High accumulation of Mx2 renders limited multiplication of oncolytic herpes simplex virus-1 in human tumor cells

AbstractIncreasing studies demonstrated that oncolytic activities of oHSV-1 are limited to the capacity of virus replicating in tumors. In order to potentiate the oHSV-1 oncolytic activity and expand the application of oHSV-1 treatment in multiple types of tumors, it is critical to explore the potential factors or mechanisms mediating tumor resistance to oHSV-1 infection. Here we evaluated the levels of oHSV-1 multiplication in various tumor cell lines and showed that glioblastoma cell line A172 had the lowest virus yields but intrinsically accumulated the highest levels of Mx2 protein. Subsequently we demonstrated that genetic depletion of Mx2 specifically enhanced oHSV-1 productive replication in A172 cells through promoting the nuclear translocation of uncoated viral genomic DNA and down-regulating innate antiviral response. In the further investigation, we found that Mx2 knockdown could alter the intrinsic mRNA accumulation of diverse sets innate immune genes in A172 cells, in particular DHX36 and MyD88. Mx2 depletion led to a decrease in mRNA levels of MyD88 and DHX36 in A172 cells and MyD88/DHX36 knockdown increased virus yield in A172 cells and decreased the production of IFNα, activation of IRF3 activity and NF-κB signaling in A172 cells. This shed new lights on understanding the roles of some intrinsic antiviral genes in oHSV-1 resistance, facilitating to offer potential targets to improve oHSV-1 oncolytic efficacy and develop candidates of biomarkers to predict the efficiency of oHSV-1 multiplication in tumors.

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Herpes Simplex Virus 1 UL24 Abrogates the DNA Sensing Signal Pathway by Inhibiting NF-κB Activation

Journal of Virology ◽

10.1128/jvi.00025-17 ◽

2017 ◽

Vol 91 (7) ◽

Cited By ~ 35

Author(s):

Haiyan Xu ◽

Chenhe Su ◽

Angela Pearson ◽

Christopher H. Mody ◽

Chunfu Zheng

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Cyclic Gmp ◽

Nuclear Translocation ◽

Signal Pathway ◽

Promoter Activation ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

First Time ◽

Hsv 1

ABSTRACT Cyclic GMP-AMP synthase (cGAS) is a newly identified DNA sensor that recognizes foreign DNA, including the genome of herpes simplex virus 1 (HSV-1). Upon binding of viral DNA, cGAS produces cyclic GMP-AMP, which interacts with and activates stimulator of interferon genes (STING) to trigger the transcription of antiviral genes such as type I interferons (IFNs), and the production of inflammatory cytokines. HSV-1 UL24 is widely conserved among members of the herpesviruses family and is essential for efficient viral replication. In this study, we found that ectopically expressed UL24 could inhibit cGAS-STING-mediated promoter activation of IFN-β and interleukin-6 (IL-6), and UL24 also inhibited interferon-stimulatory DNA-mediated IFN-β and IL-6 production during HSV-1 infection. Furthermore, UL24 selectively blocked nuclear factor κB (NF-κB) but not IFN-regulatory factor 3 promoter activation. Coimmunoprecipitation analysis demonstrated that UL24 bound to the endogenous NF-κB subunits p65 and p50 in HSV-1-infected cells, and UL24 was also found to bind the Rel homology domains (RHDs) of these subunits. Furthermore, UL24 reduced the tumor necrosis factor alpha (TNF-α)-mediated nuclear translocation of p65 and p50. Finally, mutational analysis revealed that the region spanning amino acids (aa) 74 to 134 of UL24 [UL24(74–134)] is responsible for inhibiting cGAS-STING-mediated NF-κB promoter activity. For the first time, UL24 was shown to play an important role in immune evasion during HSV-1 infection. IMPORTANCE NF-κB is a critical component of the innate immune response and is strongly induced downstream of most pattern recognition receptors (PRRs), leading to the production of IFN-β as well as a number of inflammatory chemokines and interleukins. To establish persistent infection, viruses have evolved various mechanisms to counteract the host NF-κB pathway. In the present study, for the first time, HSV-1 UL24 was demonstrated to inhibit the activation of NF-κB in the DNA sensing signal pathway via binding to the RHDs of the NF-κB subunits p65 and p50 and abolishing their nuclear translocation.

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Recombinant human tumor necrosis factor alpha regulates c-myc expression in HL-60 cells at the level of transcription

Blood ◽

10.1182/blood.v70.1.200.bloodjournal701200 ◽

1987 ◽

Vol 70 (1) ◽

pp. 200-205

Author(s):

A Tobler ◽

D Johnston ◽

HP Koeffler

Keyword(s):

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Human Tumor ◽

Tnf Alpha ◽

Mrna Levels ◽

Human Tumor Necrosis Factor ◽

Mrna Accumulation ◽

Necrosis Factor Alpha ◽

Factor Alpha ◽

Necrosis Factor

Recombinant human tumor necrosis factor alpha (TNF alpha) effectively inhibits clonal growth of leukemic cells from patients and several cell lines, including the promyelocytic HL-60 cells. Decreased expression of the c-myc oncogene is linked to growth arrest and terminal cellular differentiation. The present study characterizes the effect of TNF alpha on the regulation of the c-myc gene in HL-60 cells. TNF alpha (100 U/mL) rapidly inhibited messenger RNA (mRNA) accumulation of c-myc with a 50% reduction in less than one hour. Dose-response studies showed that a 50% reduction of c-myc mRNA occurred in the range of 15 U/mL. In vitro nuclear run-on experiments showed that this decrease of c-myc-mRNA accumulation was the result of a reduced rate of transcription of c-myc by TNF alpha. Further studies demonstrated that TNF alpha did not post-transcriptionally alter levels of c-myc mRNA, and the inhibitory action of TNF alpha on c-myc expression in HL-60 cells did not depend on new protein synthesis. In the conditions of all the experiments, TNF alpha did not affect cell viability. By contrast, TNF alpha (500 U/mL) did not decrease mRNA levels of c-myc in an HL-60 variant cell line whose growth was not inhibited by TNF alpha; also TNF alpha (500 U/mL) increased c-myc-mRNA levels in normal fibroblasts whose growth is known to be stimulated by TNF alpha. These findings, in concert with prior studies, show a close association between growth inhibition of HL-60 cells and decreased levels of mRNA coding for c-myc.

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Characterization of the nuclear import mechanisms of HSV-1 UL31

Biological Chemistry ◽

10.1515/hsz-2015-0299 ◽

2016 ◽

Vol 397 (6) ◽

pp. 555-561 ◽

Cited By ~ 11

Author(s):

Mingsheng Cai ◽

Jiang Si ◽

Xiaowei Li ◽

Zhancheng Zeng ◽

Meili Li

Keyword(s):

Nuclear Import ◽

Nuclear Translocation ◽

Fluorescent Microscopy ◽

Multiple Roles ◽

Live Cells ◽

Biological Functions ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Hsv 1

Abstract As an important protein, UL31 has been demonstrated to play multiple roles in herpes simplex virus 1 (HSV-1) replication. Previous studies showed that UL31 predominantly locates in the nucleus in chemical fixed cells and live cells, however, the determining mechanisms for its nuclear translocation is not clear. In the present study, by utilizing live cells fluorescent microscopy and co-immunoprecipitation assays, the nuclear import of UL31 was characterized to be dependent on Ran-, importin α1- and transportin-1-mediated pathway. Therefore, these results will promote the understanding of UL31-mediated biological functions in HSV-1 infection cycle.

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Activated MEK Suppresses Activation of PKR and Enables Efficient Replication and In Vivo Oncolysis by Δγ134.5 Mutants of Herpes Simplex Virus 1

Journal of Virology ◽

10.1128/jvi.80.3.1110-1120.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1110-1120 ◽

Cited By ~ 72

Author(s):

Kerrington D. Smith ◽

James J. Mezhir ◽

Kai Bickenbach ◽

Jula Veerapong ◽

Jean Charron ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Tumor Cells ◽

Cell Lines ◽

Human Tumor ◽

Dominant Negative ◽

Herpes Simplex Virus 1 ◽

Simplex Virus

ABSTRACT Herpes simplex virus mutants lacking the γ134.5 gene are not destructive to normal tissues but are potent cytolytic agents in human tumor cells in which the activation of double-stranded RNA-dependent protein kinase (PKR) is suppressed. Thus, replication of a Δγ134.5 mutant (R3616) in 12 genetically defined cancer cell lines correlates with suppression of PKR but not with the genotype of RAS. Extensive analyses of two cell lines transduced with either dominant negative MEK (dnMEK) or constitutively active MEK (caMEK) indicated that in R3616 mutant-infected cells dnMEK enabled PKR activation and decreased virus yields, whereas caMEK suppressed PKR and enabled better viral replication and cell destruction in transduced cells in vitro or in mouse xenografts. The results indicate that activated MEK mediates the suppression of PKR and that the status of MEK predicts the ability of Δγ134.5 mutant viruses to replicate in and destroy tumor cells.

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Herpes Simplex Virus 1 Protein Kinase US3 Hyperphosphorylates p65/RelA and Dampens NF-κB Activation

Journal of Virology ◽

10.1128/jvi.03394-13 ◽

2014 ◽

Vol 88 (14) ◽

pp. 7941-7951 ◽

Cited By ~ 56

Author(s):

Kezhen Wang ◽

Liwen Ni ◽

Shuai Wang ◽

Chunfu Zheng

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Protein Kinase ◽

Immune Responses ◽

Kinase Activity ◽

Nuclear Translocation ◽

Interleukin 8 ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Hsv 1

ABSTRACTNuclear factor κB (NF-κB) plays important roles in innate immune responses by regulating the expression of a large number of target genes involved in the immune and inflammatory response, apoptosis, cell proliferation, differentiation, and survival. To survive in the host cells, viruses have evolved multiple strategies to evade and subvert the host immune response. Herpes simplex virus 1 (HSV-1) bears a large DNA genome, with the capacity to encode many different viral proteins to counteract the host immune responses. In the present study, we demonstrated that HSV-1 protein kinase US3 significantly inhibited NF-κB activation and decreased the expression of inflammatory chemokine interleukin-8 (IL-8). US3 was also shown to hyperphosphorylate p65 at serine 75 and block its nuclear translocation. Two US3 mutants, K220M and D305A, still interacted with p65; however, they could not hyperphosphorylate p65, indicating that the kinase activity of US3 was indispensable for the function. The attenuation of NF-κB activation by HSV-1 US3 protein kinase may represent a critical adaptation to enable virus persistence within the host.IMPORTANCEThis study demonstrated that HSV-1 protein kinase US3 significantly inhibited NF-κB activation and decreased the expression of inflammatory chemokine interleukin-8 (IL-8). US3 hyperphosphorylated p65 at serine 75 to inhibit NF-κB activation. The kinase activity of US3 was indispensable for its hyperphosphorylation of p65 and abrogation of the nuclear translocation of p65. The present study elaborated a novel mechanism of HSV-1 US3 to evade the host innate immunity.

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Recombinant human tumor necrosis factor alpha regulates c-myc expression in HL-60 cells at the level of transcription

Blood ◽

10.1182/blood.v70.1.200.200 ◽

1987 ◽

Vol 70 (1) ◽

pp. 200-205 ◽

Cited By ~ 13

Author(s):

A Tobler ◽

D Johnston ◽

HP Koeffler

Keyword(s):

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Human Tumor ◽

Tnf Alpha ◽

Mrna Levels ◽

Human Tumor Necrosis Factor ◽

Mrna Accumulation ◽

Necrosis Factor Alpha ◽

Factor Alpha ◽

Necrosis Factor

Abstract Recombinant human tumor necrosis factor alpha (TNF alpha) effectively inhibits clonal growth of leukemic cells from patients and several cell lines, including the promyelocytic HL-60 cells. Decreased expression of the c-myc oncogene is linked to growth arrest and terminal cellular differentiation. The present study characterizes the effect of TNF alpha on the regulation of the c-myc gene in HL-60 cells. TNF alpha (100 U/mL) rapidly inhibited messenger RNA (mRNA) accumulation of c-myc with a 50% reduction in less than one hour. Dose-response studies showed that a 50% reduction of c-myc mRNA occurred in the range of 15 U/mL. In vitro nuclear run-on experiments showed that this decrease of c-myc-mRNA accumulation was the result of a reduced rate of transcription of c-myc by TNF alpha. Further studies demonstrated that TNF alpha did not post-transcriptionally alter levels of c-myc mRNA, and the inhibitory action of TNF alpha on c-myc expression in HL-60 cells did not depend on new protein synthesis. In the conditions of all the experiments, TNF alpha did not affect cell viability. By contrast, TNF alpha (500 U/mL) did not decrease mRNA levels of c-myc in an HL-60 variant cell line whose growth was not inhibited by TNF alpha; also TNF alpha (500 U/mL) increased c-myc-mRNA levels in normal fibroblasts whose growth is known to be stimulated by TNF alpha. These findings, in concert with prior studies, show a close association between growth inhibition of HL-60 cells and decreased levels of mRNA coding for c-myc.

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Herpes Simplex Virus 1 Serine/Threonine Kinase US3 Hyperphosphorylates IRF3 and Inhibits Beta Interferon Production

Journal of Virology ◽

10.1128/jvi.02355-13 ◽

2013 ◽

Vol 87 (23) ◽

pp. 12814-12827 ◽

Cited By ~ 74

Author(s):

Shuai Wang ◽

Kezhen Wang ◽

Rongtuan Lin ◽

Chunfu Zheng

Keyword(s):

Innate Immunity ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Nuclear Translocation ◽

Sendai Virus ◽

Ectopic Expression ◽

Herpes Simplex Virus 1 ◽

Host Innate Immunity ◽

Simplex Virus ◽

Hsv 1

Viral infection initiates a series of signaling cascades that lead to the transcription of interferons (IFNs), finally inducing interferon-stimulated genes (ISGs) to eliminate viruses. Viruses have evolved a variety of strategies to modulate host IFN-mediated immune responses. Herpes simplex virus 1 (HSV-1) US3, a Ser/Thr kinase conserved in alphaherpesviruses, was previously reported to counteract host innate immunity; however, the molecular mechanism is elusive. In this study, we report that US3 blocks IFN-β production by hyperphosphorylating IFN regulatory factor 3 (IRF3). Ectopic expression of US3 protein significantly inhibited Sendai virus (SeV)-mediated activation of IFN-β and IFN-stimulated response element (ISRE) promoters and the transcription of IFN-β, ISG54, and ISG56. US3 was also shown to block SeV-induced dimerization and nuclear translocation of IRF3. The kinase activity was indispensable for its inhibitory function, as kinase-dead (KD) US3 mutants K220M and D305A could not inhibit IFN-β production. Furthermore, US3 interacted with and hyperphosphorylated IRF3 at Ser175 to prevent IRF3 activation. Finally, the US3 KD mutant viruses were constructed and denoted K220M or D305A HSV-1, respectively. Cells and mice infected with both mutant viruses produced remarkably larger amounts of IFN-β than those infected with wild-type HSV-1. For the first time, these findings provide convincing evidence that US3 hyperphosphorylates IRF3, blocks the production of IFN-β, and subverts host innate immunity.

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Assembly of VP26 in herpes simplex virus-1 capsid implicated by 3-D structure of recombinant capsid

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140579 ◽

1995 ◽

Vol 53 ◽

pp. 840-841

Author(s):

Z. Hong Zhou ◽

Jing He ◽

Joanita Jakana ◽

J. D. Tatman ◽

Frazer J. Rixon ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

3D Structure ◽

Structure Study ◽

Herpes Simplex Virus 1 ◽

Shell Proteins ◽

Life Threatening ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

Herpes simplex virus-1 (HSV-1) is a ubiquitous virus which is implicated in diseases ranging from self-curing cold sores to life-threatening infections. The 2500 Å diameter herpes virion is composed of a glycoprotein spike containing, lipid envelope, enclosing a protein layer (the tegument) in which is embedded the capsid (which contains the dsDNA genome). The B-, and A- and C-capsids, representing different morphogenetic stages in HSV-1 infected cells, are composed of 7, and 5 structural proteins respectively. The three capsid types are organized in similar T=16 icosahedral shells with 12 pentons, 150 hexons, and 320 connecting triplexes. Our previous 3D structure study at 26 Å revealed domain features of all these structural components and suggested probable locations for the outer shell proteins, VP5, VP26, VP19c and VP23. VP5 makes up most of both pentons and hexons. VP26 appeared to bind to the VP5 subunit in hexon but not to that in penton.

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