scholarly journals Intracorneal delivery of HSV-targeting CRISPR/Cas9 mRNA prevents herpetic stromal keratitis

2020 ◽  
Author(s):  
Di Yin ◽  
Sikai Ling ◽  
Dawei Wang ◽  
Dai Yao ◽  
Hao Jiang ◽  
...  
Keyword(s):  
Recurrent Infection ◽  
Trigeminal Ganglia ◽  
Herpetic Stromal Keratitis ◽  
Infection Models ◽  
Viral Genes ◽  
Stromal Keratitis ◽  
Simplex Virus ◽  
Cas9 Mrna ◽  
Hsv 1

ABSTRACTHerpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current treatments for HSV-1 do not eliminate the virus and are incapable of modulating the virus reservoir. Here, we target HSV-1 genome directly using mRNA-carrying lentiviral particle (mLP) that simultaneously delivers spCas9 mRNA and two viral genes-targeting gRNAs (designated HSV-1-erasing lentiviral particles, HELP). We showed HELP efficiently blocked HSV-1 replication in both acute and recurrent infection models, and prevented occurrence of herpetic stromal keratitis (HSK). We further showed retrograde transportation of HELP from corneas to trigeminal ganglia (TG) where HSV-1 established latency and found evidence of HELP modulating herpes reservoir. Additionally, the potent antiviral activity of HELP was also replicable in human-derived corneas. These results strongly support clinical development of HELP as a new antiviral therapy and may accelerate mRNA-based CRISPR therapeutics.

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 Level of Herpes Simplex Virus Type 1 Latency Correlates with Severity of Corneal Scarring and Exhaustion of CD8+ T Cells in Trigeminal Ganglia of Latently Infected Mice

2008 ◽  
Vol 83 (5) ◽  
pp. 2246-2254 ◽  
Author(s):  
Kevin R. Mott ◽  
Catherine J. Bresee ◽  
Sariah J. Allen ◽  
Lbachir BenMohamed ◽  
Steven L. Wechsler ◽  
...  
Keyword(s):  
T Cells ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Infected Individual ◽  
Trigeminal Ganglia ◽  
Corneal Scarring ◽  
Latently Infected ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT A hallmark of infection with herpes simplex virus type 1 (HSV-1) is the establishment of latency in ganglia of the infected individual. During the life of the latently infected individual, the virus can occasionally reactivate, travel back to the eye, and cause recurrent disease. Indeed, a major cause of corneal scarring (CS) is the scarring induced by HSV-1 following reactivation from latency. In this study, we evaluated the relationship between the amount of CS and the level of the HSV-1 latency-associated transcript (LAT) in trigeminal ganglia (TG) of latently infected mice. Our results suggested that the amount of CS was not related to the amount of virus replication following primary ocular HSV-1 infection, since replication in the eyes was similar in mice that did not develop CS, mice that developed CS in just one eye, and mice that developed CS in both eyes. In contrast, mice with no CS had significantly less LAT, and thus presumably less latency, in their TG than mice that had CS in both eyes. Higher CS also correlated with higher levels of mRNAs for PD-1, CD4, CD8, F4/80, interleukin-4, gamma interferon, granzyme A, and granzyme B in both cornea and TG. These results suggest that (i) the immunopathology induced by HSV-1 infection does not correlate with primary virus replication in the eye; (ii) increased CS appears to correlate with increased latency in the TG, although the possible cause-and-effect relationship is not known; and (iii) increased latency in mouse TG correlates with higher levels of PD-1 mRNA, suggesting exhaustion of CD8+ T cells.

scholarly journals In Vivo Changes in the Patterns of Chromatin Structure Associated with the Latent Herpes Simplex Virus Type 1 Genome in Mouse Trigeminal Ganglia Can Be Detected at Early Times after Butyrate Treatment

2007 ◽  
Vol 81 (23) ◽  
pp. 13248-13253 ◽  
Author(s):  
Donna M. Neumann ◽  
Partha S. Bhattacharjee ◽  
Nicole V. Giordani ◽  
David C. Bloom ◽  
James M. Hill
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Sodium Butyrate ◽  
Herpes Simplex Virus Type ◽  
Trigeminal Ganglia ◽  
Butyrate Treatment ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT During herpes simplex virus type 1 (HSV-1) latency in mouse dorsal root ganglia (DRG), chromatin associated with the latency-associated transcript (LAT) region of the viral genome is hyperacetylated at lysines 9 and 14 of histone 3 [H3(K9, K14)], while lytic genes are hypoacetylated. Explanted DRG exhibit a pattern of deacetylation of the LAT enhancer followed by acetylation of the ICP0 promoter at early times postexplant. Recently, we reported that sodium butyrate induced in vivo reactivation of HSV-1 in latent mice. In this study, we assessed the effect of sodium butyrate on the chromatin patterns of latent and butyrate-treated mouse trigeminal ganglia (TG) via chromatin immunoprecipitation (ChIP). We detected deacetylation of acetyl H3(K9, K14) of the LAT promoter and LAT enhancer regions as early as 0.5 h post-butyrate treatment, and this deacetylation corresponded to an increase in the acetylation of the lytic promoters ICP0 and ICP4 at 0.5 h and 1 h post-butyrate treatment, respectively. This is the first study to combine in vivo reactivation with the examination of the HSV-1 genome through ChIP assays at early times after the introduction of in vivo reactivation stimuli.

scholarly journals Towards an Understanding of the Herpes Simplex Virus Type 1 Latency-Reactivation Cycle

2010 ◽  
Vol 2010 ◽  
pp. 1-18 ◽  
Author(s):  
Guey-Chuen Perng ◽  
Clinton Jones
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Productive Infection ◽  
Viral Gene ◽  
Trigeminal Ganglia ◽  
Simplex Virus ◽  
Hsv 1

Infection by herpes simplex virus type 1 (HSV-1) can cause clinical symptoms in the peripheral and central nervous system. Recurrent ocular shedding can lead to corneal scarring and vision loss making HSV-1 a leading cause of corneal blindness due to an infectious agent. The primary site of HSV-1 latency is sensory neurons within trigeminal ganglia. Periodically, reactivation from latency occurs resulting in virus transmission and recurrent disease. During latency, the latency-associated transcript (LAT) is abundantly expressed. LAT expression is important for the latency-reactivation cycle in animal models, in part, because it inhibits apoptosis, viral gene expression, and productive infection. A novel transcript within LAT coding sequences (AL3) and small nonprotein coding RNAs are also expressed in trigeminal ganglia of latently infected mice. In this review, an update of viral factors that are expressed during latency and their potential roles in regulating the latency-reactivation cycle is discussed.

scholarly journals The Dominant-Negative Herpes Simplex Virus Type 1 (HSV-1) Recombinant CJ83193 Can Serve as an Effective Vaccine against Wild-Type HSV-1 Infection in Mice

2004 ◽  
Vol 78 (11) ◽  
pp. 5756-5765 ◽  
Author(s):  
Hanka Augustinova ◽  
Daniela Hoeller ◽  
Feng Yao
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Dominant Negative ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT By selectively regulating the expression of the trans-dominant-negative mutant polypeptide UL9-C535C, of herpes simplex virus type 1 (HSV-1) origin binding protein UL9 with the tetracycline repressor (tetR)-mediated gene switch, we recently generated a novel replication-defective and anti-HSV-specific HSV-1 recombinant, CJ83193. The UL9-C535C peptides expressed by CJ83193 can function as a potent intracellular therapy against its own replication, as well as the replication of wild-type HSV-1 and HSV-2 in coinfected cells. In this report, we demonstrate that CJ83193 cannot initiate acute productive infection in corneas of infected mice nor can it reactivate from trigeminal ganglia of mice latently infected by CJ83193 in a mouse ocular model. Given that CJ83193 is capable of expressing the viral α, β, and γ1 genes but little or no γ2 genes, we tested the vaccine potential of CJ83193 against HSV-1 infection in a mouse ocular model. Our studies showed that immunization with CJ83193 significantly reduced the yields of challenge HSV in the eyes and trigeminal ganglia on days 3, 5, and 7 postchallenge. Like in mice immunized with the wild-type HSV-1 strain KOS, immunization of mice with CJ83193 prevents the development of keratitis and encephalitis induced by corneal challenge with wild-type HSV-1 strain mP. Delayed-type hypersensitivity (DTH) assays demonstrate that CJ83193 can elicit durable cell-mediated immunity at the same level as that of wild-type HSV-1 and is more effective than that induced by d27, an HSV-1 ICP27 deletion mutant. Moreover, mice immunized with CJ83193 developed strong, durable HSV-1-neutralizing antibodies at levels at least twofold higher than those induced by d27. The results presented in this report have shed new light on the development of effective HSV viral vaccines that encode a unique safety mechanism capable of inhibiting the mutant's own replication and that of wild-type virus.

scholarly journals Overexpression of Interleukin-2 by a Recombinant Herpes Simplex Virus Type 1 Attenuates Pathogenicity and Enhances Antiviral Immunity

2002 ◽  
Vol 76 (18) ◽  
pp. 9069-9078 ◽  
Author(s):  
Homayon Ghiasi ◽  
Yanira Osorio ◽  
Guey-Chuen Perng ◽  
Anthony B. Nesburn ◽  
Steven L. Wechsler
Keyword(s):  
T Cells ◽  
Interleukin 2 ◽  
Parental Virus ◽  
Wild Type Virus ◽  
Ocular Infection ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The expression of interleukin-2 (IL-2) has been implicated in the modulation of the outcome of ocular infection with herpes simplex virus type 1 (HSV-1); however, its effects remain controversial. To clarify the role of IL-2, we constructed a recombinant HSV-1 (HSV-IL-2) that expresses two copies of the murine IL-2 gene under the control of the latency-associated transcript (LAT) promoter of HSV-1 in a LAT-negative virus. In tissue culture, the replication of the HSV-IL-2 was 100-fold lower than that of the wild-type virus at a low multiplicity of infection (MOI). Addition of recombinant anti-IL-2 polyclonal antibody markedly enhanced HSV-IL-2 replication in tissue culture. In the 7-day period after ocular infection of BALB/c mice, the replication of HSV-IL-2 was significantly lower than that of wild-type virus in tear cultures, whole eyes, and brain, but was equivalent to wild-type replication in the trigeminal ganglia. Ocular challenge of BALB/c mice with HSV-IL-2 alone, at an MOI that resulted in only 13% survival when parental virus was used, was associated with 90% survival. This decrease in virulence was further shown to be attributable to the expression of IL-2 by coinfection of mice with HSV-IL-2 and the parental virus. This resulted in a decrease in virulence of the parental virus (5% survival when administered alone versus 50% survival on coinfection with HSV-IL-2). The survival of HSV-IL-2-infected mice was compromised by depletion of either IL-2, CD4+, or CD8+ T cells (50% survival) and abolished completely by depletion of both T-cell subtypes. Moreover, depletion of CD4+ T cells, CD8+ T cells, or both increased the titers of HSV-IL-2 in the tears, eyes, trigeminal ganglia, and brains of infected mice, so that titers were equivalent to or higher than that of the parental virus. These results suggest that IL-2 expression by recombinant HSV-1 reduces virulence and that depletion of IL-2 or T cells increases virulence in HSV-1-infected mice.

scholarly journals The Transgenic ICP4 Promoter Is Activated in Schwann Cells in Trigeminal Ganglia of Mice Latently Infected with Herpes Simplex Virus Type 1

2001 ◽  
Vol 75 (21) ◽  
pp. 10401-10408 ◽  
Author(s):  
Naomi S. Taus ◽  
William J. Mitchell
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Schwann Cells ◽  
Latent Infection ◽  
Early Gene ◽  
Trigeminal Ganglia ◽  
Latently Infected ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of sensory ganglia, including those of the trigeminal ganglia. Latent viral infection has been hypothesized to be regulated by restriction of viral immediate-early gene expression in neurons. Numerous in situ hybridization studies in mice and in humans have shown that transcription from the HSV-1 genome in latently infected neurons is limited to the latency-associated transcripts. In other studies, immediate-early gene (ICP4) transcripts have been detected by reverse transcription-PCR (RT-PCR) in homogenates of latently infected trigeminal ganglia of mice. We used reporter transgenic mice containing the HSV-1(F) ICP4 promoter fused to the coding sequence of the β-galactosidase gene to determine whether neurons in latently infected trigeminal ganglia activated the ICP4 promoter. Mice were inoculated via the corneal route with HSV-1(F). At 5, 11, 23, and 37 days postinfection (dpi), trigeminal ganglia were examined for β-galactosidase-positive cells. The numbers of β-galactosidase-positive neurons and nonneuronal cells were similar at 5 dpi. The number of positive neurons decreased at 11 dpi and returned to the level of mock-inoculated transgenic controls at 23 and 37 dpi. The number of positive nonneuronal cells increased at 11 and 23 dpi and remained elevated at 37 dpi. Viral proteins were detected in neurons and nonneuronal cells in acutely infected ganglia, but were not detected in latently infected ganglia. Colabeling experiments confirmed that the transgenic ICP4 promoter was activated in Schwann cells during latent infection. These findings suggest that the cells that express the HSV-1 ICP4 gene in latently infected ganglia are not neurons.

scholarly journals The CoREST/REST Repressor Is both Necessary and Inimical for Expression of Herpes Simplex Virus Genes

mBio ◽  
2010 ◽  
Vol 2 (1) ◽  
Author(s):  
Guoying Zhou ◽  
Du Te ◽  
Bernard Roizman
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Lysine Specific Demethylase ◽  
Repressor Complex ◽  
Viral Genes ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTHerpes simplex virus type 1 encodes three sets of genes, α, β, and γ, whose expression is sequentially ordered in a cascade fashion. The transactivators of α genes comprise virion protein 16 (VP16) and the cellular proteins octamer binding protein 1 (Oct1) and host factor 1 (HCF1). Efficient transition from α to β gene expression requires the α protein ICP0 (infected cell protein 0). Earlier studies have shown that this protein binds to CoREST and displaces HDAC1 from the CoREST/REST/lysine-specific demethylase 1 (LSD1) repressor complex. Ultimately, the components of the repressor complex are translocated at least in part into the cytoplasm. A key event in activation of α genes is the recruitment of LSD1 to demethylate histones bound to the α gene promoters. LSD1 is unstable in the absence of its partner, CoREST, and raises the question of whether both CoREST and REST are involved in the initiation of transcription of the α genes. Here we show that CoREST or REST small interfering RNAs (siRNAs) destabilize CoREST, REST, LSD1, and Sin3A, another component of the repressor complex. In cells transfected with REST or CoREST siRNA, the accumulation of α proteins and mRNAs is delayed in comparison to those of mock-transfected or control siRNA-transfected cells. The LSD1/CoREST/REST compressor complex is thus sequentially necessary and subsequently inimical for viral gene expression.IMPORTANCEHerpes simplex virus type 1 (HSV-1) is globally regulated at the following four checkpoints: (i) the activation of α (immediate-early) genes, (ii) the point of transition from α to β and γ gene expression, (iii) the silencing of viral genes for the establishment of the latent state, and (iv) the reactivation of viral genes on termination of latency. Earlier studies showed that the transition from α to β and γ gene expression involves the suppression of the HDAC1/lysine-specific demethylase 1 (LSD1)/CoREST/REST (HLCR) repressor complex. More recently, this laboratory reported evidence suggesting that in sensory neurons, HSV-1 hijacks the HLCR complex to silence itself for the establishment of latency. This report extends the observation that LSD1 is required for expression of the α genes to show that the HLCR complex is involved in this process. Thus, HSV-1 has evolved an intimate relationship with the HCLR complex to regulate its gene expression and, by extension, its fundamental interactions with its human host.

scholarly journals Gene Array Analysis Reveals Changes in Peripheral Nervous System Gene Expression following Stimuli That Result in Reactivation of Latent Herpes Simplex Virus Type 1: Induction of Transcription Factor Bcl-3

2001 ◽  
Vol 75 (20) ◽  
pp. 9909-9917 ◽  
Author(s):  
Dimitra Tsavachidou ◽  
Wawrzyniec Podrzucki ◽  
John Seykora ◽  
Shelley L. Berger
Keyword(s):  
Nervous System ◽  
Transcription Factors ◽  
Herpes Simplex ◽  
Gene Array ◽  
Trigeminal Ganglia ◽  
Array Analysis ◽  
Gene Array Analysis ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The earliest events within the peripheral mammalian nervous system that cause herpes simplex virus type 1 (HSV-1) to reactivate from latency are unknown but are highly likely to include altered regulation of cellular transcription factors. Using gene array analysis, we have examined the changes that occur in cellular mRNA levels in mouse trigeminal ganglia following explantation, a stimulus that results in HSV-1 reactivation from latency. We have detected both increased and decreased expression levels of particular cellular transcripts, which include RNAs encoding neuronal factors, transcription factors, and factors involved in the cell cycle. Among the transcription factors that are upregulated is Bcl-3, a coactivator for NFκB. We have confirmed these increases in Bcl-3 transcription levels using reverse transcription-PCR and S1 nuclease protection assays. In addition, we have shown Bcl-3 upregulation at the protein level. Importantly, Bcl-3 RNA levels were found to increase specifically in neuronal cells within the trigeminal ganglia. We discuss a potential role for this factor in upregulating ICP0 transcription, which is an important viral event for initiation of HSV-1 reactivation.

scholarly journals Herpes Simplex Virus Type 1 Latency-Associated Transcript Expression Protects Trigeminal Ganglion Neurons from Apoptosis

2005 ◽  
Vol 79 (14) ◽  
pp. 9019-9025 ◽  
Author(s):  
Francisco J. Branco ◽  
Nigel W. Fraser
Keyword(s):  
T Cells ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Trigeminal Ganglia ◽  
Trigeminal Ganglion Neurons ◽  
Simplex Virus ◽  
Ganglion Neurons ◽  
Hsv 1

ABSTRACT Upon infection of murine trigeminal ganglia with herpes simplex virus type 1 (HSV-1), an immune response is initiated resulting in significant infiltration of CD8+ T cells. Previous investigators have observed a lack of apoptosis in HSV-1 trigeminal ganglia even in the presence of cytotoxic immune cells. To determine the role of the latency-associated transcript (LAT) in inhibiting apoptosis, we examined mice during acute and latent infection with HSV-1 (strain 17 or a LAT-negative deletion mutant strain 17 N/H) by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and fluorescence-activated cell sorting (FACS). FACS analysis revealed CD8+ T cells in the trigeminal ganglia by day 7, with more being present in 17- than 17 N/H-infected trigeminal ganglia (6.22% versus 3.5%) and a decrease in number through day 30 (2.7% to 1.2%). To detect apoptotic CD8+ T cells, sections were assayed by TUNEL and stained for CD8+ T cells. By day 7, ∼10% of CD8+ T cells in both 17- and 17 N/H-infected trigeminal ganglia had undergone apoptosis. By day 30, 58% and 74% of all T cells had undergone apoptosis in 17- and 17 N/H-infected trigeminal ganglia, respectively. Furthermore, no HSV strain 17-infected trigeminal ganglion neurons were apoptotic, but 0.087% of 17ΔSty and 0.98% of 17 N/H-infected neurons were apoptotic. We conclude that the antiapoptotic effect of LAT appears to require the LAT promoter, with most of the antiapoptotic effect mapping within the StyI (+447) to the HpaI (+1667) region and a minor contribution from the upstream StyI (+76) to StyI (+447) region.

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