of viral glycoproteins present in different structures projecting from the virion envelope. Antibodies specific for the glycoprotein designated gB bound to the most prominent spikes, which were about 14 nm long and, in side view, had a flattened T-shaped top. Antibodies specific for gC bound to structures that, in some instances, appeared to extend as much as 24 nm from the surface of the envelope and were too thin to resolve. Antibodies specific for gD bound to structures that extended as much as 8 to 10 nm from the surface of the envelope. The gB spikes were invariably clustered, usually in protrusions of the envelope varying from small bulbous distentions to long tail-like projections. The gC components were randomly distributed and widely spaced and the gD components were irregularly clustered in patterns distinct from those of the gB spikes. These three glycoproteins therefore form structures that are different in size, morphology and distribution in the envelope.
Journal of Virology, 2001
Examination of the three-dimensional structure of intact herpes simplex virus type 1 (HSV-1) virions had revealed that the icosahedrally symmetrical interaction between the tegument and capsid involves the pentons but not the hexons (Z. 73:3210-3218, 1999). To account for this, we postulated that the presence of the small capsid protein, VP26, on top of the hexons was masking potential binding sites and preventing tegument attachment. We have now tested this hypothesis by determining the structure of virions lacking VP26. Apart from the obvious absence of VP26 from the capsids, the structures of the VP26 minus and wild-type virions were essentially identical. Notably, they showed the same tegument attachment patterns, thereby demonstrating that VP26 is not responsible for the divergent tegument binding properties of pentons and hexons.
Electron Tomography of Nascent Herpes Simplex Virus Virions
Journal of Virology, 2007
Cells infected with herpes simplex virus type 1 (HSV-1) were conventionally embedded or freeze substituted after high-pressure freezing and stained with uranyl acetate. Electron tomograms of capsids attached to or undergoing envelopment at the inner nuclear membrane (INM), capsids within cytoplasmic vesicles near the nuclear membrane, and extracellular virions revealed the following phenomena. (i) Nucleocapsids undergoing envelopment at the INM, or B capsids abutting the INM, were connected to thickened patches of the INM by fibers 8 to 19 nm in length and <5 nm in width. The fibers contacted both fivefold symmetrical vertices (pentons) and sixfold symmetrical faces (hexons) of the nucleocapsid, although relative to the respective frequencies of these subunits in the capsid, fibers engaged pentons more frequently than hexons. (ii) Fibers of similar dimensions bridged the virion envelope and surface of the nucleocapsid in perinuclear virions. (iii) The tegument of perinuclear virions was considerably less dense than that of extracellular virions; connecting fibers were observed in the former case but not in the latter. (iv) The prominent external spikes emanating from the envelope of extracellular virions were absent from perinuclear virions. (v) The virion envelope of perinuclear virions appeared denser and thicker than that of extracellular virions. (vi) Vesicles near, but apparently distinct from, the nuclear membrane in single sections were derived from extensions of the perinuclear space as seen in the electron tomograms. These observations suggest very different mechanisms of tegumentation and envelopment in extracellular compared with perinuclear virions and are consistent with application of the final tegument to unenveloped nucleocapsids in a compartment(s) distinct from the perinuclear space.
Journal of Virology, 2008
To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as -galactosidase expression cassettes. When the BAC pHSV1(17 ؉ )blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17 ؉ )blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17 ؉ )blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.
Intervirology, 2013
Background and Objective: During herpesvirus envelopment capsids, tegument polypeptides and membrane proteins assemble at the site of budding, and a cellular lipid bilayer becomes refashioned into a spherical envelope. A web of interactions between tegument proteins and the cytoplasmic tails of viral glycoproteins play a critical role in this process. We have previously demonstrated that for herpes simplex virus (HSV)-1 the cytoplasmic tail of glycoprotein H (gH) binds the tegument protein VP16. The HSV and pseudorabies virus (PRV) genomes are essentially collinear, and individual gene products show significant sequence homology. However, the demarcation of function often differs between PRV and HSV proteins. The goal of this study was to determine whether PRV gH and VP16 interact in a manner similar to their homologs in HSV. Methods: A fusion protein pull-down assay was performed in which a PRV gH cytoplasmic tail-glutathione S-transferase fusion protein, bound to glutathione-Sepha...
Characterization of the UL20 Protein……………………………………………57 The UL20 ORF…………………………………………………………..57 Membrane Topology of UL20p………………………………………….57 Interdependence with gK for Transport………………………………….58 Function of UL20p in the HSV-1 Lifecycle……………………………..59 Characterization of the UL11 Protein……………………………………………60 Function of the UL11 Protein in the HSV-1 Lifecycle…………………..61 Characterization of the UL16 Protein……………………………………………61 Function of the UL16 Protein in the HSV-1 Life Cycle…………………61 REFERENCES…………………………………………………………………………..63 CHAPTER 2: THE UL20 PROTEIN FUNCTIONS PRECEDE AND ARE REQUIRED FOR UL11 FUNCTIONS IN HERPES SIMPLEX VIRUS TYPE-1 (HSV-1) CYTOPLASMIC VIRION ENVELOPMENT ……………………………………………………………………..93 Introduction………………………………………………………………………93 Materials and Methods…………………………………………………………...95 Cells, viruses, and plasmids………………………………………………95 Construction of HSV-1 mutants with deletions of the UL11, and/or UL20 genes (pYEbac102, pYEbac102ΔUL11, pYEbac102ΔUL20, and pYEBac102ΔUL11ΔUL20)………………………………………………96 Confirmation of the targeted mutations in pYEbac102 DNA…………….97 Transfection of HSV-1 BAC DNAs………………………………………98 One step growth kinetics of YEbac102 mutants…………………………..98 Electron microscopy……………………………………………………….99 Confocal Microscopy………………………………………………….....99 Results…………………………………………………………………………..100 Construction of the HSV-1 BACs pYEbac102ΔUL11 and pYEbac102ΔUL11ΔUL20……………………………………………...100 PCR-based confirmation of the pYEbac102ΔUL11 and pYEbac102ΔUL11ΔUL20 genotypes…………………………………..101 Production of infectious virus from pYEbac102-based constructs……..104 Plaque morphology and replication kinetics of HSV-1 YEbac102 mutants………………………………………………………………….105 v Ultrastructural characterization of the YEbac102ΔUL11 and YEbac102ΔUL11ΔUL20 mutant viruses……………………………….106 UL11 and UL20 are independently transported to the TGN…………...111 Discussion………………………………………………………………………115 REFERENCES…………………………………………………………………………120 CHAPTER 3: HERPES SIMPLEX VIRUS TYPE-1 (HSV-1) UL16 IS REQUIRED FOR EFFICIENT NUCLEAR EGRESS AND CYTOPLASMIC ENVELOPMENT ...……………124 Introduction……………………………………………………………………..124 Materials and Methods………………………………………………………….126 Cells, viruses, and plasmids…………………………………………….126 PCR primer design……………………………………………………...126 Construction of HSV-1 mutants containing deletions of the UL16 or UL11 gene (pYEBacΔUL16, pYEBacΔUL11)………………………………..127 Transfection of HSV-1 BAC DNAs……………………………………129 One step growth kinetics and plaque morphology of YEbac102 mutants………………………………………………………………….129 Electron microscopy……………………………………………………130 Results…………………………………………………………………………..130 Construction of the HSV-1 BAC pYEbacΔUL16……………………...130 Production of infectious virus from pYEbac102-based constructs……..132 Plaque morphology and replication kinetics of HSV-1 YEbac102 mutants………………………………………………………………….132 Ultrastructural characterization of the YEbac102ΔUL16 and YEbac102ΔUL11 mutant viruses………………………………………135 Discussion………………………………………………………………………135 REFERENCES…………………………………………………………………………140 CHAPTER 4: CONCLUDING REMARKS…………………………………………………...143 Summary………………………………………………………………………..143 Current and future research……………………………………………………..146 REFERENCES…………………………………………………………………………149 APPENDIX: ADDITIONAL WORK……...…………..………………………………………153 DISCLAIMER………………………………………………………………………….153 INTRODUCTION……………………………………………………………………...153 MATERIALS AND METHODS……………………………………………………….156 Cells and viruses………………………………………………………………..156 Plasmids………………………………………………………………………...156 UL20 complementation assay for infectious virus production…………………157 UL20 complementation assay for virus induced cell-to-cell fusion……………158 RESULTS………………………………………………………………………………158 Mutagenesis of HSV-1 UL20…………………………………………………..158 Complementation assay for infectious virus production……………………….159 Complementation assay for virus induced cell-to-cell fusion…………………..