scholarly journals Mutational analysis of the virion host shutoff gene (UL41) of herpes simplex virus (HSV): characterization of HSV type 1 (HSV-1)/HSV-2 chimeras.

1997 ◽  
Vol 71 (10) ◽  
pp. 7157-7166 ◽  
Author(s):  
D N Everly ◽  
G S Read
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Mutational Analysis ◽  
Virion Host Shutoff ◽  
Host Shutoff ◽  
Simplex Virus ◽  
Hsv 1

scholarly journals Site-Directed Mutagenesis of the Virion Host Shutoff Gene (UL41) of Herpes Simplex Virus (HSV): Analysis of Functional Differences between HSV Type 1 (HSV-1) and HSV-2 Alleles

1999 ◽  
Vol 73 (11) ◽  
pp. 9117-9129 ◽  
Author(s):  
David N. Everly ◽  
G. Sullivan Read
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Site Directed Mutagenesis ◽  
Viral Mrnas ◽  
Virion Host Shutoff ◽  
Host Shutoff ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT During lytic herpes simplex virus (HSV) infections, the HSV virion host shutoff protein (UL41) accelerates the turnover of host and viral mRNAs. Although the UL41 polypeptides from HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. In a previous study, we identified three regions of the HSV-2 UL41 polypeptide (amino acids 1 to 135, 208 to 243, and 365 to 492) that enhance the activity of KOS when substituted for the corresponding portions of the KOS protein (D. N. Everly, Jr., and G. S. Read, J. Virol. 71:7157–7166, 1997). These results have been extended through the analysis of more than 50 site-directed mutants of UL41 in which selected HSV-2 amino acids were introduced into an HSV-1 background and HSV-1 amino acids were introduced into the HSV-2 allele. The HSV-2 amino acids R22 and E25 were found to contribute dramatically to the greater activity of the HSV-2 allele, as did the HSV-2 amino acids A396 and S423. The substitution of six HSV-2 amino acids between residues 210 and 242 enhanced the HSV-1 activity to a lesser extent. In most cases, individual substitutions or the substitution of combinations of fewer than all six amino acids reduced the UL41 activity to less than that of KOS. The results pinpoint several type-specific amino acids that are largely responsible for the greater activity of the UL41 polypeptide of HSV-2. In addition, several spontaneous mutations that abolish detectable UL41 activity were identified.

scholarly journals Disruption of Virion Host Shutoff Activity Improves the Immunogenicity and Protective Capacity of a Replication-Incompetent Herpes Simplex Virus Type 1 Vaccine Strain

2000 ◽  
Vol 74 (23) ◽  
pp. 11137-11144 ◽  
Author(s):  
Brian J. Geiss ◽  
Tracy J. Smith ◽  
David A. Leib ◽  
Lynda A. Morrison
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Protective Capacity ◽  
Virion Host Shutoff ◽  
Host Shutoff ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The virion host shutoff (vhs) protein encoded by herpes simplex virus type 1 (HSV-1) destabilizes both viral and host mRNAs. An HSV-1 strain with a mutation in vhs is attenuated in virulence and induces immune responses in mice that are protective against corneal infection with virulent HSV-1, but it has the capacity to establish latency. Similarly, a replication-incompetent HSV-1 strain with a mutation in ICP8 elicits an immune response protective against corneal challenge, but it may be limited in viral antigen production. We hypothesized therefore that inactivation of vhs in an ICP8− virus would yield a replication-incompetent mutant with enhanced immunogenicity and protective capacity. In this study, a vhs−/ICP8− HSV-1 mutant was engineered. BALB/c mice were immunized with incremental doses of the vhs−/ICP8− double mutant or vhs−or ICP8− single mutants, or the mice were mock immunized, and protective immunity against corneal challenge with virulent HSV-1 was assessed. Mice immunized with the vhs−/ICP8− mutant showed prechallenge serum immunoglobulin G titers comparable to those immunized with replication-competent vhs− virus and exceed those of mice immunized with the ICP8− single mutant. Following corneal challenge, the degrees of protection against ocular disease, weight loss, encephalitis, and establishment of latency were similar for vhs−/ICP8− and vhs−virus-vaccinated mice. Moreover, the double deleted vhs−/ICP8− virus protected mice better in all respects than the single deleted ICP8− mutant virus. The data indicate that inactivation of vhs in a replication-incompetent virus significantly enhances its protective efficacy while retaining its safety for potential human vaccination. Possible mechanisms of enhanced immunogenicity are discussed.

scholarly journals The Splicing History of an mRNA Affects Its Level of Translation and Sensitivity to Cleavage by the Virion Host Shutoff Endonuclease during Herpes Simplex Virus Infections

2016 ◽  
Vol 90 (23) ◽  
pp. 10844-10856 ◽  
Author(s):  
Jouliana Sadek ◽  
G. Sullivan Read
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Mrna Splicing ◽  
Exon Junction ◽  
Virion Host Shutoff ◽  
History Of ◽  
Host Shutoff ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTDuring lytic herpes simplex virus (HSV) infections, the virion host shutoff (Vhs) (UL41) endoribonuclease degrades many cellular and viral mRNAs. In uninfected cells, spliced mRNAs emerge into the cytoplasm bound by exon junction complexes (EJCs) and are translated several times more efficiently than unspliced mRNAs that have the same sequence but lack EJCs. Notably, most cellular mRNAs are spliced, whereas most HSV mRNAs are not. To examine the effect of splicing on gene expression during HSV infection, cells were transfected with plasmids harboring an unspliced renilla luciferase (RLuc) reporter mRNA or RLuc constructs with introns near the 5′ or 3′ end of the gene. After splicing of intron-containing transcripts, all three RLuc mRNAs had the same primary sequence. Upon infection in the presence of actinomycin D, spliced mRNAs were much less sensitive to degradation by copies of Vhs from infecting virions than were unspliced mRNAs. During productive infections (in the absence of drugs), RLuc was expressed at substantially higher levels from spliced than from unspliced mRNAs. Interestingly, the stimulatory effect of splicing on RLuc expression was significantly greater in infected than in uninfected cells. The translational stimulatory effect of an intron during HSV-1 infections could be replicated by artificially tethering various EJC components to an unspliced RLuc transcript. Thus, the splicing history of an mRNA, and the consequent presence or absence of EJCs, affects its level of translation and sensitivity to Vhs cleavage during lytic HSV infections.IMPORTANCEMost mammalian mRNAs are spliced. In contrast, of the more than 80 mRNAs harbored by herpes simplex virus 1 (HSV-1), only 5 are spliced. In addition, synthesis of the immediate early protein ICP27 causes partial inhibition of pre-mRNA splicing, with the resultant accumulation of both spliced and unspliced versions of some mRNAs in the cytoplasm. A common perception is that HSV-1 infection necessarily inhibits the expression of spliced mRNAs. In contrast, this study demonstrates two instances in which pre-mRNA splicing actually enhances the synthesis of proteins from mRNAs during HSV-1 infections. Specifically, splicing stabilized an mRNA against degradation by copies of the Vhs endoribonuclease from infecting virions and greatly enhanced the amount of protein synthesized from spliced mRNAs at late times after infection. The data suggest that splicing, and the resultant presence of exon junction complexes on an mRNA, may play an important role in gene expression during HSV-1 infections.

scholarly journals Mutational Analysis of the Herpes Simplex Virus Type 1 DNA Packaging Protein UL33

2009 ◽  
Vol 83 (17) ◽  
pp. 8938-8945 ◽  
Author(s):  
Frauke Beilstein ◽  
Martin R. Higgs ◽  
Nigel D. Stow
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Mutational Analysis ◽  
Dna Packaging ◽  
Simplex Virus

ABSTRACT The UL33 protein of herpes simplex virus type 1 (HSV-1) is thought to be a component of the terminase complex that mediates the cleavage and packaging of viral DNA. In this study we describe the generation and characterization of a series of 15 UL33 mutants containing insertions of five amino acids located randomly throughout the 130-residue protein. Of these mutants, seven were unable to complement the growth of the UL33-null virus dlUL33 in transient assays and also failed to support the cleavage and packaging of replicated amplicon DNA into capsids. The insertions in these mutants were clustered between residues 51 and 74 and between 104 and 116, within the most highly conserved regions of the protein. The ability of the mutants to interact with the UL28 component of the terminase was assessed in immunoprecipitation and immunofluorescence assays. All four mutants with insertions between amino acids 51 and 74 were impaired in this interaction, whereas two of the three mutants in the second region (with insertions at positions 111 and 116) were not affected. These data indicate that the ability of UL33 to interact with UL28 is probably necessary, but not sufficient, to support viral growth and DNA packaging.

scholarly journals Characterization of Pseudorabies Virus (PrV) Cleavage-Encapsidation Proteins and Functional Complementation of PrV pUL32 by the Homologous Protein of Herpes Simplex Virus Type 1

2009 ◽  
Vol 83 (8) ◽  
pp. 3930-3943 ◽  
Author(s):  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Tobias Leege ◽  
Thomas C. Mettenleiter
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Pseudorabies Virus ◽  
Gene Products ◽  
Homologous Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Cleavage and encapsidation of newly replicated herpes simplex virus type 1 (HSV-1) DNA requires several essential viral gene products that are conserved in sequence within the Herpesviridae. However, conservation of function has not been analyzed in greater detail. For functional characterization of the UL6, UL15, UL28, UL32, and UL33 gene products of pseudorabies virus (PrV), the respective deletion mutants were generated by mutagenesis of the virus genome cloned as a bacterial artificial chromosome (BAC) in Escherichia coli and propagated in transgenic rabbit kidney cells lines expressing the deleted genes. Neither of the PrV mutants was able to produce plaques or infectious progeny in noncomplementing cells. DNA analyses revealed that the viral genomes were replicated but not cleaved into monomers. By electron microscopy, only scaffold-containing immature but not DNA-containing mature capsids were detected in the nuclei of noncomplementing cells infected with either of the mutants. Remarkably, primary envelopment of empty capsids at the nuclear membrane occasionally occurred, and enveloped tegument-containing light particles were formed in the cytoplasm and released into the extracellular space. Immunofluorescence analyses with monospecific antisera of cells transfected with the respective expression plasmids indicated that pUL6, pUL15, and pUL32 were able to enter the nucleus. In contrast, pUL28 and pUL33 were predominantly found in the cytoplasm. Only pUL6 could be unequivocally identified and localized in PrV-infected cells and in purified virions, whereas the low abundance or immunogenicity of the other proteins hampered similar studies. Yeast two-hybrid analyses revealed physical interactions between the PrV pUL15, pUL28, and pUL33 proteins, indicating that, as in HSV-1, a tripartite protein complex might catalyze cleavage and encapsidation of viral DNA. Whereas the pUL6 protein is supposed to form the portal for DNA entry into the capsid, the precise role of the UL32 gene product during this process remains to be elucidated. Interestingly, the defect of UL32-negative PrV could be completely corrected in trans by the homologous protein of HSV-1, demonstrating similar functions. However, trans-complementation of UL32-negative HSV-1 by the PrV protein was not observed.

scholarly journals Therapeutic Immunization with a Virion Host Shutoff-Defective, Replication-Incompetent Herpes Simplex Virus Type 1 Strain Limits Recurrent Herpetic Ocular Infection

2002 ◽  
Vol 76 (8) ◽  
pp. 3615-3625 ◽  
Author(s):  
Tammie L. Keadle ◽  
Lynda A. Morrison ◽  
Jessica L. Morris ◽  
Jay S. Pepose ◽  
Patrick M. Stuart
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Ocular Infection ◽  
Therapeutic Vaccines ◽  
Corneal Infection ◽  
Virion Host Shutoff ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Immunization of mice with herpes simplex virus type 1 (HSV-1) mutant viruses containing deletions in the gene for virion host shutoff (vhs) protein diminishes primary and recurrent corneal infection with wild-type HSV-1. vhs mutant viruses are severely attenuated in vivo but establish latent infections in sensory neurons. A safer HSV-1 mutant vaccine strain, Δ41Δ29, has combined vhs and replication (ICP8−) deficits and protects BALB/c mice against primary corneal infection equivalent to a vhs− strain (BGS41). Here, we tested the hypothesis that Δ41Δ29 can protect as well as BGS41 in a therapeutic setting. Because immune response induction varies with the mouse and virus strains studied, we first determined the effect of prophylactic Δ41Δ29 vaccination on primary ocular infection of NIH inbred mice with HSV-1 McKrae, a model system used to evaluate therapeutic vaccines. In a dose-dependent fashion, prophylactic Δ41Δ29 vaccination decreased postchallenge tear film virus titers and ocular disease incidence and severity while eliciting high levels of HSV-specific antibodies. Adoptive transfer studies demonstrated a dominant role for immune serum and a lesser role for immune cells in mediating prophylactic protection. Therapeutically, vaccination with Δ41Δ29 effectively reduced the incidence of UV-B-induced recurrent virus shedding in latently infected mice. Therapeutic Δ41Δ29 and BGS41 vaccination decreased corneal opacity and delayed-type hypersensitivity responses while elevating antibody titers, compared to controls. These data indicate that replication is not a prerequisite for generation of therapeutic immunity by live HSV mutant virus vaccines and raise the possibility that genetically tailored replication-defective viruses may make effective and safe therapeutic vaccines.

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