Simultaneous Deletion of Pseudorabies Virus Tegument Protein UL11 and Glycoprotein M Severely Impairs Secondary Envelopment

Kopp, Martina; Granzow, Harald; Fuchs, Walter; Klupp, Barbara G.; Mettenleiter, Thomas C.

doi:10.1128/jvi.78.6.3024-3034.2004

Cited by 73 publications

(78 citation statements)

References 38 publications

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“…They consist of tegument, which apparently initiates and completes secondary envelopment by budding into trans-Golgi vesicles without the need for a capsid. Formation of these capsidless particles, however, requires tegument and envelope proteins, because L-particle formation is blocked when specific viral tegument proteins, such as pUL11, and envelope proteins, e.g., gM, are absent (39). L-particles and dense bodies are heterogeneous in size but clearly resemble complete viral particles.…”

Section: Discussionmentioning

confidence: 99%

Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins

Klupp

Granzow²,

Fuchs³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Although the nuclear envelope is a dynamic structure that disassembles and reforms during mitosis, the formation of membranous vesicles derived from the nuclear envelope has not yet been described in noninfected cells. However, during herpesvirus maturation, intranuclear capsids initiate transit to the cytosol for final maturation by budding at the inner nuclear membrane. Two conserved herpesvirus proteins are required for this primary envelopment, designated in the alphaherpesviruses as pUL31 and pUL34. Here, we show that simultaneous expression of pUL31 and pUL34 of the alphaherpesvirus pseudorabies virus in stably transfected rabbit kidney cells resulted in the formation of vesicles in the perinuclear space that resemble primary envelopes without a nucleocapsid. They contain pUL31 and pUL34 as shown by immunolabeling and are derived from the nuclear envelope. Thus, coexpression of only two conserved herpesvirus proteins without any other viral factor is sufficient to induce the formation of vesicles from the nuclear membrane. This argues for the contribution of cellular factors in this process either recruited from their natural cytoplasmic location or not yet identified as components of the nuclear compartment.nuclear envelope ͉ primary envelopment ͉ pseudorabies virus ͉ herpesvirus egress H erpesvirus particles are complex macromolecular assemblies consisting of Ͼ30 virally encoded proteins, which make up four morphologically distinct structures, core, capsid, tegument, and envelope. Herpesvirus morphogenesis proceeds in two different cellular compartments. While capsid assembly and DNA packaging take place in the nuclei of infected cells, acquisition of the majority of tegument and final envelopment occur in the cytoplasm. To gain access to the cytoplasm the nucleocapsid has to traverse the nuclear lamina and the inner and outer nuclear membranes, because the diameter of the nuclear pores is too small to allow exit of the Ϸ110-nm particle. Although alternative mechanisms for nuclear egress have been proposed, most data support a model that entails primary envelopment by budding of capsids at the inner nuclear membrane resulting in the formation of primary virions in the perinuclear space whose envelope then fuses with the outer nuclear membrane to release the capsid into the cytoplasm (reviewed in ref. 1).The molecular mechanism of this budding, scission, and fusion reaction is unknown. Glycoproteins gB and gH, which are essential for fusion of the viral envelope with the plasma membrane during entry (2) as well as for cell-cell spread, are not required for virion formation indicating that a different molecular mechanism is responsible for fusion during nuclear egress (3). Cellular fusion processes involving the nuclear membranes occur during mitosis. However, the fusion machinery involved is unknown. Soluble N-ethylmaleimide sensitive factor attachment protein receptors ( Two conserved herpesvirus proteins, in the alphaherpesviruses designated as pUL31 and pUL34, are required for nuclear egress of herpesv...

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Section: Discussionmentioning

confidence: 99%

Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins

Klupp

Granzow²,

Fuchs³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

165

205

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“…In agreement with previous investigations of UL37-deleted HSV-1 and PrV (Desai et al, 2001;Klupp et al, 2001), non-enveloped nucleocapsids of HSV-1DUL37 accumulated in the cytosol, forming characteristic clusters. However, these clusters lacked the electron-dense material associated with aggregates of nucleocapsids that accumulate in the absence of HSV-1 or PrV pUL11 and gM (Kopp et al, 2004;Leege et al, 2009), PrV gE/gI and gM (Brack et al, 1999) or HSV-1 gE/gI and gD (Farnsworth et al, 2003), indicating that tegumentation of nucleocapsids in these clusters had not yet occurred or did not proceed beyond a very early stage.…”

Section: Discussionmentioning

confidence: 99%

“…In both cell lines infected with either UL37 deletion mutant, clusters of nonenveloped nucleocapsids were detected in the cytoplasm (Figs 5c, f and 6c, f), and enveloped virions were found only rarely. However, the cytoplasmic nucleocapsids were not associated with electron-dense tegument material, as in cells infected for example with PrV unable to express glycoproteins E/I and M (Brack et al, 1999) or with PrV and HSV-1 lacking pUL11 and gM (Kopp et al, 2004;Leege et al, 2009). Nucleocapsids at the inner nuclear membrane or primary virions in the perinuclear cleft were present in HSV-1DUL37[86-1035]-infected cells (Fig.…”

Section: Lack Of Trans-complementation Of Prv and Hsv-1 Ul37 Deletionmentioning

confidence: 99%

Phenotypic similarities and differences between UL37-deleted pseudorabies virus and herpes simplex virus type 1

Leege

Granzow

Fuchs

et al. 2009

Journal of General Virology

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In the absence of the tegument protein pUL37, virion formation of pseudorabies virus (PrV) and herpes simplex virus type 1 (HSV-1) is severely impaired. Non-enveloped nucleocapsids accumulate in clusters in the cytoplasm, whereas only a few enveloped particles can be detected. Although a contribution of pUL37 to nuclear egress of HSV-1 has been suggested, the nuclear stages of morphogenesis are not impaired in PrV-DUL37-infected cells. Moreover, HSV-1 pUL37 has been described as essential for replication, whereas PrV is able to replicate productively without pUL37, although to lower titres than wild-type virus. Thus, there may be functional differences between the respective pUL37 proteins. This study compared the phenotypes of UL37-deleted PrV and HSV-1 in parallel assays, using a novel pUL37 deletion mutant of HSV-1 strain KOS, HSV-1DUL37 . Aggregates of seemingly 'naked' nucleocapsids were present in the cytoplasm of African green monkey (Vero) or rabbit kidney (RK13) cells infected with HSV-1DUL37 or PrV-DUL37. Nuclear retention of nucleocapsids was not observed in either virus. However, in contrast to PrV-DUL37, HSV-1DUL37[86-1035] was unable to replicate productively in, and to form plaques on, either Vero or RK13 cells. Transcomplementation of respective deletion mutants with the heterologous pUL37 did not ensue. These data demonstrate that the conserved pUL37 in HSV-1 and PrV have similar but distinct functions.

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“…A role for UL11 in secondary envelopment was further highlighted by the phenotype of a mutant PrV that simultaneously lacked gM and UL11. In cells infected with this virus mutant, no infectious enveloped virions were produced but huge accumulations of capsids embedded in tegument were observed (25). Absence of the UL11 homolog in human cytomegalovirus blocked intracytoplasmic virion formation (40).…”

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confidence: 99%

Functional Analysis of the Pseudorabies Virus UL51 Protein

Klupp

Granzow

Klopfleisch

et al. 2005

J Virol

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Homologs of the UL51 protein of herpes simplex virus have been identified in all herpesvirus subfamilies, but until now, no function has been assigned to any of them. To investigate function of the UL51 gene product of the alphaherpesvirus pseudorabies virus (PrV), we isolated and analyzed a mutant lacking the major part of the open reading frame, PrV-⌬UL51F, and a rescuant. One-step growth analysis of PrV-⌬UL51F revealed only slightly reduced titers, but plaque size was notably diminished and reached only approximately 30% the plaque size of wild-type PrV. Ultrastructurally, intracytoplasmic capsids were found in large numbers either without envelope or in different stages of envelopment, indicating that secondary envelopment in the cytoplasm was less efficient. However, neuroinvasion in the mouse trigeminal pathway after intranasal infection was only slightly delayed. A PrV UL11 mutant also showed a defect in secondary envelopment (M. Kopp, H. Granzow, W. Fuchs, B. G. Klupp, E. Mundt, A. Karger, and T. C. Mettenleiter, J. Virol. 77:5339-5351, 2003). Since both proteins are part of the viral tegument and are predicted to be membrane associated, they may serve similar, possibly redundant functions during viral morphogenesis. Therefore, we also isolated a mutant simultaneously lacking UL51 and UL11. This mutant exhibited further reduced plaque size compared to the single-deletion mutants, but viral titers were comparable to those for the UL11 mutant. In electron microscopic analyses, the observed defect in secondary envelopment was similar to that found in the UL11 single-deletion mutant. In conclusion, both conserved tegument proteins, either singly or in combination, are involved in virion morphogenesis in the cytoplasm but are not essential for viral replication in vitro and in vivo.

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Simultaneous Deletion of Pseudorabies Virus Tegument Protein UL11 and Glycoprotein M Severely Impairs Secondary Envelopment

Cited by 73 publications

References 38 publications

Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins

Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins

Phenotypic similarities and differences between UL37-deleted pseudorabies virus and herpes simplex virus type 1

Functional Analysis of the Pseudorabies Virus UL51 Protein

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