Competition and complementation between thymidine kinase-negative and wild-type herpes simplex virus during co-infection of mouse trigeminal ganglia

Laboratory strains of herpes simplex virus lacking thymidine kinase (TK) cannot replicate acutely to detectable levels in mouse trigeminal ganglia and do not reactivate from latency. However, many pathogenic clinical isolates that are resistant to the antiviral drug acyclovir are heterogeneous populations of TK-negative (TK−) and TK-positive (TK+) viruses. To recapitulate this in vivo, mice were infected with mixtures of wild-type virus and a recombinant TK− mutant in various ratios. Following co-infection, the replication, number of latent viral genomes and reactivation efficiency of TK+ virus in trigeminal ganglia were reduced in a manner related to the amount of TK− virus in the inoculum. TK+ virus did not always complement the acute replication or increase the number of latent viral genomes of TK− mutant in mouse ganglia. Even so, TK+ virus could still confer the pathogenic phenotype to a TK− mutant, somehow providing sufficient TK activity in trans to permit a TK− mutant to reactivate from latently infected ganglia.

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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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Accumulation of Viral Transcripts and DNA during Establishment of Latency by Herpes Simplex Virus

Journal of Virology ◽

10.1128/jvi.72.2.1177-1185.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 1177-1185 ◽

Cited By ~ 62

Author(s):

Martha F. Kramer ◽

Shun-Hua Chen ◽

David M. Knipe ◽

Donald M. Coen

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Latent Infection ◽

Acute Infection ◽

Mutant Virus ◽

Wild Type Virus ◽

Wild Type ◽

Simplex Virus

ABSTRACT Latent infection of mice with wild-type herpes simplex virus is established during an acute phase of ganglionic infection in which there is abundant viral replication and productive-cycle gene expression. Thymidine kinase-negative mutants establish latent infections but are severely impaired for acute ganglionic replication and productive-cycle gene expression. Indeed, by in situ hybridization assays, acute infection by these mutants resembles latency. To assess events during establishment of latency by wild-type and thymidine kinase-negative viruses, we quantified specific viral nucleic acid sequences in mouse trigeminal ganglia during acute ganglionic infection by using sensitive PCR-based assays. Through 32 h postinfection, viral DNA and transcripts representative of the three kinetic classes of productive-cycle genes accumulated to comparable levels in wild-type- and mutant-infected ganglia. At 48 and 72 h, although latency-associated transcripts accumulated to comparable levels in ganglia infected with wild-type or mutant virus, levels of DNA accumulating in wild-type-infected ganglia exceeded those in mutant-infected ganglia by 2 to 3 orders of magnitude. Coincident with this increase in DNA, wild-type-infected ganglia exhibited abundant expression of productive-cycle genes and high titers of infectious progeny. Nevertheless, the levels of productive-cycle RNAs expressed by mutant virus during acute infection greatly exceeded those expressed by wild-type virus during latency. The results thus distinguish acute infection of ganglia by a replication-compromised mutant from latent infection and may have implications for mechanisms of latency.

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In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Journal of Virology ◽

10.1128/jvi.72.7.5351-5359.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5351-5359 ◽

Cited By ~ 86

Author(s):

Thandavarayan Nagashunmugam ◽

John Lubinski ◽

Liyang Wang ◽

Lester T. Goldstein ◽

Benjamin S. Weeks ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Immunoglobulin G ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE339, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE339 expresses gE and gI, is FcγR−, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE339 causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR− mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE339-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.

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Mutation Spectra of Herpes Simplex Virus Type 1 Thymidine Kinase Mutants

Journal of Virology ◽

10.1128/jvi.76.11.5822-5828.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5822-5828 ◽

Cited By ~ 13

Author(s):

Qiaosheng Lu ◽

Ying T. Hwang ◽

Charles B. C. Hwang

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Wild Type Virus ◽

Exonuclease Activity ◽

Wild Type ◽

Simplex Virus

ABSTRACT To examine whether the exonuclease activity intrinsic to the polymerase (Pol) of herpes simplex virus type 1 can influence the mutational spectra, we applied the denaturing gradient gel electrophoresis (DGGE) system combined with sequencing to characterize thymidine kinase mutants isolated from both the wild-type virus and a mutant deficient in exonuclease activity, Y7. Wild-type viruses produced predominately frameshift mutations (67%), whereas Y7 replicated a significantly lower proportion of frameshifts (21%; P < 0.005). Furthermore, the majority of substitutions were transitional changes in both groups, although they distributed differently. The implications of these findings are discussed.

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Expression of Extremely Low Levels of Thymidine Kinase from an Acyclovir-Resistant Herpes Simplex Virus Mutant Supports Reactivation from Latently Infected Mouse Trigeminal Ganglia

Journal of Virology ◽

10.1128/jvi.00484-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 8356-8360 ◽

Cited By ~ 17

Author(s):

Michael I. Besecker ◽

Caroline L. Furness ◽

Donald M. Coen ◽

Anthony Griffiths

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Infected Mouse ◽

Laboratory Strain ◽

Trigeminal Ganglia ◽

Wild Type ◽

Latently Infected ◽

Low Levels ◽

Simplex Virus

ABSTRACT A single-cytosine-deletion in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. A laboratory strain engineered to carry this mutation did not generate sufficient TK activity for detection by plaque autoradiography, which detected 0.25% wild-type activity. However, a drug sensitivity assay suggested that extremely low levels of TK are generated by this virus. The virus was estimated to express 0.09% of wild-type TK activity via a ribosomal frameshift 24 nucleotides upstream of the mutation. Remarkably, this appeared to be sufficient active TK to support a low level of reactivation from latently infected mouse trigeminal ganglia.

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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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In vivo fitness and virulence of a drug-resistant herpes simplex virus 1 mutant

Journal of General Virology ◽

10.1099/vir.0.82787-0 ◽

2007 ◽

Vol 88 (5) ◽

pp. 1410-1414 ◽

Cited By ~ 7

Author(s):

Jean M. Pesola ◽

Donald M. Coen

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Antiviral Drug ◽

Mutant Virus ◽

Drug Resistant ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Wild Type Parent

Two important issues regarding a virus mutant that is resistant to an antiviral drug are its ability to replicate in animal hosts (in vivo fitness) relative to other genetic variants, including wild type, and its ability to cause disease. These issues have been investigated for a herpes simplex virus 1 mutant that is resistant to thiourea compounds, which inhibit encapsidation of viral DNA. Following corneal inoculation of mice, the mutant virus replicated very similarly to its wild-type parent in the eye, trigeminal ganglion and brain. The mutant virus was as lethal to mice as its wild-type parent following this route of inoculation. Indeed, it exhibited increased virulence. Thus, unlike most drug-resistant virus mutants, this mutant retained in vivo fitness and virulence.

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Quantitative analysis of herpes simplex virus DNA and transcriptional activity in ganglia of mice latently infected with wild-type and thymidine kinase-deficient viral strains

Journal of General Virology ◽

10.1099/0022-1317-75-9-2469 ◽

1994 ◽

Vol 75 (9) ◽

pp. 2469-2474 ◽

Cited By ~ 19

Author(s):

B. Slobedman ◽

S. Efstathiou ◽

A. Simmons

Keyword(s):

Herpes Simplex Virus ◽

Quantitative Analysis ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Transcriptional Activity ◽

Wild Type ◽

Latently Infected ◽

Simplex Virus

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gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes

Molecular and Cellular Biology ◽

10.1128/mcb.5.3.518-528.1985 ◽

1985 ◽

Vol 5 (3) ◽

pp. 518-528

Author(s):

S Silver ◽

B Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Dna Synthesis ◽

Thymidine Kinase ◽

Viral Dna ◽

Bamhi Fragment ◽

Viral Genomes ◽

Chimeric Genes ◽

Simplex Virus ◽

Hsv 1

True gamma or gamma 2 genes, unlike alpha, beta, and gamma 1 (beta gamma) genes of herpes simplex virus 1 (HSV-1), stringently require viral DNA synthesis for their expression. We report that gamma 2 genes resident in cells were induced in trans by infection with HSV-1 but that the induction did not require amplification of either the resident gene or the infecting viral genome. Specifically, to test the hypothesis that expression of these genes is amplification dependent, we constructed two sets of gamma 2-thymidine kinase (TK) chimeric genes. The first (pRB3038) consisted of the promoter-regulatory region and a portion of 5'-transcribed noncoding region of the domain of a gamma 2 gene identified by Hall et al. (J. Virol. 43:594-607) in the HSV-1(F) BamHI fragment D' to the 5'-transcribed noncoding and coding regions of the TK gene. The second (pRB3048) contained, in addition, an origin of HSV-1 DNA replication. Cells transfected with either the first or second construct and selected for the TK+ phenotype were then tested for TK induction after superinfection with HSV-1(F) delta 305, containing a deletion in the coding sequences of the TK gene, and viruses containing, in addition, a ts lesion in the alpha 4 regulatory protein (ts502 delta 305) or in the beta 8 major DNA-binding protein (tsHA1 delta 305). The results were as follows: induction by infection with TK- virus of chimeric TK genes with or without an origin of DNA replication was dependent on functional alpha 4 protein but not on viral DNA synthesis; the resident chimeric gene in cells selected for G418 (neomycin) resistance was regulated in the same fashion; the chimeric gene recombined into the viral DNA was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis; the gamma 2-chimeric genes resident in the host and in viral genomes were transcribed from the donor BamHI fragment D' containing the promoter-regulatory domain of the gamma 2 gene. The significance of the differential regulation of gamma 2 genes in the environments of host and viral genomes by viral trans-acting factors is discussed.

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