Herpesviruses and Intermediate Filaments: Close Encounters with the Third Type (original) (raw)
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Journal of Virology, 2007
The biogenesis of multivesicular bodies (MVBs) is topologically equivalent to virion budding. Hence, a number of viruses exploit the MVB pathway to build their envelope and exit from the cell. By expression of dominant negative forms of Vps4 and Vps24, two components of the MVB pathway, we observed an impairment in infectious herpes simplex virus (HSV) assembly/egress, in agreement with a recent report showing the involvement in HSV envelopment of Vps4, the MVB-specific ATPase (C. M. Crump, C. Yates, and T. Minson, J. Virol. 81:7380-7387). Furthermore, HSV infection resulted in morphological changes to MVBs. Glycoprotein B (gB), one of the most highly conserved glycoproteins across the Herpesviridae family, was sorted to MVB membranes. In cells expressing the dominant negative form of Vps4, the site of intracellular gB accumulation was altered; part of gB accumulated as an endoglycosidase H-sensitive immature form at a calreticulin-positive compartment, indicating that gB traffic was dependent on a functional MVB pathway. gB was ubiquitinated in both infected and transfected cells. Ubiquitination was in part dependent on ubiquitin lysine 63, a signal for cargo sorting to MVBs. Partial deletion of the gB cytoplasmic tail resulted in a dramatic reduction of ubiquitination, as well as of progeny virus assembly and release to the extracellular compartment. Thus, HSV envelopment/egress and gB intracellular trafficking are dependent on functional MVB biogenesis. Our data support the view that the sorting of gB to MVB membranes may represent a critical step in HSV envelopment and egress and that modified MVB membranes constitute a platform for HSV cytoplasmic envelopment or that MVB components are recruited to the site(s) of envelopment.
Host Cell Signatures of the Envelopment Site within Beta-Herpes Virions
International Journal of Molecular Sciences
Beta-herpesvirus infection completely reorganizes the membrane system of the cell. This system is maintained by the spatiotemporal arrangement of more than 3000 cellular proteins that continuously adapt the configuration of membrane organelles according to cellular needs. Beta-herpesvirus infection establishes a new configuration known as the assembly compartment (AC). The AC membranes are loaded with virus-encoded proteins during the long replication cycle and used for the final envelopment of the newly formed capsids to form infectious virions. The identity of the envelopment membranes is still largely unknown. Electron microscopy and immunofluorescence studies suggest that the envelopment occurs as a membrane wrapping around the capsids, similar to the growth of phagophores, in the area of the AC with the membrane identities of early/recycling endosomes and the trans-Golgi network. During wrapping, host cell proteins that define the identity and shape of these membranes are captu...
Proceedings of the National Academy of Sciences, 2013
The herpesvirus virion is a multilayered structure consisting of a DNA-filled capsid, tegument, and envelope. Detailed reconstructions of the capsid are possible based on its icosahedral symmetry, but the surrounding tegument and envelope layers lack regular architecture. To circumvent limitations of symmetry-based ultrastructural reconstruction methods, a fluorescence approach was developed using single-particle imaging combined with displacement measurements at nanoscale resolution. An analysis of 11 tegument and envelope proteins defined the composition and plasticity of symmetric and asymmetric elements of the virion architecture. The resulting virion protein map ascribes molecular composition to density profiles previously acquired by traditional ultrastructural methods, and provides a way forward to examine the dynamics of the virion architecture during infection.
Herpes Simplex Virus Tegument Protein VP16 Is a Component of Primary Enveloped Virions
Journal of Virology, 2006
Immunogold electron microscopy was used to determine whether the tegument proteins VP13/14, VP22, and VP16 of herpes simplex virus type 1 (HSV1) are components of primary enveloped virions. Whereas VP13/14 and VP22 were not detected in virus particles in the perinuclear space and were present in only mature extracellular virions, VP16 was acquired prior to primary envelopment of the virus at the inner nuclear membrane. This finding highlights potential similarities and differences between HSV1 and the related alphaherpesvirus, pseudorabies virus, in which the homologues of all three of these tegument proteins are not incorporated into the virion until secondary envelopment.
The way out: what we know and do not know about herpesvirus nuclear egress
Cellular Microbiology, 2013
To access the final maturation compartment, intranuclear capsids have to cross the nuclear envelope which represents a formidable barrier. They do so by budding at the inner nuclear membrane, thereby forming a primary enveloped particle residing in the perinuclear cleft. Formation of primary envelopes is driven by a heterodimeric complex of two conserved herpesviral proteins, designated in the herpes simplex virus nomenclature as pUL34, a tail-anchored transmembrane protein located in the nuclear envelope, and pUL31. This nuclear egress complex recruits viral and cellular kinases to soften the nuclear lamina and allowing access of capsids to the inner nuclear membrane. How capsids are recruited to the budding site and into the primary virus particle is still not completely understood, nor is the composition of the primary enveloped virion in the perinuclear cleft. Fusion of the primary envelope with the outer nuclear membrane then results in translocation of the capsid to the cytosol. This fusion event is clearly different from fusion during infectious entry of free virions into target cells in that it does not require the conserved essential core herpesvirus fusion machinery. Nuclear egress can thus be viewed as a vesicle (primary envelope)mediated transport of cargo (capsids) through the nuclear envelope, a process which had been unique in cell biology. Only recently has a similar process been identified in Drosophila for nuclear egress of large ribonucleoprotein complexes. Thus, herpesviruses appear to subvert a hitherto cryptic cellular pathway for translocation of capsids from the nucleus to the cytosol.
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…………………..
Journal of Virology, 2006
Assembly of herpes simplex viruses (HSV) is a poorly understood process involving multiple redundant interactions between large number of tegument and envelope proteins. We have previously shown (G. E. Lee, G. A. Church, and D. W. Wilson, J. Virol. 77:2038-2045, 2003) that the virion host shutoff (Vhs) tegument protein is largely insoluble in HSV-infected cells and is also stably associated with membranes. Here we demonstrate that both insolubility and stable membrane binding are stimulated during the course of an HSV infection. Furthermore, we have found that the amino-terminal 42 residues of Vhs are sufficient to mediate membrane association and tegument incorporation when fused to a green fluorescent protein (GFP) reporter. Particle incorporation correlates with sorting to cytoplasmic punctate structures that may correspond to sites of HSV assembly. We conclude that the amino terminus of Vhs mediates targeting to sites of HSV assembly and to the viral tegument.
Intranuclear formation of filaments in herpesvirus hominis infection of mice
Archiv f�r die gesamte Virusforschung, 1967
In several investigations of cells infected with the herpesviruses of various species, nuclei have been shown to contain filamentous and granular structures in addition to virions. Watraeh (1) reported finding filaments of uniform diameter in cultured chicken embryo kidney cells infected with the avian herpesvirus, infectious taryngotracheitis virus, and Reczko et al. (2) demonstrated rather irregular bundles of threadlike structures in nuclei of swine kidney cells infected with equine rhinopneumonitis virus. Similar hollow appearing fibrils have been reported by Ruebner et al. (3) in studies of mouse salivary glands infected with mouse cytomegalovirus. In addition, Morgan et al. ( in 1959 showed that, in nuclei of cultured cells, herpesvirus hominis infection was associated with some accumulation of filamentous forms and especially of granular aggregates. By curtailing viral biosynthetic pathways with p-fluorophenylalanine, Chitwood and Bracken (5) presented evidence indicating that filaments, granules and other bizarre forms actually do represent anomalously accumulated viral subunits. In the present investigation, the presence of intranuclear filaments is demonstrated in neurons of mice infected with herpesvirus hominis. It would appear that this human virus like other herpesviruses, may be assembled aberrantly in vivo as well as in vitro.