scholarly journals Pseudorabies Virus UL37 Gene Product Is Involved in Secondary Envelopment

2001 ◽  
Vol 75 (19) ◽  
pp. 8927-8936 ◽  
Author(s):  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Egbert Mundt ◽  
Thomas C. Mettenleiter
Keyword(s):  
Gene Product ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Ordered Structure ◽  
Wild Type ◽  
Tegument Proteins ◽  
Outer Leaflet ◽  
Perinuclear Space ◽  
Viral Glycoproteins ◽  
Capsid Maturation

ABSTRACT Herpesvirus envelopment is a two-step process which includes acquisition of a primary envelope resulting from budding of intranuclear capsids through the inner nuclear membrane. Fusion with the outer leaflet of the nuclear membrane releases nucleocapsids into the cytoplasm, which then gain their final envelope by budding intotrans-Golgi vesicles. It has been shown that the UL34 gene product is required for primary envelopment of the alphaherpesvirus pseudorabies virus (PrV) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063–10073, 2000). For secondary envelopment, several virus-encoded PrV proteins are necessary, including glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364–5372, 1999). We show here that the product of the UL37 gene of PrV, which is a constituent of mature virions, is involved in secondary envelopment. Replication of a UL37 deletion mutant, PrV-ΔUL37, was impaired in normal cells; this defect could be complemented on cells stably expressing UL37. Ultrastructural analysis demonstrated that intranuclear capsid maturation and budding of capsids into and release from the perinuclear space were unimpaired. However, secondary envelopment was drastically reduced. Instead, apparently DNA-filled capsids accumulated in the cytoplasm in large aggregates similar to those observed in the absence of glycoproteins E/I and M but lacking the surrounding electron-dense tegument material. Although displaying an ordered structure, capsids did not contact each other directly. We postulate that the UL37 protein is necessary for correct addition of other tegument proteins, which are required for secondary envelopment. In the absence of the UL37 protein, capsids interact with each other through unknown components but do not acquire the electron-dense tegument which is normally found around wild-type capsids during and after secondary envelopment. Thus, apposition of the UL37 protein to cytoplasmic capsids may be crucial for the addition of other tegument proteins, which in turn are able to interact with viral glycoproteins to mediate secondary envelopment.

scholarly journals Primary Envelopment of Pseudorabies Virus at the Nuclear Membrane Requires the UL34 Gene Product

2000 ◽  
Vol 74 (21) ◽  
pp. 10063-10073 ◽  
Author(s):  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Thomas C. Mettenleiter
Keyword(s):  
Gene Product ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Outer Nuclear Membrane ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Perinuclear Space ◽  
Infected Cells ◽  
Mature Virus

ABSTRACT Primary envelopment of several herpesviruses has been shown to occur by budding of intranuclear capsids through the inner nuclear membrane. By subsequent fusion of the primary envelope with the outer nuclear membrane, capsids are released into the cytoplasm and gain their final envelope by budding into vesicles in thetrans-Golgi area. We show here that the product of the UL34 gene of pseudorabies virus, an alphaherpesvirus of swine, is localized in transfected and infected cells in the nuclear membrane. It is also detected in the envelope of virions in the perinuclear space but is undetectable in intracytoplasmic and extracellular enveloped virus particles. Conversely, the tegument protein UL49 is present in mature virus particles and absent from perinuclear virions. In the absence of the UL34 protein, acquisition of the primary envelope is blocked and neither virus particles in the perinuclear space nor intracytoplasmic capsids or virions are observed. However, light particles which label with the anti-UL49 serum are formed in the cytoplasm. We conclude that the UL34 protein is required for primary envelopment, that the primary envelope is biochemically different from the final envelope in that it contains the UL34 protein, and that perinuclear virions lack the tegument protein UL49, which is present in mature virions. Thus, we provide additional evidence for a two-step envelopment process in herpesviruses.

scholarly journals The Pseudorabies Virus US3 Protein Is a Component of Primary and of Mature Virions

2004 ◽  
Vol 78 (3) ◽  
pp. 1314-1323 ◽  
Author(s):  
Harald Granzow ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Immunoelectron Microscopy ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Gene Products ◽  
Tegument Proteins ◽  
Outer Leaflet ◽  
Nuclear Egress ◽  
Intracytoplasmic Inclusions

ABSTRACT Herpesviruses acquire a primary envelope by budding of capsids at the inner leaflet of the nuclear membrane. They then traverse into the cytoplasm after fusion of the primary envelope with the outer leaflet of the nuclear membrane. In the alphaherpesvirus pseudorabies virus (PrV), the latter process is impaired when the US3 protein is absent. Acquisition of final tegument and envelope occurs in the cytoplasm. Besides the capsid components, only the UL31 and UL34 gene products of PrV have unequivocally been shown to be part of primary enveloped virions, whereas they lack several tegument proteins present in mature virions (reviewed by T. C. Mettenleiter, J. Virol. 76:1537-1547, 2002). Using immunoelectron microscopy, we show that the US3 protein is present in primary enveloped as well as in mature virions. It is also detectable in intracytoplasmic inclusions produced in the absence of other viral tegument components or envelope-associated glycoproteins. In particular, inclusions formed in the absence of the inner tegument protein UL37 contained the US3 protein. Thus, the US3 protein is a tegument component of both forms of enveloped alphaherpes virions. We hypothesize that US3 protein in primary virions modulates deenvelopment at the outer leaflet of the nuclear membrane and is either lost from primary virions during nuclear egress and subsequently reacquired early during tegumentation or is retained during transit of the nucleocapsid through the nuclear membrane.

scholarly journals Effect of the pseudorabies virus US3 protein on nuclear membrane localization of the UL34 protein and virus egress from the nucleus

2001 ◽  
Vol 82 (10) ◽  
pp. 2363-2371 ◽  
Author(s):  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Thomas C. Mettenleiter
Keyword(s):  
Immunoelectron Microscopy ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Intracellular Localization ◽  
Outer Leaflet ◽  
Perinuclear Space ◽  
Virus Morphogenesis ◽  
Simplex Virus ◽  
Virus Egress

The alphaherpesvirus UL34 protein is necessary for the primary envelopment of intranuclear capsids at the inner leaflet of the nuclear membrane. In herpes simplex virus type 1, the UL34 protein is exclusively phosphorylated by the protein kinase encoded by the non-essential US3 gene. To investigate the effect of the pseudorabies virus (PrV) US3 product on the intracellular localization of the UL34 protein and on virus morphogenesis, PrV US3 deletion mutants were isolated and characterized. Immunofluorescence analyses demonstrated that in the absence of the US3 protein, the localization of the UL34 polypeptide to the nuclear membrane was not as pronounced as that seen with US3, although immunoelectron microscopy indicated the presence of the UL34 protein in both leaflets of the nuclear membrane. Ultrastructurally, an accumulation of enveloped virions in the perinuclear space in large invaginations of the inner nuclear membrane was observed, which were shown by immunoelectron microscopy to contain the UL34 protein, but not glycoproteins gB or gC. Thus, the US3 protein appears to be involved in the de-envelopment of perinuclear virions by fusion with the outer leaflet of the nuclear membrane. Surprisingly, no difference in the phosphorylation of the PrV UL34 protein was observed in the presence or absence of the US3 kinase. Therefore, the observed effects of the PrV US3 protein on the intracellular localization of the UL34 protein and on virus morphogenesis are probably not due to the phosphorylation of the UL34 protein by the US3 kinase.

scholarly journals The Interacting UL31 and UL34 Gene Products of Pseudorabies Virus Are Involved in Egress from the Host-Cell Nucleus and Represent Components of Primary Enveloped but Not Mature Virions

2002 ◽  
Vol 76 (1) ◽  
pp. 364-378 ◽  
Author(s):  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Nikolaus Osterrieder ◽  
Thomas C. Mettenleiter
Keyword(s):  
Gene Product ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Physical Interaction ◽  
Wild Type ◽  
Virus Particles ◽  
Inner Nuclear Membrane ◽  
Enveloped Virus ◽  
Extracellular Virus ◽  
Ul31 Gene

ABSTRACT A 2.6-kbp fragment of the pseudorabies virus (PrV) genome was sequenced and shown to contain the homologues of the highly conserved herpesvirus genes UL31 and UL32. By use of a monospecific antiserum, the UL31 gene product was identified as a nuclear protein with an apparent molecular mass of 29 kDa. For functional analysis, UL31 was deleted by mutagenesis in Escherichia coli of an infectious full-length clone of the PrV genome. The resulting virus mutants were deficient in plaque formation, and titers were reduced more than 100-fold from those of wild-type PrV. Ultrastructural analyses demonstrated that capsid maturation and DNA packaging were not affected. However, neither budding at the inner nuclear membrane nor cytoplasmic or extracellular virus particles were observed. These replication defects were similar to those of a UL34 deletion mutant (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063–10073, 2000) and could be completely repaired in a cell line which constitutively expresses the UL31 protein. Yeast two-hybrid studies revealed that a UL31 fusion protein specifically interacts with plasmids of a PrV genome library expressing the N-terminal part of UL34. Vice versa, UL34 selected UL31-encoding plasmids from the library. Immunofluorescence studies and immune electron microscopy demonstrated that in cells infected with wild-type PrV, both proteins accumulate at the nuclear membrane, whereas in the absence of UL34 the UL31 protein is dispersed throughout the nucleus. Like the UL34 protein, the UL31 gene product is a component of enveloped virus particles within the perinuclear space and absent from mature virions. Our findings suggest that physical interaction between these two virus proteins might be a prerequisite for primary envelopment of PrV at the inner nuclear membrane and that this envelope is removed by fusion with the outer nuclear membrane.

scholarly journals Essential Function of the Pseudorabies Virus UL36 Gene Product Is Independent of Its Interaction with the UL37 Protein

2004 ◽  
Vol 78 (21) ◽  
pp. 11879-11889 ◽  
Author(s):  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Thomas C. Mettenleiter
Keyword(s):  
Gene Product ◽  
Pseudorabies Virus ◽  
Rabbit Kidney ◽  
Tegument Protein ◽  
Wild Type ◽  
Coding Region ◽  
Ordered Structures ◽  
Ca 50 ◽  
Essential Function ◽  
Full Length Clone

ABSTRACT The large tegument protein encoded by the UL36 gene of pseudorabies virus (PrV) physically interacts with the product of the adjacent UL37 gene (B. G. Klupp, W. Fuchs, H. Granzow, R. Nixdorf, and T. C. Mettenleiter, J. Virol. 76:3065-3071, 2002). To analyze UL36 function, two PrV recombinants were generated by mutagenesis of an infectious PrV full-length clone in Escherichia coli: PrV-ΔUL36F exhibited a deletion of virtually the complete UL36 coding region, whereas PrV-UL36BSF contained two in-frame deletions of 238 codons spanning the predicted UL37 binding domain. Coimmunoprecipitation experiments confirmed that the mutated gene product of PrV-UL36BSF did not interact with the UL37 protein. Like the previously described PrV-ΔUL37 (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 75:8927-8936, 2001) but in contrast to PrV-ΔUL36F, PrV-UL36BSF was able to replicate in rabbit kidney (RK13) cells, although maximum virus titers were reduced ca. 50-fold and plaque diameters were reduced by ca. 45% compared to wild-type PrV. PrV-ΔUL36F was able to productively replicate after repair of the deleted gene or in a trans-complementing cell line. Electron microscopy of infected RK13 cells revealed that PrV-UL36BSF and phenotypically complemented PrV-ΔUL36F were capable of nucleocapsid formation and egress from the nucleus by primary envelopment and deenvelopment at the nuclear membrane. However, reenvelopment of nucleocapsids in the cytoplasm was blocked. Only virus-like particles without capsids were released efficiently from cells. Interestingly, cytoplasmic nucleocapsids of PrV-UL36BSF but not of PrV-ΔUL36F were found in large ordered structures similar to those which had previously been observed with PrV-ΔUL37. In summary, our results demonstrate that the interaction between the UL36 and UL37 proteins is important but not strictly essential for the formation of secondary enveloped, infectious PrV particles. Furthermore, UL36 possesses an essential function during virus replication which is independent of its ability to bind the UL37 protein.

scholarly journals Herpes Simplex Virus Tegument Protein VP16 Is a Component of Primary Enveloped Virions

2006 ◽  
Vol 80 (5) ◽  
pp. 2582-2584 ◽  
Author(s):  
Raquel Naldinho-Souto ◽  
Helena Browne ◽  
Tony Minson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Immunogold Electron Microscopy ◽  
Virus Particles ◽  
Tegument Proteins ◽  
Perinuclear Space ◽  
Simplex Virus

ABSTRACT 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.

scholarly journals Influence of Tegument Proteins of Pseudorabies Virus on Neuroinvasion and Transneuronal Spread in the Nervous System of Adult Mice after Intranasal Inoculation

2004 ◽  
Vol 78 (6) ◽  
pp. 2956-2966 ◽  
Author(s):  
Robert Klopfleisch ◽  
Jens P. Teifke ◽  
Walter Fuchs ◽  
Martina Kopp ◽  
Barbara G. Klupp ◽  
...  
Keyword(s):  
Cell Culture ◽  
Nervous System ◽  
Pseudorabies Virus ◽  
Single Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Survival Times ◽  
Intranasal Infection ◽  
Tegument Proteins ◽  
Adult Mice

ABSTRACT Pseudorabies virus (PrV) is a neurotropic alphaherpesvirus that, after intranasal infection of adult mice, enters peripheral neurons and propagates to the central nervous system. In recent years we have analyzed the contribution of virus-encoded glycoproteins to neuroinvasion and transneuronal spread (reviewed in T. C. Mettenleiter, Virus Res. 92:197-206, 2003). We now extend our studies to analyze the role of tegument proteins in these processes. To this end, PrV mutants unable to express the UL11, UL37, UL46, UL47, and UL48 tegument proteins, as well as the corresponding rescued viruses, were intranasally instilled into 6- to 8-week-old CD1 strain mice. First, mean survival times were determined which showed that mice infected with the UL46 deletion mutant succumbed to the disease as early as wild-type PrV-infected animals. Survival times increased in the order: PrV-ΔUL47-, PrV-ΔUL11-, and PrV-ΔUL48-infected animals, a finding which parallels the growth phenotype of these viruses in cell culture. In contrast, none of the PrV-ΔUL37-infected animals died. Upon closer histological examination, all viruses except PrV-ΔUL37 were able to infect the nasal cavity and propagate to first- and second-order neurons as shown by two-color immunofluorescence. However, neuroinvasion was delayed in PrV-ΔUL47, PrV-ΔUL11, and PrV-ΔUL48, a finding that correlated with the extended survival times. Surprisingly, whereas PrV-ΔUL48 and PrV-ΔUL37 replicated to similar titers in cell culture which were ∼500-fold lower than those of wild-type virus, after intranasal infection of mice PrV-ΔUL48 was able to infect areas of the brain like wild-type PrV, although only after a considerably longer time period. In contrast, PrV-ΔUL37 was not able to enter neurons and was restricted to the infection of single cells in the nasal respiratory epithelium. Thus, our data demonstrate the importance of herpesviral tegument proteins in neuronal infection and show a different contribution of tegument proteins to the neuroinvasion phenotype of a neurotropic alphaherpesvirus.

scholarly journals Physical Interaction between Envelope Glycoproteins E and M of Pseudorabies Virus and the Major Tegument Protein UL49

2002 ◽  
Vol 76 (16) ◽  
pp. 8208-8217 ◽  
Author(s):  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Christoph Hengartner ◽  
Alexandra Brack ◽  
...  
Keyword(s):  
Gene Product ◽  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Physical Interaction ◽  
Virus Maturation ◽  
Virus Particles ◽  
Tegument Proteins ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT Envelope glycoprotein M (gM) and the complex formed by glycoproteins E (gE) and I (gI) are involved in the secondary envelopment of pseudorabies virus (PrV) particles in the cytoplasm of infected cells. In the absence of the gE-gI complex and gM, envelopment is blocked and capsids surrounded by tegument proteins accumulate in the cytoplasm (A. R. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Here we demonstrate by yeast two-hybrid analyses that the cytoplasmic domains of gE and gM specifically interact with the C-terminal part of the UL49 gene product of PrV, which represents a major tegument protein and which is homologous to VP22 of herpes simplex virus type 1. However, deletion of the UL49 gene from PrV had only minor effects on viral replication, and ultrastructural analyses of infected cells confirmed that virus maturation and egress, including secondary envelopment in the cytoplasm, were not detectably affected by the absence of UL49. Moreover, the UL49 gene product was shown to be dispensable for virion localization of gE and gM, and mutants lacking either gE or gM incorporated the UL49 protein efficiently into virus particles. In contrast, a PrV mutant with deletions of gE-gI and gM failed to incorporate the UL49 protein despite apparently unaltered intracytoplasmic UL49 expression. In summary, we describe specific interactions between herpesvirus envelope and tegument proteins which may play a role in secondary envelopment during herpesvirus virion maturation.

scholarly journals Efficient Incorporation of Tegument Proteins pUL46, pUL49, and pUS3 into Pseudorabies Virus Particles Depends on the Presence of pUL21

2006 ◽  
Vol 81 (2) ◽  
pp. 1048-1051 ◽  
Author(s):  
Kathrin Michael ◽  
Barbara G. Klupp ◽  
Axel Karger ◽  
Thomas C. Mettenleiter
Keyword(s):  
Virus Particle ◽  
Protein Interactions ◽  
Pseudorabies Virus ◽  
Structural Proteins ◽  
Tegument Protein ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Virus Particles ◽  
Tegument Proteins ◽  
Drastic Decrease

ABSTRACT The mature virion of the alphaherpesvirus pseudorabies virus (PrV) contains a minimum of 31 structural proteins which are recruited into the virus particle by a network of protein-protein interactions which is only incompletely understood. We show here that deletion of the tegument protein pUL21 resulted in a drastic decrease in the incorporation of the pUL46, pUL49, and pUS3 tegument components into mature virions. Moreover, the attenuated PrV strain Bartha (PrV-Ba), which, among other defects, carries mutations in pUL21, also fails to package pUL46, pUL49, and pUS3 efficiently. By the reconstitution of wild-type pUL21 expression to PrV-Ba and the transfer of mutated PrV-Ba pUL21 into wild-type PrV, we demonstrate that this phenotype is due to the mutated pUL21.

scholarly journals Function of Torsin AAA+ ATPases in Pseudorabies Virus Nuclear Egress

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 738
Author(s):  
Julia E. Hölper ◽  
Barbara G. Klupp ◽  
G. W. Gant Luxton ◽  
Kati Franzke ◽  
Thomas C. Mettenleiter
Keyword(s):  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Gene Knockout ◽  
Early Time ◽  
Wild Type ◽  
Mutant Proteins ◽  
Nuclear Egress ◽  
Cytoplasmic Transport ◽  
Virion Envelope ◽  
Aaa Atpases

Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.

Sign in / Sign up

Export Citation Format

Share Document