scholarly journals Regions of the Herpes Simplex Virus Type 1 Latency-Associated Transcript That Protect Cells from Apoptosis In Vitro and Protect Neuronal Cells In Vivo

2002 ◽  
Vol 76 (2) ◽  
pp. 717-729 ◽  
Author(s):  
Maryam Ahmed ◽  
Martin Lock ◽  
Cathie G. Miller ◽  
Nigel W. Fraser
Keyword(s):  
Herpes Simplex ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Exon 1 ◽  
Induced Apoptosis ◽  
Simplex Virus ◽  
In Cells

ABSTRACT Recent studies have suggested that the latency-associated transcript (LAT) region of herpes simplex virus type 1 (HSV-1) is effective at blocking virus-induced apoptosis both in vitro and in the trigeminal ganglia of acutely infected rabbits (Inman et al., J. Virol. 75:3636–3646, 2001; Perng et al., Science 287:1500–1503, 2000). By transfecting cells with a construct expressing the Pst-Mlu segment of the LAT, encompassing the LAT exon 1, the stable 2.0-kb intron, and 5′ part of exon 2, we confirmed that this region was able to diminish the onset of programmed cell death initiated by anti-Fas and camptothecin treatment. In addition, caspase 8-induced apoptosis was specifically inhibited in cells expressing the Pst-Mlu LAT fragment. To further delineate the minimal region of LAT that is necessary for this antiapoptotic function, LAT mutants were used in our cotransfection assays. In HeLa cells, the plasmids lacking exon sequences were the least effective at blocking apoptosis. However, similar to previous work (Inman et al., op. cit.), our data also indicated that the 5′ end of the stable 2.0-kb LAT intron appeared to contribute to the promotion of cell survival. Furthermore, cells productively infected with the 17N/H LAT mutant virus, a virus deleted in the LAT promoter, exon 1, and about half of the intron, exhibited a greater degree of DNA fragmentation than cells infected with wild-type HSV-1. These data support the finding that the exon 1 and 2.0-kb intron region of the LAT transcription unit display an antiapoptotic function both in transfected cells and in the context of the virus infection in vitro. In trigeminal ganglia of mice acutely infected with the wild-type virus, 17, and 17ΔSty, a virus lacking most of exon 1, apoptosis was not detected in cells that were positive for virus particles. However, dual staining was observed in cells from mice infected with 17N/H virus, indicating that the LAT antiapoptotic function demonstrated in cells transfected by LAT-expressing constructs may also play a role in protecting cells from virus-induced apoptosis during acute viral infection in vivo.

scholarly journals Immediate-Early Expression of the Herpes Simplex Virus Type 1 ICP27 Transcript Is Not Critical for Efficient Replication In Vitro or In Vivo

2004 ◽  
Vol 78 (19) ◽  
pp. 10470-10478 ◽  
Author(s):  
Aixu Sun ◽  
G. V. Devi-Rao ◽  
M. K. Rice ◽  
L. W. Gary ◽  
D. C. Bloom ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Immediate Early ◽  
Wild Type ◽  
Early Expression ◽  
Simplex Virus

ABSTRACT We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.

scholarly journals Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo

2003 ◽  
Vol 77 (2) ◽  
pp. 1382-1391 ◽  
Author(s):  
Michiko Tanaka ◽  
Hiroyuki Kagawa ◽  
Yuji Yamanashi ◽  
Tetsutaro Sata ◽  
Yasushi Kawaguchi
Keyword(s):  
Vero Cells ◽  
Infectious Molecular Clone ◽  
Wild Type ◽  
Molecular Clone ◽  
Viral Genes ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT In recent years, several laboratories have reported on the cloning of herpes simplex virus type 1 (HSV-1) genomes as bacterial artificial chromosomes (BACs) in Escherichia coli and on procedures to manipulate these genomes by using the bacterial recombination machinery. However, the HSV-BACs reported so far are either replication incompetent or infectious, with a deletion of one or more viral genes due to the BAC vector insertion. For use as a multipurpose clone in research on HSV-1, we attempted to generate infectious HSV-BACs containing the full genome of HSV-1 without any loss of viral genes. Our results were as follows. (i) E. coli (YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAC flanked by loxP sites was inserted into the intergenic region between UL3 and UL4 was constructed. (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii) The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by coinfection of Vero cells with YK304 and a recombinant adenovirus, AxCANCre, expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus, the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulence in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics, and a recombinant virus carrying amino acid substitutions in both copies of the α0 gene was generated. pYEbac102 will have multiple applications to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.

scholarly journals In vivo and in vitro reactivation impairment of a herpes simplex virus type 1 latency-associated transcript variant in a rabbit eye model.

1991 ◽  
Vol 65 (12) ◽  
pp. 6989-6993 ◽  
Author(s):  
M D Trousdale ◽  
I Steiner ◽  
J G Spivack ◽  
S L Deshmane ◽  
S M Brown ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Eye Model ◽  
Rabbit Eye ◽  
Simplex Virus

scholarly journals Point Mutations in Herpes Simplex Virus Type 1 oriL, but Not in oriS, Reduce Pathogenesis during Acute Infection of Mice and Impair Reactivation from Latency

2006 ◽  
Vol 80 (1) ◽  
pp. 440-450 ◽  
Author(s):  
John W. Balliet ◽  
Priscilla A. Schaffer
Keyword(s):  
Herpes Simplex ◽  
Point Mutations ◽  
Tear Film ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT In vitro studies of herpes simplex virus type 1 (HSV-1) viruses containing mutations in core sequences of the viral origins of DNA replication, oriL and oriS, that eliminate the ability of these origins to initiate viral-DNA synthesis have demonstrated little or no effect on viral replication in cultured cells, leading to the conclusion that the two types of origins are functionally redundant. It remains unclear, therefore, why origins that appear to be redundant are maintained evolutionarily in HSV-1 and other neurotropic alphaherpesviruses. To test the hypothesis that oriL and oriS have distinct functions in the HSV-1 life cycle in vivo, we determined the in vivo phenotypes of two mutant viruses, DoriL-ILR and DoriS-I, containing point mutations in oriL and oriS site I, respectively, that eliminate origin DNA initiation function. Following corneal inoculation of mice, tear film titers of DoriS-I were reduced relative to wild-type virus. In all other tests, however, DoriS-I behaved like wild-type virus. In contrast, titers of DoriL-ILR in tear film, trigeminal ganglia (TG), and hindbrain were reduced and mice infected with DoriL-ILR exhibited greatly reduced mortality relative to wild-type virus. In the TG explant and TG cell culture models of reactivation, DoriL-ILR reactivated with delayed kinetics and, in the latter model, with reduced efficiency relative to wild-type virus. Rescuant viruses DoriL-ILR-R and DoriS-I-R behaved like wild-type virus in all tests. These findings demonstrate that functional differences exist between oriL and oriS and reveal a prominent role for oriL in HSV-1 pathogenesis.

scholarly journals Antiviral effect of oryzacystatin, a proteinase inhibitor in rice, against herpes simplex virus type 1 in vitro and in vivo.

1995 ◽  
Vol 39 (4) ◽  
pp. 846-849 ◽  
Author(s):  
H Aoki ◽  
T Akaike ◽  
K Abe ◽  
M Kuroda ◽  
S Arai ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Rice Seed ◽  
Simplex Virus ◽  
Hsv 1

Oryzacystatin (OC) is the first-described cystatin originating from rice seed; it consists of two molecular species, OC-I and OC-II, which have antiviral action against poliovirus in vitro (H. Kondo, S. Ijiri, K. Abe, H. Maeda, and S. Arai, FEBS Lett. 299:48-50, 1992). In the experiments reported here, we investigated the effects of OC-I and OC-II on the replication of herpes simplex virus type 1 (HSV-1) in vitro and in vivo. HSV-1 was inoculated onto monolayers of monkey kidney epithelial cells (CV-1 cells) at a multiplicity of infection of 0.1 PFU per cell. After adsorption of the virus onto cells, the cultures were incubated in the presence of either OC-I or OC-II in the concentration range of 1.0 to 300 microM, and the supernatant virus yield was quantitated at 24 h. The effective concentration for 90% inhibition of HSV-1 was 14.8 microM, while a cytotoxic effect on CV-1 cells without infection of HSV-1 was not observed below 500 microM OC-I. Therefore, the apparent in vitro chemotherapeutic index was estimated to be more than 33. In the mouse model of HSV-1-induced keratitis and encephalopathy, topical administration of OC-I to the mouse cornea produced a significant decrease in virus production in the cornea (mean virus yields: 3.11 log10 PFU in the treated group and 4.37 log10 PFU in the control group) and significant improvement in survival rates (P = 0.01). The in vivo antiherpetic effect of OC-I was comparable to that of acyclovir, indicating that topical treatment of HSV-1 infection in humans with OC-I might be possible. Our data also suggest the importance of some thiol proteinases, which may be derived from either the host's cells or HSV-1, during the replication process of HSV-1.

scholarly journals Herpes Simplex Virus Type 1 Thymidine Kinase Sequence Fused to the lacZ Gene Increases Levels of β-Galactosidase Activity per Genome of High-Capacity but Not First-Generation Adenoviral Vectors In Vitro and In Vivo

2008 ◽  
Vol 83 (4) ◽  
pp. 2004-2010 ◽  
Author(s):  
M. Puntel ◽  
R. J. Barrett ◽  
S. Mondkar ◽  
V. Saxena ◽  
K. M. Kroeger ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transgene Expression ◽  
First Generation ◽  
High Capacity ◽  
Adenoviral Vectors ◽  
Simplex Virus

ABSTRACT Increased transgene expression per vector genome is an important goal in the optimization of viral vectors for gene therapy. Herein we demonstrate that herpes simplex virus type 1 (HSV1) thymidine kinase (TK) gene sequences (1,131 bp) fused to the 3′ end of lacZ increase transgene expression from high-capacity adenoviral vectors (HCAd), but not from first-generation (Ad) vectors. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), in contrast, increased transgene expression levels from Ad but not HCAd vectors. The differential activity of the HSV1 TK gene and WPRE sequences was detected both in vitro and in vivo and suggests potentially different mechanisms of action or the interaction of these elements with vector genomic sequences.

scholarly journals A herpes simplex virus type 1 mutant deleted for γ34.5 and LAT kills glioma cells in vitro and is inhibited for in vivo reactivation

2001 ◽  
Vol 8 (4) ◽  
pp. 269-277 ◽  
Author(s):  
Ken Samoto ◽  
Guey-Chuen Perng ◽  
Moneeb Ehtesham ◽  
Yunhui Liu ◽  
Steven L Wechsler ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Glioma Cells ◽  
Simplex Virus

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

2006 ◽  
Vol 87 (12) ◽  
pp. 3495-3502 ◽  
Author(s):  
Shih-Heng Chen ◽  
Yu-Wen Lin ◽  
Anthony Griffiths ◽  
Wen-Yen Huang ◽  
Shun-Hua Chen
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Antiviral Drug ◽  
Wild Type Virus ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Viral Genomes ◽  
Simplex Virus

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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