A Herpes Simplex Virus DNA Polymerase Mutation That Specifically Attenuates Neurovirulence in Mice (original) (raw)
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A Point Mutation in a Herpesvirus Polymerase Determines Neuropathogenicity
PLoS Pathogens, 2007
Infection with equid herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurologic disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism resulting in an amino acid variation of the EHV-1 DNA polymerase (N752/D752) is significantly associated with the neuropathogenic potential of naturally occurring strains. To test the hypothesis that this single amino acid exchange by itself influences neuropathogenicity, we generated recombinant viruses with differing polymerase sequences. Here we show that the N752 mutant virus caused no neurologic signs in the natural host, while the D752 virus was able to cause inflammation of the central nervous system and ataxia. Neurologic disease induced by the D752 virus was concomitant with significantly increased levels of viremia (p ¼ 0.01), but the magnitude of virus shedding from the nasal mucosa was similar between the N752 and D752 viruses. Both viruses replicated with similar kinetics in fibroblasts and epithelial cells, but exhibited differences in leukocyte tropism. Last, we observed a significant increase (p , 0.001) in sensitivity of the N752 mutant to aphidicolin, a drug targeting the viral polymerase. Our results demonstrate that a single amino acid variation in a herpesvirus enzyme can influence neuropathogenic potential without having a major effect on virus shedding from infected animals, which is important for horizontal spread in a population. This observation is very interesting from an evolutionary standpoint and is consistent with data indicating that the N752 DNA pol genotype is predominant in the EHV-1 population, suggesting that decreased viral pathogenicity in the natural host might not be at the expense of less efficient inter-individual transmission. Citation: Goodman LB, Loregian A, Perkins GA, Nugent J, Buckles EL, et al. (2007) A point mutation in a herpesvirus polymerase determines neuropathogenicity. PLoS Pathog 3(11): e160.
Japanese Journal of Infectious Diseases, 2013
ACV r) mutants were generated from plaque-purified ACVsensitive herpes simplex virus type 1 (HSV-1) by culturing the virus in Vero cells in the presence of 2amino-7-(1,3-dihydroxy-2-propoxymethyl) purine (S2242). Three DNA polymerase (DNApol)-associated ACV r HSV-1 generated under ACV selection in a previous study (Suzutani, T., Ishioka, K., De Clercq, E., et al., Antimicrob. Agents Chemother., 47, 1707-1713, 2003) were also included. The sensitivity of the mutants to other antivirals and their neurovirulence were determined. The treatment efficacy of ACV and ganciclovir (GCV) against ACV r HSV-1 infections was evaluated in mice. Amino acid substitutions were demonstrated in conserved regions II and III in DNApol in 5 of the 6 mutants, while the other substitution was located in non-conserved regions. DNApol-associated ACV r clones showed cross-resistance to foscarnet, penciclovir, and vidarabine but were sensitive or hypersensitive to GCV, brivudin, sorivudine, and spongothymidine. The ACV r clone with an N815S mutation in DNApol showed similar neurovirulence to that of the parent virus; however, those with other mutations showed attenuation. GCV was effective in the treatment of the ACV r clone with similar virulence to that of parent HSV-1, while ACV was less effective in mice. These results indicate the importance of the characterization of HSV-1 isolates for the proper treatment of HSV-1 infections exhibiting ACV-resistance. Japan, for providing us with BVaraU.
Journal of Virology, 2003
Herpes simplex virus type 1 (HSV-1) DNA polymerase contains several conserved regions within the polymerase domain. The conserved regions I, II, III, V, and VII have been shown to have functional roles in the interaction with deoxynucleoside triphosphates (dNTPs) and DNA. However, the role of conserved region VI in DNA replication has remained unclear due, in part, to the lack of a well-characterized region VI mutant. In this report, recombinant viruses containing a point mutation (L774F) within the conserved region VI were constructed. These recombinant viruses were more susceptible to aphidicolin and resistant to both foscarnet and acyclovir, compared to the wild-type KOS strain. Marker transfer experiments demonstrated that the L774F mutation conferred the altered drug sensitivities. Furthermore, mutagenesis assays demonstrated that L774F recombinant viruses containing the supF marker gene, which was integrated within the thymidine kinase locus ( tk ), exhibited increased fidelit...
Virology, 1985
Five herpes simplex virus mutants containing temperature-sensitive mutations in the gene for the major DNA-binding protein were assayed for their sensitivities to the DNA polymerase inhibitors aphidicolin and phosphonoacetic acid (PAA). Four of the mutants (tsA1, tsA15, tsA24, and tsA42) exhibited altered sensitivity to one or both of the inhibitors relative to the wild-type parent. In tsA1, a mutation or mutations conferring aphidicolin and PAA hypersensitivity were mapped by corescue with the temperature-sensitivity marker of tsA1 to a region of the DNA-binding protein locus, between map coordinates 0.385 and 0.398. The mutation conferring PAA hypersensitivity in t&24 similarly corescued with the tsA24 temperature-sensitivity marker, mapping to the DNA-binding protein locus between coordinates 0.398 and 0.413. Thus, mutations outside the DNA polymerase locus and within the DNA-binding protein locus can confer altered sensitivity to certain DNA polymerase inhibitors.
Journal of general …, 1989
The herpes simplex virus type 2 (HG52) deletion variant JH2604 is avirulent (LD50 > 107 p.f.u./mouse) for mice compared to the parental wild-type virus (LDs0 < 102 p.f.u./mouse) and fails to replicate in vivo. JH2604 has a 1488 bp deletion within the 3 kb BamHI v fragment (0 to 0-02 and 0.81 to 0.83 map units) which removes one copy of the 17 bp direct repeat DR1 element of the 'a' sequence and terminates 522 bp upstream of the 5' end of the immediate early gene 1. In vivo selection after transfection of intact JH2604 DNA with the BamHI g (v + u) joint fragment of HG52 results in the isolation of wild-type virus with an LDs0 of < 10 z p.f.u./mouse. These results show that a 1488 bp sequence within the terminal portion of the genome long repeat region confers neurovirulence on strain HG52.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1996
Herpes simplex viruses that lack ICP34.5 are neuroattenuated and are presently being considered for cancer and gene therapy in the nervous system. Previously, we documented the focal presence of the latency-associated transcripts (LATs) in the hippocampi of immunocompromised mice after intracranial (IC) inoculation of an ICP34.5-deficient virus called strain 1716. To characterize further the biological properties of strain 1716 in the CNS of immunocompetent mice, we determined the extent of viral gene expression in different cell types and regions of the CNS after stereotactic IC inoculation of this virus. At survival times of > 30 d after inoculation, we found that (1) infectious virus was not detectable by titration and immunohistochemical studies; (2) neurons harbored virus as demonstrated by the detection of the LATs by in situ hybridization (ISH); (3) transcripts expressed during the lytic cycle of infection were not detected by ISH; and (4) subsets of neurons were selective...
Unusual regulation of expression of the herpes simplex virus DNA polymerase gene
Journal of Virology, 1993
During herpes simplex virus infection, expression of the viral DNA polymerase (pol) gene is regulated temporally as an early (beta) gene and is additionally down-regulated at late times at the level of translation (D. R. Yager, A. I. Marcy, and D. M. Coen, J. Virol. 64:2217-2225, 1990). To examine the role of viral DNA synthesis in pol regulation, we studied pol expression during infections in which viral DNA synthesis was blocked, either by using drugs that inhibit Pol or ribonucleotide reductase or by using viral mutants with lesions in either the pol or a primase-helicase subunit gene. Under any of these conditions, the level of cytoplasmic pol mRNA was reduced. This reduction was first seen at approximately the time DNA synthesis begins and, when normalized to levels of other early mRNAs, became as great as 20-fold late in infection. The reduction was also observed in the absence of the adjacent origin of replication, oriL. Thus, although pol mRNA accumulated as expected for an ...