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 mRNA Decay during Herpes Simplex Virus (HSV) Infections: Protein-Protein Interactions Involving the HSV Virion Host Shutoff Protein and Translation Factors eIF4H and eIF4A

2005 ◽  
Vol 79 (15) ◽  
pp. 9651-9664 ◽  
Author(s):  
Pinghui Feng ◽  
David N. Everly ◽  
G. Sullivan Read
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Translation Initiation ◽  
Mammalian Cells ◽  
Translation Initiation Factor ◽  
Site Directed Mutagenesis ◽  
Initiation Factor ◽  
Virion Host Shutoff ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein of herpes simplex virus accelerates the degradation of both host and viral mRNAs. In so doing, it helps redirect the cell from host to viral protein synthesis and facilitates the sequential expression of different viral genes. Vhs interacts with the cellular translation initiation factor eIF4H, and several point mutations that abolish its mRNA degradative activity also abrogate its ability to bind eIF4H. In addition, a complex containing bacterially expressed Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein has RNase activity. eIF4H shares a region of sequence homology with eIF4B, and it appears to be functionally similar in that both stimulate the RNA helicase activity of eIF4A, a component of the mRNA cap-binding complex eIF4F. We show that eIF4H interacts physically with eIF4A in the yeast two-hybrid system and in GST pull-down assays and that the two proteins can be coimmunoprecipitated from mammalian cells. Vhs also interacts with eIF4A in GST pull-down and coimmunoprecipitation assays. Site-directed mutagenesis of Vhs and eIF4H revealed residues of each that are important for their mutual interaction, but not for their interaction with eIF4A. Thus, Vhs, eIF4H, and eIF4A comprise a group of proteins, each of which is able to interact directly with the other two. Whether they interact simultaneously as a tripartite complex or sequentially is unclear. The data suggest a mechanism for linking the degradation of an mRNA to its translation and for targeting Vhs to mRNAs and to regions of translation initiation.

scholarly journals Amino Acids 143 to 150 of the Herpes Simplex Virus Type 1 Scaffold Protein Are Required for the Formation of Portal-Containing Capsids

2008 ◽  
Vol 82 (13) ◽  
pp. 6778-6781 ◽  
Author(s):  
Jamie B. Huffman ◽  
William W. Newcomb ◽  
Jay C. Brown ◽  
Fred L. Homa
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Scaffold Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) portal is composed of a dodecamer of UL6 protein molecules whose incorporation into the capsid is mediated by interaction with the HSV-1 UL26.5 scaffold protein. Previous results with an in vitro capsid assembly assay demonstrated that nine amino acids (amino acids 143 to 151) of the UL26.5 protein are required for its interaction with UL6 and for incorporation of the portal complex into capsids. In the present study an HSV-1 mutant, bvFH411, was isolated and contained a deletion that removed the codons for UL26.5 amino acids 143 to 150. The mutant virus failed to produce infectious virus in noncomplementing cells, and only B capsids that contained only minor amounts of portal protein were made. These data corroborate our previous in vitro studies and demonstrate that amino acids 143 to 150 of UL26.5 are required for the formation of portal-containing HSV-1 capsids.

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.

scholarly journals The Virion Host Shutoff Endonuclease (UL41) of Herpes Simplex Virus Interacts with the Cellular Cap-Binding Complex eIF4F

2010 ◽  
Vol 84 (13) ◽  
pp. 6886-6890 ◽  
Author(s):  
Heidi G. Page ◽  
G. Sullivan Read
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Translation Initiation ◽  
Viral Mrnas ◽  
Translation Factors ◽  
Virion Host Shutoff ◽  
Cap Binding Complex ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT The herpes simplex virus Vhs endonuclease degrades host and viral mRNAs. Isolated Vhs cuts any RNA at many sites. Yet, within cells, it targets mRNAs and cuts at preferred sites, including regions of translation initiation. Previous studies have shown that Vhs binds the translation factors eIF4A and eIF4H. Here, we show that Vhs binds the cap-binding complex eIF4F. Association with eIF4F correlated with the ability of Vhs to bind eIF4A but not eIF4H. All Vhs proteins that degrade mRNAs associated with eIF4F. However, simply tethering an active endonuclease to eIF4F is not sufficient to degrade mRNAs. Binding to eIF4H may also be required.

scholarly journals Proteolytic Cleavage of the Amino Terminus of the UL15 Gene Product of Herpes Simplex Virus Type 1 Is Coupled with Maturation of Viral DNA into Unit-Length Genomes

1999 ◽  
Vol 73 (10) ◽  
pp. 8338-8348 ◽  
Author(s):  
Brandy Salmon ◽  
Dorothy Nalwanga ◽  
Ying Fan ◽  
Joel D. Baines
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Amino Terminus ◽  
Viral Dna ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The UL15 gene of herpes simplex virus type 1 (HSV-1), like UL6, UL17, UL28, UL32, and UL33, is required for cleavage of concatameric DNA into genomic lengths and for packaging of cleaved genomes into preformed capsids. A previous study indicated that the UL15 gene encodes minor capsid proteins. In the present study, we have shown that the amino-terminal 509 amino acids of the UL15-encoded protein are sufficient to confer capsid association inasmuch as a carboxyl-terminally truncated form of the UL15-encoded protein with an M r of approximately 55,000 readily associated with capsids. This and previous studies have shown that, whereas three UL15-encoded proteins with apparent M rs of 83,000, 80,000, and 79,000 associated with wild-type B capsids, only the full-length 83,000-M r protein associated with B capsids purified from cells infected with viruses lacking functional UL6, UL17, UL28, UL32, and UL33 genes (B. Salmon and J. D. Baines, J. Virol. 72:3045–3050, 1998). Thus, all viral mutants that fail to cleave viral DNA into genomic-length molecules also fail to produce capsid-associated UL15 80,000- and 79,000-M r proteins. In contrast, the 80,000- and 79,000-M r proteins were readily detected in capsids purified from cells infected with a UL25 null virus that cleaves, but does not package, DNA. The conclusion that the amino terminus of the 83,000-M r protein is truncated to produce the 80,000- and/or 79,000-M r protein was supported by the following observations. (i) Whereas the C termini of the 83,000-, 80,000-, and 79,000-M r proteins are identical, immunoreactivity dependent on the first 35 amino acids of the UL15 83,000-M r protein was absent from the 80,000- and 79,000-M r proteins. (ii) The 79,000- and 80,000-M r proteins were detected in capsids from cells infected with HSV-1(UL15M36V), an engineered virus encoding valine rather than methionine at codon 36. Thus, initiation at codon 36 is unlikely to account for production of the 80,000- and/or 79,000-M r protein. Taken together, these data strongly suggest that capsid-associated UL15-encoded protein is proteolytically cleaved near the N terminus and indicate that this modification is tightly linked to maturation of genomic DNA.

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