scholarly journals Effect of Deletion of the Ribonucleotide Reductase Gene in Wild Type and Virion Associated Host Shutoff (vhs-1) Mutant Herpes Simplex Virus-1 on Viral Proliferation and Infected-Carcinoma Cell Cultures Growth

2020 ◽  
Author(s):  
Pnina Schlesinger ◽  
Niza Frenkel
Keyword(s):  
Glioblastoma Multiforme ◽  
Cell Cultures ◽  
Ribonucleotide Reductase ◽  
Viral Vector ◽  
Green Fluorescence Protein ◽  
Vero Cells ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Host Shutoff ◽  
Simplex Virus

Glioblastoma multiforme is the most prevalent and deadliest form of glioma and brain cancer, with a very poor prognosis. In an effort to develop an oncolytic viral vector for the treatment of Glioblastoma multiforme, we replaced the UL39 and UL40 genes encoding ribonucleotide reductase (RR) with green fluorescence protein and luciferase genes in wild type KOS and in the virion host shutoff mutant vhs-1, resulting in strains KOS-RR and Vhs-RR, respectively. KOS-RR and Vhs-RR caused death of infected U87 Glioblastoma multiforme cell cultures within one day after infection, whereas KOS and vhs-1-infected cells were more viable. All four viral strains caused apoptotic DNA laddering in infected H1299 lung cancer cells, while only Vhs-RR caused apoptosis in U87 cell cultures. Vhs-RR gave higher yields on U87 than on Vero cells, while it barely proliferated on non-dividing Goiter cells. These results indicate that Vhs-RR proliferates well in actively growing U87 Glioblastoma multiforme cells, causing their death in a mechanism involving apoptosis, while sparing non-dividing cells. Therefore, Vhs-RR is a promising candidate for oncolytic treatment of brain tumor malignancies.

scholarly journals Herpes Simplex Virus 1 Mutant with Point Mutations inUL39Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Heba H. Mostafa ◽  
Thornton W. Thompson ◽  
Adam J. Konen ◽  
Steve D. Haenchen ◽  
Joshua G. Hilliard ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Protein Levels ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTIn the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations inUL39, which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypesin vivo, we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role ofUL39in KOS-NA's decreased pathogenicity, theUL39mutations were recombined into HSV-1 (generating UL39mut), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection.IMPORTANCEHSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in greater detail the events that modulate HSV-1 replication and pathogenesis. In the current study, we identified a neuroattenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also known as ICP6). This mutant form of ICP6 was responsible for the attenuation of KOS-NAin vivoand resulted in diminished ICP6 protein levels and antiapoptotic effect. Thus, we have determined that subtle alteration of the UL39 gene regulates expression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially making KOS-NA a promising vaccine candidate against HSV-1.

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 ICP0, ICP4, or VP16 Expressed from Adenovirus Vectors Induces Reactivation of Latent Herpes Simplex Virus Type 1 in Primary Cultures of Latently Infected Trigeminal Ganglion Cells

2001 ◽  
Vol 75 (13) ◽  
pp. 6143-6153 ◽  
Author(s):  
William P. Halford ◽  
Clinton D. Kemp ◽  
Jennifer A. Isler ◽  
David J. Davido ◽  
Priscilla A. Schaffer
Keyword(s):  
Cell Cultures ◽  
Primary Cultures ◽  
Adenovirus Vector ◽  
Vero Cells ◽  
Wild Type ◽  
Latently Infected ◽  
Adenovirus Vectors ◽  
Simplex Virus ◽  
Mutant Forms ◽  
Hsv 1

ABSTRACT In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TG) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240–3249, 2001). The present study was undertaken to determine whether ICP0 is sufficient to trigger HSV-1 reactivation in latently infected TG cells. To test this hypothesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element (TRE) promoter were constructed. Similar adenovirus vectors encoding wild-type ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OBP), and wild-type and mutant forms of VP16 were also constructed. The TRE promoter was induced by coinfection of Vero cells with the test vector and an adenovirus vector that expresses the reverse tetracycline-regulated transactivator in the presence of doxycycline. Northern blot analysis demonstrated that transcription of the OBP gene in the adenovirus expression vector increased as a function of doxycycline concentration over a range of 0.1 to 10 μM. Likewise, Western blot analysis demonstrated that addition of 3 μM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fold increase in OBP expression. Wild-type forms of ICP0, ICP4, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability to complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild-type forms of ICP0, ICP4, or VP16 induced reactivation of HSV-1 in 86% ± 5%, 86% ± 5%, and 97% ± 5% of TG cell cultures, respectively (means ± standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% ± 5%, 8% ± 0%, 0% ± 0%, and 13% ± 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation. Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular and viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.

scholarly journals Herpes Simplex Virus 1 UL34 Protein Regulates the Global Architecture of the Endoplasmic Reticulum in Infected Cells

2017 ◽  
Vol 91 (12) ◽  
Author(s):  
Fumio Maeda ◽  
Jun Arii ◽  
Yoshitaka Hirohata ◽  
Yuhei Maruzuru ◽  
Naoto Koyanagi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Null Mutation ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Nuclear Egress ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Upon herpes simplex virus 1 (HSV-1) infection, the CD98 heavy chain (CD98hc) is redistributed around the nuclear membrane (NM), where it promotes viral de-envelopment during the nuclear egress of nucleocapsids. In this study, we attempted to identify the factor(s) involved in CD98hc accumulation and demonstrated the following: (i) the null mutation of HSV-1 UL34 caused specific dispersion throughout the cytoplasm of CD98hc and the HSV-1 de-envelopment regulators, glycoproteins B and H (gB and gH); (ii) as observed with CD98hc, gB, and gH, wild-type HSV-1 infection caused redistribution of the endoplasmic reticulum (ER) markers calnexin and ERp57 around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of these markers; (iii) the ER markers colocalized efficiently with CD98hc, gB, and gH in the presence and absence of UL34 in HSV-1-infected cells; (iv) at the ultrastructural level, wild-type HSV-1 infection caused ER compression around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of the ER; and (v) the UL34-null mutation significantly decreased the colocalization efficiency of lamin protein markers of the NM with CD98hc and gB. Collectively, these results indicate that HSV-1 infection causes redistribution of the ER around the NM, with resulting accumulation of ER-associated CD98hc, gB, and gH around the NM and that UL34 is required for ER redistribution, as well as for efficient recruitment to the NM of the ER-associated de-envelopment factors. Our study suggests that HSV-1 induces remodeling of the global ER architecture for recruitment of regulators mediating viral nuclear egress to the NM. IMPORTANCE The ER is an important cellular organelle that exists as a complex network extending throughout the cytoplasm. Although viruses often remodel the ER to facilitate viral replication, information on the effects of herpesvirus infections on ER morphological integrity is limited. Here, we showed that HSV-1 infection led to compression of the global ER architecture around the NM, resulting in accumulation of ER-associated regulators associated with nuclear egress of HSV-1 nucleocapsids. We also identified HSV-1 UL34 as a viral factor that mediated ER remodeling. Furthermore, we demonstrated that UL34 was required for efficient targeting of these regulators to the NM. To our knowledge, this is the first report showing that a herpesvirus remodels ER global architecture. Our study also provides insight into the mechanism by which the regulators for HSV-1 nuclear egress are recruited to the NM, where this viral event occurs.

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 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

Assembly of VP26 in herpes simplex virus-1 inferred from structures of wild-type and recombinant capsids

1995 ◽  
Vol 2 (11) ◽  
pp. 1026-1030 ◽  
Author(s):  
Z. Hong Zhou ◽  
Jing He ◽  
Joanita Jakana ◽  
Jacqueline D. Tatman ◽  
Frazer J. Rixon ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus
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