Incorporation of green fluorescent protein into the essential envelope glycoprotein B of herpes simplex virus type 1

ABSTRACT Hammerhead ribozymes were designed to target mRNA of several essential herpes simplex virus type 1 (HSV-1) genes. A ribozyme specific for the late gene UL20 was packaged in an adenovirus vector (Ad-UL20 Rz) and evaluated for its capacity to inhibit the viral replication of several HSV-1 strains, including that of the wild-type HSV-1 (17syn+ and KOS) and several acycloguanosine-resistant strains (PAAr5, tkLTRZ1, and ACGr4) in tissue culture. The Ad-UL20 Rz was also tested for its ability to block an HSV-1 infection, using the mouse footpad model. Mouse footpads were treated with either the Ad-UL20 Rz or an adenoviral vector expressing green fluorescent protein (Ad-GFP) and then infected immediately thereafter with 104 PFU of HSV-1 strain 17syn+. Ad-UL20 ribozyme treatment consistently led to a 90% rate of protection for mice from lethal HSV-1 infection, while the survival rate in the control groups was less than 45%. Consistent with this protective effect, treatment with the Ad-UL20 Rz reduced the viral DNA load in the feet, the dorsal root ganglia, and the spinal cord relative to that of the Ad-GFP-treated animals. This study suggests that ribozymes targeting essential genes of the late kinetic class may represent a new therapeutic strategy for inhibiting HSV infection.

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Live Visualization of Herpes Simplex Virus Type 1 Compartment Dynamics

Journal of Virology ◽

10.1128/jvi.02431-07 ◽

2008 ◽

Vol 82 (10) ◽

pp. 4974-4990 ◽

Cited By ~ 36

Author(s):

Anna Paula de Oliveira ◽

Daniel L. Glauser ◽

Andrea S. Laimbacher ◽

Regina Strasser ◽

Elisabeth M. Schraner ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Fusion Protein ◽

Viral Replication ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Fluorescent Protein ◽

Simplex Virus ◽

Hsv 1

ABSTRACT We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments—capsid, tegument, and envelope—fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.

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The Latency-Associated Transcript Gene Enhances Establishment of Herpes Simplex Virus Type 1 Latency in Rabbits

Journal of Virology ◽

10.1128/jvi.74.4.1885-1891.2000 ◽

2000 ◽

Vol 74 (4) ◽

pp. 1885-1891 ◽

Cited By ~ 79

Author(s):

Guey-Chuen Perng ◽

Susan M. Slanina ◽

Ada Yukht ◽

Homayon Ghiasi ◽

Anthony B. Nesburn ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Fluorescent Protein ◽

Ocular Infection ◽

Latently Infected ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The latency-associated transcript (LAT) gene the only herpes simplex virus type 1 (HSV-1) gene abundantly transcribed during neuronal latency, is essential for efficient in vivo reactivation. Whether LAT increases reactivation by a direct effect on the reactivation process or whether it does so by increasing the establishment of latency, thereby making more latently infected neurons available for reactivation, is unclear. In mice, LAT-negative mutants appear to establish latency in fewer neurons than does wild-type HSV-1. However, this has not been confirmed in the rabbit, and the role of LAT in the establishment of latency remains controversial. To pursue this question, we inserted the gene for the enhanced green fluorescent protein (EGFP) under control of the LAT promoter in a LAT-negative virus (ΔLAT-EGFP) and in a LAT-positive virus (LAT-EGFP). Sixty days after ocular infection, trigeminal ganglia (TG) were removed from the latently infected rabbits, sectioned, and examined by fluorescence microscopy. EGFP was detected in significantly more LAT-EGFP-infected neurons than ΔLAT-EGFP-infected neurons (4.9% versus 2%, P < 0.0001). The percentages of EGFP-positive neurons per TG ranged from 0 to 4.6 for ΔLAT-EGFP and from 2.5 to 11.1 for LAT-EGFP (P = 0.003). Thus, LAT appeared to increase neuronal latency in rabbit TG by an average of two- to threefold. These results suggest that LAT enhances the establishment of latency in rabbits and that this may be one of the mechanisms by which LAT enhances spontaneous reactivation. These results do not rule out additional LAT functions that may be involved in maintenance of latency and/or reactivation from latency.

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A Null Mutation in the UL36 Gene of Herpes Simplex Virus Type 1 Results in Accumulation of Unenveloped DNA-Filled Capsids in the Cytoplasm of Infected Cells

Journal of Virology ◽

10.1128/jvi.74.24.11608-11618.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11608-11618 ◽

Cited By ~ 161

Author(s):

Prashant J. Desai

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Cell Lines ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Fluorescent Protein ◽

Null Mutation ◽

Infected Cells ◽

Simplex Virus

ABSTRACT The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated KΔUL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with KΔUL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of KΔUL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In KΔUL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in KΔUL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.

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The role of a single N-linked glycosylation site for a functional epitope of herpes simplex virus type 1 envelope glycoprotein gC

Glycobiology ◽

10.1093/glycob/9.1.73 ◽

1999 ◽

Vol 9 (1) ◽

pp. 73-81 ◽

Cited By ~ 11

Author(s):

S. Olofsson ◽

A. Bolmstedt ◽

M. Biller ◽

K. Mardberg ◽

J. Leckner ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Envelope Glycoprotein ◽

Glycosylation Site ◽

Simplex Virus

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Herpes Simplex Virus Type 1 UL34 Gene Product Is Required for Viral Envelopment

Journal of Virology ◽

10.1128/jvi.74.1.117-129.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 117-129 ◽

Cited By ~ 153

Author(s):

Richard J. Roller ◽

Yuping Zhou ◽

Renee Schnetzer ◽

John Ferguson ◽

Diana DeSalvo

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Gene Product ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Recombinant Virus ◽

Fluorescent Protein ◽

Electron Microscopic ◽

Simplex Virus

ABSTRACT The herpes simplex virus type 1 UL34 gene encodes a protein that is conserved in all human herpesviruses. The association of the UL34 protein with membranes in the infected cell and its expression as a gamma-1 gene suggest a role in maturation or egress of the virus particle from the cell. To determine the function of this gene product, we have constructed a recombinant virus that fails to express the UL34 protein. This recombinant virus, in which the UL34 protein coding sequence has been replaced by green fluorescent protein, forms minute plaques and replicates in single-step growth experiments to titers 3 to 5 log orders of magnitude lower than wild-type or repair viruses. On Vero cells, the deletion virus synthesizes proteins of all kinetic classes in normal amounts. Electron microscopic and biochemical analyses show that morphogenesis of the deletion virus proceeds normally to the point of formation of DNA-containing nuclear capsids, but electron micrographs show no enveloped virus particles in the cytoplasm or at the surface of infected cells, suggesting that the UL34 protein is essential for efficient envelopment of capsids.

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Structural analysis of precursor and product forms of type-common envelope glycoprotein D (CP-1 antigen) of herpes simplex virus type 1.

Journal of Virology ◽

10.1128/jvi.31.3.608-620.1979 ◽

1979 ◽

Vol 31 (3) ◽

pp. 608-620 ◽

Cited By ~ 21

Author(s):

R J Eisenberg ◽

C Hydrean-Stern ◽

G H Cohen

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Structural Analysis ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Envelope Glycoprotein ◽

Common Envelope ◽

Simplex Virus ◽

Product Forms

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Localization of Herpes Simplex Virus Type 1 UL37 in the Golgi Complex Requires UL36 but Not Capsid Structures

Journal of Virology ◽

10.1128/jvi.00956-08 ◽

2008 ◽

Vol 82 (22) ◽

pp. 11354-11361 ◽

Cited By ~ 46

Author(s):

Prashant Desai ◽

Gerry L. Sexton ◽

Eugene Huang ◽

Stanley Person

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Golgi Complex ◽

Herpes Simplex Virus Type ◽

Fluorescent Protein ◽

Vero Cells ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) UL37 gene encodes a 120-kDa polypeptide which resides in the tegument structure of the virion and is important for morphogenesis. The goal of this study was to use green fluorescent protein (GFP) to follow the fate of UL37 within cells during the normal course of virus replication. GFP was inserted in frame at the C terminus of UL37 to generate a fluorescent-protein-tagged UL37 polypeptide. A virus designated K37eGFP, which replicated normally on Vero cells, was isolated and was shown to express the fusion polypeptide. When cells infected with this virus were examined by confocal microscopy, the fluorescence was observed to be predominantly cytoplasmic. As the infection progressed, fluorescence began to accumulate in a juxtanuclear structure. Mannosidase II and giantin were observed to colocalize with UL37eGFP at these structures, as judged by immunofluorescence assays. Therefore, UL37 traffics to the Golgi complex during infection. A VP26mRFP marker (red fluorescent protein fused to VP26) was recombined into K37eGFP, and when cells infected with this “dual-color” virus were examined, colocalization of the red (capsid) and green (UL37) fluorescence in the Golgi structure was observed. Null mutations in VP5 (ΔVP5), which abolished capsid assembly, and in UL36 (Δ36) were recombined into the K37eGFP virus genome. In cells infected with K37eGFP/ΔVP5, localization of UL37eGFP to the Golgi complex was similar to that for the parental virus (K37eGFP), indicating that trafficking of UL37eGFP to the Golgi complex did not require capsid structures. Confocal analysis of cells infected with K37eGFP/Δ36 showed that, in the absence of UL36, accumulation of UL37eGFP at the Golgi complex was not evident. This indicates an interaction between these two proteins that is important for localization of UL37 in the Golgi complex and thus possibly for cytoplasmic envelopment of the capsid. This is the first demonstration of a functional role for UL36:UL37 interaction in HSV-1-infected cells.

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