Correlation between precolonization of trigeminal ganglia by attenuated strains of pseudorabies virus and resistance to wild-type virus latency.

ABSTRACT Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.

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Role of Pseudorabies Virus Us3 Protein Kinase during Neuronal Infection

Journal of Virology ◽

10.1128/jvi.00352-06 ◽

2006 ◽

Vol 80 (13) ◽

pp. 6387-6398 ◽

Cited By ~ 27

Author(s):

L. M. Olsen ◽

T. H. Ch'ng ◽

J. P. Card ◽

L. W. Enquist

Keyword(s):

Protein Kinase ◽

Pseudorabies Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Spread Of Infection ◽

Viral Components ◽

Axonal Targeting ◽

Null Mutants

ABSTRACT The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.

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UL54-Null Pseudorabies Virus Is Attenuated in Mice but Productively Infects Cells in Culture

Journal of Virology ◽

10.1128/jvi.80.2.769-784.2006 ◽

2006 ◽

Vol 80 (2) ◽

pp. 769-784 ◽

Cited By ~ 17

Author(s):

Jennifer A. Schwartz ◽

Elizabeth E. Brittle ◽

Ashley E. Reynolds ◽

Lynn W. Enquist ◽

Saul J. Silverstein

Keyword(s):

Protein Synthesis ◽

Pseudorabies Virus ◽

Host Protein ◽

Wild Type Virus ◽

Cell Protein ◽

Transcriptional Level ◽

Dependent Manner ◽

Type Virus ◽

Wild Type ◽

In Cells

ABSTRACT The pseudorabies virus (PRV) UL54 homologs are important multifunctional proteins with roles in shutoff of host protein synthesis, transactivation of virus and cellular genes, and regulation of splicing and translation. Here we describe the first genetic characterization of UL54. We constructed UL54 null mutations in a PRV bacterial artificial chromosome using sugar suicide and λRed allele exchange systems. Surprisingly, UL54 is dispensable for growth in tissue culture but exhibits a small-plaque phenotype that can be complemented in trans by both the herpes simplex virus type 1 ICP27 and varicella-zoster virus open reading frame 4 proteins. Deletion of UL54 in the virus vJSΔ54 had no effect on the ability of the virus to shut off host cell protein synthesis but did affect virus gene expression. The glycoprotein gC accumulated to lower levels in cells infected with vJSΔ54 compared to those infected with wild-type virus, while gK levels were undetectable. Other late gene products, gB, gE, and Us9, accumulated to higher levels than those seen in cells infected with wild-type virus in a multiplicity-dependent manner. DNA replication is also reduced in cells infected with vJSΔ54. UL54 appears to regulate UL53 and UL52 at the transcriptional level as their respective RNAs are decreased in cells infected with vJSΔ54. Interestingly, vJSΔ54 is highly attenuated in a mouse model of PRV infection. Animals infected with vJSΔ54 survive twice as long as animals infected with wild-type virus, and this results in delayed accumulation of virus-specific antigens in skin, dorsal root ganglia, and spinal cord tissues.

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Pseudorabies virus particles lacking tegument proteins pUL11 or pUL16 incorporate less full-length pUL36 than wild-type virus, but specifically accumulate a pUL36 N-terminal fragment

Journal of General Virology ◽

10.1099/vir.0.82168-0 ◽

2006 ◽

Vol 87 (12) ◽

pp. 3503-3507 ◽

Cited By ~ 17

Author(s):

Kathrin Michael ◽

Sindy Böttcher ◽

Barbara G. Klupp ◽

Axel Karger ◽

Thomas C. Mettenleiter

Keyword(s):

Protein Interactions ◽

Pseudorabies Virus ◽

Single Gene ◽

Full Length ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Protein Protein Interactions ◽

Tegument Proteins ◽

Terminal Fragment

Proteins of the virion tegument of alphaherpesviruses are involved in protein–protein interactions, which play important roles in virus morphogenesis. Seven single-gene deletion mutants of Pseudorabies virus were analysed for alterations in the overall composition of the virion beyond the loss of the targeted protein. The UL36 protein (pUL36) was present in equal amounts in wild-type virions and mutants lacking pUL21, pUL49, pUL51, pUS3 or pUS8. Virions lacking pUL11 or pUL16 incorporated less full-length pUL36 than wild-type particles, but contained increased amounts of an N-terminal fragment of pUL36 that is present only in traces in wild-type virus and the other mutants.

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Failure of Thymidine Kinase-Negative Herpes Simplex Virus To Reactivate from Latency following Efficient Establishment

Journal of Virology ◽

10.1128/jvi.78.1.520-523.2004 ◽

2004 ◽

Vol 78 (1) ◽

pp. 520-523 ◽

Cited By ~ 32

Author(s):

Shih-Heng Chen ◽

Angela Pearson ◽

Donald M. Coen ◽

Shun-Hua Chen

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Wild Type Virus ◽

Trigeminal Ganglia ◽

Type Virus ◽

Wild Type ◽

Viral Loads ◽

Simplex Virus

ABSTRACT Thymidine kinase-negative mutants of herpes simplex virus did not reactivate from latency in mouse trigeminal ganglia, even when their latent viral loads were comparable to those that permitted reactivation by wild-type virus. Thus, reduced establishment of latency does not suffice to account for the failure to reactivate.

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The Polyserine Tract of Herpes Simplex Virus ICP4 Is Required for Normal Viral Gene Expression and Growth in Murine Trigeminal Ganglia

Journal of Virology ◽

10.1128/jvi.72.9.7115-7124.1998 ◽

1998 ◽

Vol 72 (9) ◽

pp. 7115-7124 ◽

Cited By ~ 20

Author(s):

Patricia A. Bates ◽

Neal A. DeLuca

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Productive Infection ◽

Wild Type Virus ◽

Trigeminal Ganglia ◽

Type Virus ◽

Wild Type ◽

Low Levels ◽

Simplex Virus

ABSTRACT ICP4 of herpes simplex virus (HSV) is essential for productive infection due to its central role in the regulation of HSV transcription. This study identified a region of ICP4 that is not required for viral growth in culture or at the periphery of experimentally inoculated mice but is critical for productive growth in the trigeminal ganglia. This region of ICP4 encompasses amino acids 184 to 198 and contains 13 nearly contiguous serine residues that are highly conserved among the alphaherpesviruses. A mutant in which this region is deleted (ΔSER) was able to grow on the corneas of mice and be transported back to the trigeminal ganglia. ΔSER did not grow in the trigeminal ganglia but did express low levels of several immediate-early (ICP4 and ICP27) and early (thymidine kinase [tk] and UL42) genes. It expressed very low levels of the late gC gene and did not appear to replicate DNA. This pattern of gene expression was similar to that observed for a tk mutant,dlsptk. Both ΔSER and dlsptk expressed higher levels of the latency-associated transcript (LAT) per genome earlier in infected ganglia than did the wild-type virus, KOS. However, infected ganglia from all three viruses accumulated the same level of LAT per genome at 30 days postinfection (during latency). The data suggest that the polyserine tract of ICP4 provides an activity that is required for lytic infection in ganglia to progress to viral DNA synthesis and full lytic gene expression. In the absence of this activity, higher levels of LAT per genome accumulate earlier in infection than with wild-type virus.

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Pseudorabies Virus Glycoprotein gK Is a Virion Structural Component Involved in Virus Release but Is Not Required for Entry

Journal of Virology ◽

10.1128/jvi.72.3.1949-1958.1998 ◽

1998 ◽

Vol 72 (3) ◽

pp. 1949-1958 ◽

Cited By ~ 43

Author(s):

Barbara G. Klupp ◽

Judith Baumeister ◽

Petra Dietz ◽

Harald Granzow ◽

Thomas C. Mettenleiter

Keyword(s):

Structural Component ◽

Pseudorabies Virus ◽

Virus Assembly ◽

Protein A ◽

Rabbit Antiserum ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Infected Cells ◽

Hsv 1

ABSTRACT The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560–5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S-transferase–gK fusion protein expressed inEscherichia coli. In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N-glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-lacZ expression cassette into the wild-type PrV genome (PrV-gKβ) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB− genome (PrV-gKgB). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKβ was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.

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