The Herpesvirus Capsid Surface Protein, VP26, and the Majority of the Tegument Proteins Are Dispensable for Capsid Transport toward the Nucleus

ABSTRACT Upon entering a cell, alphaherpesvirus capsids are transported toward the minus ends of microtubules and ultimately deposit virus DNA within the host nucleus. The virus proteins that mediate this centripetal transport are unknown but are expected to be either viral tegument proteins, which are a group of capsid-associated proteins, or a surface component of the capsid itself. Starting with derivatives of pseudorabies virus that encode a fluorescent protein fused to a structural component of the virus, we have made a collection of 12 mutant viruses that lack either the VP26 capsid protein or an individual tegument protein. Using live-cell fluorescence microscopy, we tracked individual virus particles in axons following infection of primary sensory neurons. Quantitative analysis of the VP26-null virus indicates that this protein plays no observable role in capsid transport. Furthermore, viruses lacking tegument proteins that are nonessential for virus propagation in cell culture were also competent for axonal transport. These results indicate that a protein essential for viral propagation mediates transport of the capsid to the nucleus.

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The Capsid and Tegument of the Alphaherpesviruses Are Linked by an Interaction between the UL25 and VP1/2 Proteins

Journal of Virology ◽

10.1128/jvi.01113-07 ◽

2007 ◽

Vol 81 (21) ◽

pp. 11790-11797 ◽

Cited By ~ 105

Author(s):

Kelly Elizabeth Coller ◽

Joy I-Hsuan Lee ◽

Aki Ueda ◽

Gregory Allan Smith

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Protein Product ◽

Tegument Protein ◽

Viral Propagation ◽

Tegument Proteins ◽

Green Fluorescent ◽

Simplex Virus ◽

Carboxy Terminal ◽

Hsv 1

ABSTRACT How alphaherpesvirus capsids acquire tegument proteins remains a key question in viral assembly. Using pseudorabies virus (PRV), we have previously shown that the 62 carboxy-terminal amino acids of the VP1/2 large tegument protein are essential for viral propagation and when transiently expressed as a fusion to green fluorescent protein relocalize to nuclear capsid assemblons following viral infection. Here, we show that localization of the VP1/2 capsid-binding domain (VP1/2cbd) into assemblons is conserved in herpes simplex virus type 1 (HSV-1) and that this recruitment is specifically on capsids. Using a mutant virus screen, we find that the protein product of the UL25 gene is essential for VP1/2cbd association with capsids. An interaction between UL25 and VP1/2 was corroborated by coimmunoprecipitation from cells transiently expressing either HSV-1 or PRV proteins. Taken together, these findings suggest that the essential function of the VP1/2 carboxy terminus is to anchor the VP1/2 tegument protein to capsids. Furthermore, UL25 encodes a multifunctional capsid protein involved in not only encapsidation, as previously described, but also tegumentation.

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Identification of an Essential Domain in the Herpesvirus VP1/2 Tegument Protein: the Carboxy Terminus Directs Incorporation into Capsid Assemblons

Journal of Virology ◽

10.1128/jvi.01184-06 ◽

2006 ◽

Vol 80 (24) ◽

pp. 12086-12094 ◽

Cited By ~ 46

Author(s):

Joy I-Hsuan Lee ◽

G. W. Gant Luxton ◽

Gregory Allan Smith

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Infectious Clone ◽

Tegument Protein ◽

Epithelial Cell Line ◽

Carboxy Terminus ◽

Virus Propagation ◽

Conserved Sequence ◽

Amino Terminal ◽

Essential Domain

ABSTRACT The herpesvirus tegument is a layer of viral and cellular proteins located between the capsid and envelope of the virion. The VP1/2 tegument protein is critical for the propagation of all herpesviruses examined. Using an infectious clone of the alphaherpesvirus pseudorabies virus, we have made a collection of truncation and in-frame deletion mutations within the VP1/2 gene (UL36) and examined the resulting viruses for spread between cells. We found that the majority of the VP1/2 protein either was essential for virus propagation or did not tolerate large deletions. A recently described amino-terminal deubiquitinase-encoding domain was dispensable for alphaherpesvirus propagation, but the rate of propagation in an epithelial cell line and the frequency of transport in axons of primary sensory neurons were both reduced. We mapped one essential domain to a conserved sequence at the VP1/2 carboxy terminus and demonstrated that this domain sufficient to redirect the green fluorescent protein to capsid assemblons in nuclei of infected cells.

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A Cell Surface Protein with Herpesvirus Entry Activity (HveB) Confers Susceptibility to Infection by Mutants of Herpes Simplex Virus Type 1, Herpes Simplex Virus Type 2, and Pseudorabies Virus

Virology ◽

10.1006/viro.1998.9218 ◽

1998 ◽

Vol 246 (1) ◽

pp. 179-189 ◽

Cited By ~ 347

Author(s):

Morgyn S. Warner ◽

Robert J. Geraghty ◽

Wanda M. Martinez ◽

Rebecca I. Montgomery ◽

J.Charles Whitbeck ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Pseudorabies Virus ◽

Surface Protein ◽

Susceptibility To Infection ◽

A Cell ◽

Simplex Virus

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Translocation of Incoming Pseudorabies Virus Capsids to the Cell Nucleus Is Delayed in the Absence of Tegument Protein pUL37

Journal of Virology ◽

10.1128/jvi.02090-08 ◽

2009 ◽

Vol 83 (7) ◽

pp. 3389-3396 ◽

Cited By ~ 34

Author(s):

Mirjam Krautwald ◽

Walter Fuchs ◽

Barbara G. Klupp ◽

Thomas C. Mettenleiter

Keyword(s):

Laser Scanning ◽

Pseudorabies Virus ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Laser Scanning Microscopy ◽

Rabbit Kidney ◽

Confocal Laser ◽

Tegument Proteins ◽

Green Fluorescent ◽

Virus Mutant

ABSTRACT After fusion of the envelope of herpesvirus particles with the host cell plasma membrane, incoming nucleocapsids are transported to nuclear pores. Inner tegument proteins pUL36, pUL37, and pUS3 remain attached to the nucleocapsid after entry and therefore might mediate interactions between the nucleocapsid and cellular microtubule-associated motor proteins during transport. To assay for the role of pUL37 in this process, we constructed a pUL37-deleted pseudorabies virus mutant, PrV-ΔUL37/UL35GFP, which expresses a fusion protein of green fluorescent protein (GFP) and the nonessential small capsid protein pUL35, resulting in the formation of fluorescently labeled capsids. Confocal laser-scanning microscopy of rabbit kidney cells infected with PrV-ΔUL37/UL35GFP revealed that, whereas penetration was not affected in the absence of pUL37, nuclear translocation of incoming particles was delayed by approximately 1 h compared to PrV-UL35GFP, but not abolished. In contrast, phenotypically complemented pUL37-containing virions of PrV-ΔUL37/UL35GFP exhibited wild type-like entry kinetics. Thus, the presence of pUL37 is required for rapid nuclear translocation of incoming nucleocapsids.

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Functional Carboxy-Terminal Fluorescent Protein Fusion to Pseudorabies Virus Small Capsid Protein VP26

Journal of Virology ◽

10.1128/jvi.01193-17 ◽

2017 ◽

Vol 92 (1) ◽

Cited By ~ 1

Author(s):

Ian B. Hogue ◽

Jolie Jean ◽

Andrew D. Esteves ◽

Nikhila S. Tanneti ◽

Julian Scherer ◽

...

Keyword(s):

Nervous System ◽

Capsid Protein ◽

Particle Tracking ◽

Intracellular Transport ◽

Pseudorabies Virus ◽

Fluorescent Protein ◽

Living Cells ◽

Amino Terminus ◽

Virus Particles ◽

Carboxy Terminal

ABSTRACTFluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely used method to visualize pseudorabies virus (PRV) and herpes simplex virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy and have modest effects on virus replication and pathogenesis. Recent cryoelectron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and therefore may better tolerate fluorescent protein fusions. Here, we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells and incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single-particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport.IMPORTANCEAlphaherpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular transport, as a brighter particle will improve localization accuracy of individual particles and allow for shorter exposure times, reducing phototoxicity and improving the time resolution of particle tracking in live cells.

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

Journal of Virology ◽

10.1128/jvi.80.5.2582-2584.2006 ◽

2006 ◽

Vol 80 (5) ◽

pp. 2582-2584 ◽

Cited By ~ 51

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.

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

Journal of Virology ◽

10.1128/jvi.76.16.8208-8217.2002 ◽

2002 ◽

Vol 76 (16) ◽

pp. 8208-8217 ◽

Cited By ~ 94

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.

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Fusion of Enhanced Green Fluorescent Protein to the Pseudorabies Virus Axonal Sorting Protein Us9 Blocks Anterograde Spread of Infection in Mammalian Neurons

Journal of Virology ◽

10.1128/jvi.01204-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 10308-10311 ◽

Cited By ~ 5

Author(s):

M. G. Lyman ◽

D. Curanovic ◽

A. D. Brideau ◽

L. W. Enquist

Keyword(s):

Green Fluorescent Protein ◽

Pseudorabies Virus ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Neuronal Cultures ◽

Primary Neuronal Cultures ◽

Virus Particles ◽

Green Fluorescent ◽

Spread Of Infection ◽

Axonal Sorting

ABSTRACT Pseudorabies virus encodes a membrane protein (Us9) that is essential for the axonal sorting of virus particles within neurons and anterograde spread in the mammalian nervous system. Enhanced green fluorescent protein (GFP)-tagged Us9 mimicked the trafficking properties of the wild-type protein in nonneuronal cells. We constructed a pseudorabies virus strain that expressed Us9-GFP and tested its spread capabilities in the rat visual system and in primary neuronal cultures. We report that Us9-EGFP does not promote anterograde spread of infection and may disrupt packing of viral membrane proteins in lipid rafts, an essential step for Us9-mediated axonal sorting.

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

Journal of Virology ◽

10.1128/jvi.01801-06 ◽

2006 ◽

Vol 81 (2) ◽

pp. 1048-1051 ◽

Cited By ~ 22

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.

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