scholarly journals Replication-Competent Herpes Simplex Virus 1 Isolates Selected from Cells Transfected with a Bacterial Artificial Chromosome DNA Lacking Only the UL49 Gene Vary with Respect to the Defect in the UL41 Gene Encoding Host Shutoff RNase

2007 ◽  
Vol 81 (20) ◽  
pp. 10924-10932 ◽  
Author(s):  
Maria Teresa Sciortino ◽  
Brunella Taddeo ◽  
Maria Giuffrè-Cuculletto ◽  
Maria Antonietta Medici ◽  
Antonio Mastino ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Bacterial Artificial Chromosome ◽  
Artificial Chromosome ◽  
Vero Cells ◽  
Wild Type Virus ◽  
Herpes Simplex Virus 1 ◽  
Reading Frame ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT To generate a null UL49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the UL49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-ΔUL49 DNA yielded foci of degenerated cells that could not be expanded and a few replication-competent clones. The replication-competent viral clones derived from independent transfections yielded viruses that expressed genes with some delay, produced smaller plaques, and gave lower yields than wild-type virus. A key finding is that the independently derived replication-competent viruses lacked the virion host shutoff (vhs) activity expressed by the RNase encoded by the UL41 gene. One mutant virus expressed no vhs protein, whereas two others, derived from independent transfections, produced truncated vhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-UL49R DNA (R-UL49) accumulated a full-length vhs protein, indicating that in the parental BAC-ΔUL49 DNA, the UL41 gene was intact. We conclude that expression of the vhs protein in the absence of UL49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of UL49, to be neutralized.

scholarly journals Structure-Based Mutations in the Herpes Simplex Virus 1 Glycoprotein B Ectodomain Arm Impart a Slow-Entry Phenotype

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Qing Fan ◽  
Sarah J. Kopp ◽  
Sarah A. Connolly ◽  
Richard Longnecker
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Bacterial Artificial Chromosome ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Elevated Temperatures ◽  
Artificial Chromosome ◽  
Glycoprotein B ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus

ABSTRACTGlycoprotein B (gB) is the conserved herpesvirus fusion protein, and it is required for the entry of herpesviruses. The structure of the postfusion conformation of gB has been solved for several herpesviruses; however, the gB prefusion crystal structure and the details of how the protein refolds from a prefusion to a postfusion form to mediate fusion have not been determined. Using structure-based mutagenesis, we previously reported that three mutations (I671A, H681A, and F683A) in the C-terminal arm of the gB ectodomain greatly reduced cell-cell fusion. This fusion deficit could be rescued by the addition of a hyperfusogenic mutation, suggesting that the gB triple mutant was not misfolded. Using a bacterial artificial chromosome (BAC), we constructed two independent herpes simplex virus 1 mutant strains (gB 3A) carrying the three arm mutations. The gB 3A viruses have 200-fold smaller plaques than the wild-type virus and demonstrate remarkably delayed entry into cells. Single-step and multistep growth curves show that gB 3A viruses have delayed replication kinetics. Interestingly, incubation at 40°C promoted the entry of the gB 3A viruses. We propose that the gB 3A viruses’ entry deficit is due to a loss of interactions between residues in the gB C-terminal arm and the coiled-coil core of gB. The results suggest that the triple alanine mutation may destabilize the postfusion gB conformation and/or stabilize the prefusion gB conformation and that exposure to elevated temperatures can overcome the defect in gB 3A viruses.IMPORTANCEBecause of its complexity, the mechanism of herpesvirus entry into cells is not well understood. Our study investigated one of the most important unanswered questions about herpesvirus entry; i.e., how does the herpesvirus fusion protein gB mediate membrane fusion? gB is an essential protein that is conserved in all herpesviruses and is thought to undergo a conformational change to provide the energy to fuse the viral and cellular membranes. Using our understanding of the structure of gB, we designed mutations in the gB “arm” region that we predicted would impede gB function. We introduced these mutations into herpes simplex virus 1 by using a bacterial artificial chromosome, and the mutant virus exhibited a drastically delayed rate of entry. This entry defect was rescued by incubation at elevated temperatures, supporting a hypothesis that the engineered mutations altered the energetics of gB refolding. This study supports our hypothesis that an interaction between the gB arm and the core of gB is critical for gB refolding and the execution of membrane fusion, thus providing key details about the function of gB in herpesvirus-mediated fusion and subsequent virus entry.

scholarly journals Engagement of the Lysine-Specific Demethylase/HDAC1/CoREST/REST Complex by Herpes Simplex Virus 1

2009 ◽  
Vol 83 (9) ◽  
pp. 4376-4385 ◽  
Author(s):  
Haidong Gu ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Bodies ◽  
Wild Type Virus ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Lysine Specific Demethylase ◽  
Repressor Complex ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT Among the early events in herpes simplex virus 1 replication are localization of ICP0 in ND10 bodies and accumulation of viral DNA-protein complexes in structures abutting ND10. ICP0 degrades components of ND10 and blocks silencing of viral DNA, achieving the latter by dislodging HDAC1 or -2 from the lysine-specific demethylase 1 (LSD1)/CoREST/REST repressor complex. The role of this process is apparent from the observation that a dominant-negative CoREST protein compensates for the absence of ICP0 in a cell-dependent fashion. HDAC1 or -2 and the CoREST/REST complex are independently translocated to the nucleus once viral DNA synthesis begins. The focus of this report is twofold. First, we report that in infected cells, LSD1, a key component of the repressor complex, is partially degraded or remains stably associated with CoREST and is ultimately also translocated, in part, to the cytoplasm. Second, we examined the distribution of the components of the repressor complex and ICP8 early in infection in wild-type-virus- and ICP0 mutant virus-infected cells. The repressor component and ultimately ICP8 localize in structures that abut the ND10 nuclear bodies. There is no evidence that the two compartments fuse. We propose that ICP0 must dynamically interact with both compartments in order to accomplish its functions of degrading PML and SP100 and suppressing silencing of viral DNA through its interactions with CoREST. In turn, the remodeling of the viral DNA-protein complex enables recruitment of ICP8 and initiation of formation of replication compartments.

scholarly journals Herpes Simplex Virus 1 Counteracts Viperin via Its Virion Host Shutoff Protein UL41

2014 ◽  
Vol 88 (20) ◽  
pp. 12163-12166 ◽  
Author(s):  
G. Shen ◽  
K. Wang ◽  
S. Wang ◽  
M. Cai ◽  
M.-l. Li ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus 1 ◽  
Virion Host Shutoff ◽  
Virion Host Shutoff Protein ◽  
Host Shutoff ◽  
Simplex Virus

scholarly journals Herpes Simplex Virus 1 Induces Cytoplasmic Accumulation of TIA-1/TIAR and both Synthesis and Cytoplasmic Accumulation of Tristetraprolin, Two Cellular Proteins That Bind and Destabilize AU-Rich RNAs

2004 ◽  
Vol 78 (16) ◽  
pp. 8582-8592 ◽  
Author(s):  
Audrey Esclatine ◽  
Brunella Taddeo ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Cytoplasmic Accumulation ◽  
Simplex Virus

ABSTRACT Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the UL41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3′-to-5′ degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking UL41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking UL41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.

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