scholarly journals Composition of Pseudorabies Virus Particles Lacking Tegument Protein US3, UL47, or UL49 or Envelope Glycoprotein E

2006 ◽  
Vol 80 (3) ◽  
pp. 1332-1339 ◽  
Author(s):  
Kathrin Michael ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter ◽  
Axel Karger
Keyword(s):  
Protein Interactions ◽  
Pseudorabies Virus ◽  
Cellular Protein ◽  
Tegument Protein ◽  
Virion Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Glycoprotein E ◽  
Tegument Proteins ◽  
Glycoprotein H ◽  
The Impact

ABSTRACT Proteins located in the tegument layer of herpesvirus particles play important roles in the replicative cycle at both early and late times after infection. As major constituents of the virion, they execute important functions in particular during formation of progeny virions. These functions have mostly been elucidated by construction and analysis of mutant viruses deleted in single or multiple tegument protein-encoding genes (reviewed in the work of T. C. Mettenleiter, Virus Res. 106:167-180, 2004). However, since tegument proteins have been shown to be involved in numerous protein-protein interactions, the impact of single protein deletions on the composition of the virus particle is unknown, but they could impair correct interpretation of the results. To analyze how the absence of single virion constituents influences virion composition, we established a procedure to assay relative amounts of virion structural proteins in deletion mutants of the alphaherpesvirus Pseudorabies virus (PrV) in comparison to wild-type particles. The assay is based on the mass spectrometric quantitation of virion protein-derived peptides carrying stable isotope mass tags. After deletion of the US3, UL47, UL49, or glycoprotein E gene, relative amounts of a capsid protein (UL38), a capsid-associated protein (UL25), several tegument proteins (UL36 and UL47, if present), and glycoprotein H were unaffected, whereas the content of other tegument proteins (UL46, UL48, and UL49, if present) varied significantly. In the case of the UL48 gene product, a specific increase in incorporation of a smaller isoform was observed after deletion of the UL47 or UL49 gene, whereas a larger isoform remained unaffected. The cellular protein actin was enriched in virions of mutants deficient in any of the tegument proteins UL47, UL49, or US3. By two-dimensional gel electrophoresis multiple isoforms of host cell-derived heat shock protein 70 and annexins A1 and A2 were also identified as structural components of PrV virions.

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 CRISPR/Cas9-constructed pseudorabies virus mutants reveal the importance of UL13 in alphaherpesvirus escape from genome silencing

2020 ◽  
Author(s):  
Jolien Van Cleemput ◽  
Orkide O. Koyuncu ◽  
Kathlyn Laval ◽  
Esteban A. Engel ◽  
Lynn W. Enquist
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Pseudorabies Virus ◽  
Neuronal Cell ◽  
Gene Replacement ◽  
Productive Infection ◽  
Tegument Protein ◽  
Neuronal Cultures ◽  
Tegument Proteins

Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally-delivered genomes is facilitated by PRV tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP) and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N-terminus (PRV mTurq-VP16) or C-terminus (PRV VP16-mTurq). Live cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. Surprisingly, immunofluorescence staining of particles in mid-axons indicated that UL13 might be co-transported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing when compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as AAV-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins. Importance Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude on the indirect role of UL13 in PRV escape from latency in primary neurons and on its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing.

scholarly journals Identification of a 709-Amino-Acid Internal Nonessential Region within the Essential Conserved Tegument Protein (p)UL36 of Pseudorabies Virus

2006 ◽  
Vol 80 (19) ◽  
pp. 9910-9915 ◽  
Author(s):  
Sindy Böttcher ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Walter Fuchs ◽  
Kathrin Michael ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Deletion Mutants ◽  
Limited Information ◽  
Internal Deletion ◽  
Rich Region ◽  
Tegument Proteins ◽  
C Terminus ◽  
Simplex Virus

ABSTRACT Tegument proteins homologous to the essential herpes simplex virus type 1 UL36 gene product (p)UL36 are conserved throughout the Herpesviridae and constitute the largest herpesvirus-encoded proteins. So far, only limited information is available on their functions, which include complex formation with the (p)UL37 homologs via an N-terminal domain and a deubiquitinating activity in the extreme N terminus. For further analysis we constructed deletion mutants lacking 437, 784, 926, 1,046, 1,217, or 1,557 amino acids (aa) from the C terminus. While none of them supported replication of a pseudorabies virus (PrV) UL36 deletion mutant, a mutant polypeptide with an internal deletion from aa 2087 to 2795, which comprises a proline/alanine-rich region, fully complemented the lethal replication defect. Thus, our data indicate that the extreme C terminus of (p)UL36 fulfills an essential role in PrV replication, while a large internal portion of the C-terminal half of the protein is dispensable for replication in cell culture.

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 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 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 The UL48 Tegument Protein of Pseudorabies Virus Is Critical for Intracytoplasmic Assembly of Infectious Virions

2002 ◽  
Vol 76 (13) ◽  
pp. 6729-6742 ◽  
Author(s):  
Walter Fuchs ◽  
Harald Granzow ◽  
Barbara G. Klupp ◽  
Martina Kopp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Pseudorabies Virus ◽  
Early Gene ◽  
Tegument Protein ◽  
Western Blots ◽  
Tegument Proteins ◽  
Extracellular Virus ◽  
Virion Morphogenesis ◽  
Infected Cells ◽  
Virion Formation ◽  
Hsv 1

ABSTRACT The pseudorabies virus (PrV) homolog of the tegument protein encoded by the UL48 gene of herpes simplex virus type 1 (HSV-1) was identified by using a monospecific rabbit antiserum against a bacterial fusion protein. UL48-related polypeptides of 53, 55, and 57 kDa were detected in Western blots of infected cells and purified virions. Immunofluorescence studies demonstrated that the PrV UL48 protein is predominantly localized in the cytoplasm but is also found in the nuclei of infected cells. Moreover, it is a constituent of extracellular virus particles but is absent from primary enveloped perinuclear virions. In noncomplementing cells, a UL48-negative PrV mutant (PrV-ΔUL48) exhibited delayed growth and significantly reduced plaque sizes and virus titers, deficiencies which were corrected in UL48-expressing cells. RNA analyses indicated that, like its HSV-1 homolog, the PrV UL48 protein is involved in regulation of immediate-early gene expression. However, the most salient effect of the UL48 gene deletion was a severe defect in virion morphogenesis. Late after infection, electron microscopy of cells infected with PrV-ΔUL48 revealed retention of newly formed nucleocapsids in the cytoplasm, whereas enveloped intracytoplasmic or extracellular complete virions were only rarely observed. In contrast, capsidless particles were produced and released in great amounts. Remarkably, the intracytoplasmic capsids were labeled with antibodies against the UL36 and UL37 tegument proteins, whereas the capsidless particles were labeled with antisera directed against the UL46, UL47, and UL49 tegument proteins. These findings suggested that the UL48 protein is involved in linking capsid and future envelope-associated tegument proteins during virion formation. Thus, like its HSV-1 homolog, the UL48 protein of PrV functions in at least two different steps of the viral life cycle. The drastic inhibition of virion formation in the absence of the PrV UL48 protein indicates that it plays an important role in virion morphogenesis prior to secondary envelopment of intracytoplasmic nucleocapsids. However, the UL48 gene of PrV is not absolutely essential, and concomitant deletion of the adjacent tegument protein gene UL49 also did not abolish virus replication in cell culture.

scholarly journals Dissecting the herpesvirus architecture by targeted proteolysis

2018 ◽  
Author(s):  
Gina R. Daniel ◽  
Caitlin E. Pegg ◽  
Gregory A. Smith
Keyword(s):  
Protein Interactions ◽  
Pseudorabies Virus ◽  
Protein A ◽  
Dynamic Network ◽  
Structural Element ◽  
Particle Assembly ◽  
Tegument Proteins ◽  
Tev Protease ◽  
Economic Significance ◽  
Fluorescent Tags

ABSTRACTHerpesvirus particles have a complex architecture consisting of an icosahedral capsid that is surrounded by a lipid envelope. Connecting these two components is a layer of tegument that consists of varying amounts of twenty or more proteins. The arrangement of proteins within the tegument cannot easily be assessed and instead is inferred from tegument interactions identified in reductionist models. To better understand the tegument architecture, we have developed an approach to probe capsid-tegument interactions within extracellular viral particles by encoding tobacco etch virus (TEV) protease sites in viral structural proteins, along with distinct fluorescent tags in capsid and tegument components. In this study, TEV sites were engineered within the pUL36 large tegument protein: a critical structural element that is anchored directly on the capsid surface. Purified pseudorabies virus extracellular particles were permeabilized and TEV protease was added to selectively cleave the exposed pUL36 backbone. Interactions with the capsid were assessed in situ by monitoring the fate of the fluorescent signals following cleavage. Although several regions of pUL36 are proposed to bind capsids, pUL36 was found stably anchored to the capsid exclusively at its carboxyl terminus. Two additional tegument proteins, pUL37 and pUS3, were tethered to the capsid via pUL36 whereas the pUL16, pUL47, pUL48, and pUL49 tegument proteins were not stably bound to the capsid.IMPORTANCENeuroinvasive alphaherpesviruses produce diseases of clinical and economic significance in humans and veterinary animals, but are predominantly associated with less serious recurrent disease. Like all viruses, herpesviruses assemble a metastable particle that selectively dismantles during initial infection. This process is made more complex by the presence of a tegument layer that resides between the capsid surface and envelope. Components of the tegument are essential for particle assembly and also serve as critical effectors that promote infection upon entry into cells. How this dynamic network of protein interactions is arranged within virions is largely unknown. We present a molecular approach to dissect the tegument and with it, begin to tease apart the protein interactions that underlie this complex layer of the virion architecture.

scholarly journals Dissecting the Herpesvirus Architecture by Targeted Proteolysis

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Gina R. Daniel ◽  
Caitlin E. Pegg ◽  
Gregory A. Smith
Keyword(s):  
Protein Interactions ◽  
Pseudorabies Virus ◽  
Protein A ◽  
Dynamic Network ◽  
Structural Element ◽  
Particle Assembly ◽  
Tegument Proteins ◽  
Tev Protease ◽  
Economic Significance ◽  
Fluorescent Tags

ABSTRACTHerpesvirus particles have a complex architecture consisting of an icosahedral capsid that is surrounded by a lipid envelope. Connecting these two components is a layer of tegument that consists of various amounts of 20 or more proteins. The arrangement of proteins within the tegument cannot easily be assessed and instead is inferred from tegument interactions identified in reductionist models. To better understand the tegument architecture, we have developed an approach to probe capsid-tegument interactions of extracellular viral particles by encoding tobacco etch virus (TEV) protease sites in viral structural proteins, along with distinct fluorescent tags in capsid and tegument components. In this study, TEV sites were engineered within the pUL36 large tegument protein, a critical structural element that is anchored directly on the capsid surface. Purified pseudorabies virus extracellular particles were permeabilized, and TEV protease was added to selectively cleave the exposed pUL36 backbone. Interactions with the capsid were assessedin situby monitoring the fate of the fluorescent signals following cleavage. Although several regions of pUL36 are proposed to bind capsids, pUL36 was found stably anchored to the capsid exclusively at its carboxyl terminus. Two additional tegument proteins, pUL37 and pUS3, were tethered to the capsid via pUL36, whereas the pUL16, pUL47, pUL48, and pUL49 tegument proteins were not stably bound to the capsid.IMPORTANCENeuroinvasive alphaherpesviruses produce diseases of clinical and economic significance in humans and veterinary animals but are predominantly associated with less serious recurrent disease. Like all viruses, herpesviruses assemble a metastable particle that selectively dismantles during initial infection. This process is made more complex by the presence of a tegument layer that resides between the capsid surface and envelope. Components of the tegument are essential for particle assembly and also serve as critical effectors that promote infection upon entry into cells. How this dynamic network of protein interactions is arranged within virions is largely unknown. We present a molecular approach to dissect the tegument, and with it we begin to tease apart the protein interactions that underlie this complex layer of the virion architecture.

scholarly journals Crystal Structure of the N-Terminal Half of the Traffic Controller UL37 from Herpes Simplex Virus 1

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Andrea L. Koenigsberg ◽  
Ekaterina E. Heldwein
Keyword(s):  
Crystal Structure ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Pseudorabies Virus ◽  
Surface Region ◽  
Tegument Protein ◽  
Herpes Simplex Virus 1 ◽  
Tegument Proteins ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Inner tegument protein UL37 is conserved among all three subfamilies of herpesviruses. Studies of UL37 homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), have suggested that UL37 plays an essential albeit poorly defined role in intracellular capsid trafficking. At the same time, HSV and PRV homologs cannot be swapped, which suggests that in addition to a conserved function, UL37 homologs also have divergent virus-specific functions. Accurate dissection of UL37 functions requires detailed maps in the form of atomic-resolution structures. Previously, we reported the crystal structure of the N-terminal half of UL37 (UL37N) from PRV. Here, we report the crystal structure of HSV-1 UL37N. Comparison of the two structures reveals that UL37 homologs differ in their overall shapes, distributions of surface charges, and locations of projecting loops. In contrast, the previously identified R2 surface region is structurally conserved. We propose that within the N-terminal half of UL37, functional conservation is centered within the R2 surface region, whereas divergent structural elements pinpoint regions mediating virus-specific functions and may engage different binding partners. Together, the two structures can now serve as templates for a structure-guided exploration of both conserved and virus-specific functions of UL37. IMPORTANCE The ability to move efficiently within host cell cytoplasm is essential for replication in all viruses. It is especially important in the neuroinvasive alphaherpesviruses, such as human herpes simplex virus 1 (HSV-1), HSV-2, and veterinarian pseudorabies virus (PRV), that infect the peripheral nervous system and have to travel long distances along axons. Capsid movement in these viruses is controlled by capsid-associated tegument proteins, yet their specific roles have not yet been defined. Systematic exploration of the roles of tegument proteins in capsid trafficking requires detailed navigational charts in the form of their three-dimensional structures. Here, we determined the crystal structure of the N-terminal half of a conserved tegument protein, UL37, from HSV-1. This structure, along with our previously reported structure of the UL37 homolog from PRV, provides a much needed 3-dimensional template for the dissection of both conserved and virus-specific functions of UL37 in intracellular capsid trafficking.

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