Publications by authors named "Fuchun Zhou"

25 Publications

  • Page 1 of 1

Menstrual Dysfunction in Women With Schizophrenia During Risperidone Maintenance Treatment.

J Clin Psychopharmacol 2021 Mar-Apr 01;41(2):135-139

Background: A percentage of women patients with schizophrenia may suffer from menstrual dysfunction associated with antipsychotic medication. This study evaluated menstrual dysfunction in women with schizophrenia given maintenance risperidone, and investigated the association between menstrual dysfunction and the duration and dose of risperidone and clinical symptoms.

Methods: The data of 161 women were obtained from the Risperidone Maintenance Treatment in Schizophrenia study, including patients' characteristics, menstrual conditions, and duration and dosage of risperidone. Qualitative data regarding menstrual health were evaluated at baseline (clinical stabilization after 4-8 weeks maintenance treatment with a standard risperidone dose 4-8 mg/d) and follow-up interviews up to 52 weeks.

Results: At baseline, 73.2% (119/161) of the patients were eumenorrheic; specific rates of menstrual dysfunction were 14.3% (23/161) irregular menstruation, 6.8% (11/161) oligomenorrhea, and 5.0% (8/161) amenorrhea. At the end of follow-up, 16.0% (19/119) of those with eumenorrhea at baseline reported menstrual dysfunction. During the entire risperidone maintenance treatment, 37.9% (61/161) experienced menstrual dysfunction. The range of onset time from the beginning of risperidone treatment to menstrual dysfunction was 64 to 243 days. Risperidone dose was positively associated with menstrual dysfunction (r = 0.187, P = 0.046). The total Positive and Negative Syndrome Scale score was significantly associated with menstrual dysfunction (r = 0.274, P = 0.001).

Conclusions: Attention should be given to menstrual dysfunction of women with schizophrenia that is an adverse effect of risperidone maintenance treatment. Menstrual dysfunction may occur early or late during maintenance treatment, partly depending on the dose.ClinicalTrials.govidentifier: NCT00848432.
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http://dx.doi.org/10.1097/JCP.0000000000001344DOI Listing
February 2021

Activation of FXR by ganoderic acid A promotes remyelination in multiple sclerosis via anti-inflammation and regeneration mechanism.

Biochem Pharmacol 2021 Mar 20;185:114422. Epub 2021 Jan 20.

Yunnan University, School of Medicine and College of Life Sciences, 2 Cuihu North Road, Kunming, Yunnan 650091, China. Electronic address:

Multiple sclerosis (MS), as an inflammatory demyelinating disorder of central nervous system, is the leading cause of non-traumatic neurologic disability in young adults. The pathogenesis of MS remains unknown, however, a dysregulation of glia-neuroimmune signaling plays a key role during progressive disease stage. Most of the existing drugs are aimed at the immune system, but there is no approved drug by promoting remyelination after demyelination so far. There is a great interest in identifying novel agents for treating MS bytargeting to switch the immune imbalance from pro-inflammation and apoptosis to anti-inflammation and regeneration during remyelination phase. Here, we reported that ganoderic acid A (GAA) significantly enhanced the remyelination and rescued motor deficiency in two animal models of MS, including cuprizone-induced demyelination and myelin oligodendrocyte glycoprotein (MOG) 35-55-induced experimental autoimmune encephalomyelitis model. In these two independent MS animal models, GAA modulated neuroimmune to enhance the anti-inflammatory and regeneration markers IL-4 and BDNF, inhibited inflammatory markers IL-1β and IL-6, followed by down-regulation of microglia activation and astrocyte proliferation. Pharmacological and genetic ablation of farnesoid-X-receptor (FXR) abolished GAA-induced remyelination and restoration of motor deficiency in MS mice. Thus, GAA is a novel and potential therapeutic agent that can rescue MS neuroimmune imbalance and remyelination through an FXR receptor-dependent mechanism. Clinical investigation on the therapeutic effect of GAA in improving remyelination of the MS patients to rescue the motor function is warranted.
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http://dx.doi.org/10.1016/j.bcp.2021.114422DOI Listing
March 2021

GDF6-CD99 Signaling Regulates Src and Ewing Sarcoma Growth.

Cell Rep 2020 11;33(5):108332

Greehey Children's Cancer Research Institute, The University of Texas Health Science Center, San Antonio, TX 78229, USA; Mays Cancer Center, The University of Texas Health Science Center, San Antonio, TX 78229, USA; Department of Biochemistry and Structural Biology, The University of Texas Health Science Center, San Antonio, TX 78229, USA. Electronic address:

We report here that the autocrine signaling mediated by growth and differentiation factor 6 (GDF6), a member of the bone morphogenetic protein (BMP) family of cytokines, maintains Ewing sarcoma growth by preventing Src hyperactivation. Surprisingly, Ewing sarcoma depends on the prodomain, not the BMP domain, of GDF6. We demonstrate that the GDF6 prodomain is a ligand for CD99, a transmembrane protein that has been widely used as a marker of Ewing sarcoma. The binding of the GDF6 prodomain to the CD99 extracellular domain results in recruitment of CSK (C-terminal Src kinase) to the YQKKK motif in the intracellular domain of CD99, inhibiting Src activity. GDF6 silencing causes hyperactivation of Src and p21-dependent growth arrest. We demonstrate that two GDF6 prodomain mutants linked to Klippel-Feil syndrome are hyperactive in CD99-Src signaling. These results reveal a cytokine signaling pathway that regulates the CSK-Src axis and cancer cell proliferation and suggest the gain-of-function activity for disease-causing GDF6 mutants.
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http://dx.doi.org/10.1016/j.celrep.2020.108332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688343PMC
November 2020

Impaired Sensorimotor Gating Using the Acoustic Prepulse Inhibition Paradigm in Individuals at a Clinical High Risk for Psychosis.

Schizophr Bull 2021 01;47(1):128-137

The National Clinical Research Center for Mental Disorders and Beijing Key Laboratory of Mental Disorders and Beijing Institute for Brain Disorders Center of Schizophrenia, Beijing Anding Hospital, Capital Medical University, Beijing, China.

Many robust studies have investigated prepulse inhibition (PPI) in patients with schizophrenia. Recent evidence indicates that PPI may help identify individuals who are at clinical high risk for psychosis (CHR). Selective attention to prepulse stimulus can specifically enhance PPI in healthy subjects; however, this enhancement effect is not observed in patients with schizophrenia. Modified PPI measurement with selective attentional modulation using perceived spatial separation (PSS) condition may be a more robust and sensitive index of PPI impairment in CHR individuals. The current study investigated an improved PSSPPI condition in CHR individuals compared with patients with first-episode schizophrenia (FES) and healthy controls (HC) and evaluated the accuracy of PPI in predicting CHR from HC. We included 53 FESs, 55 CHR individuals, and 53 HCs. CHRs were rated on the Structured Interview for Prodromal Syndromes. The measures of perceived spatial co-location PPI (PSCPPI) and PSSPPI conditions were applied using 60- and 120-ms lead intervals. Compared with HC, the CHR group had lower PSSPPI level (Inter-stimulus interval [ISI] = 60 ms, P < .001; ISI = 120 ms, P < .001). PSSPPI showed an effect size (ES) between CHR and HC (ISI = 60 ms, Cohen's d = 0.91; ISI = 120 ms, Cohen's d = 0.98); on PSSPPI using 60-ms lead interval, ES grade increased from CHR to FES. The area under the receiver operating characteristic curve for PSSPPI was greater than that for PSCPPI. CHR individuals showed a PSSPPI deficit similar to FES, with greater ES and sensitivity. PSSPPI appears a promising objective approach for preliminary identification of CHR individuals.
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http://dx.doi.org/10.1093/schbul/sbaa102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825103PMC
January 2021

Defects of Gamma Oscillations in Auditory Steady-State Evoked Potential of Schizophrenia.

Shanghai Arch Psychiatry 2018 Feb;30(1):27-38

Beijing Anding Hospital, Capital Medical University, Beijing, China.

Background: Patients with schizophrenia have many cognitive deficits. Gamma oscillations exist in the human brain and are closely related to neurocognition. Auditory Steady-State Responses (ASSRs) is an electroneurophysiological index that could reflect gamma oscillations. It was found that the energy evoked by 40 Hz ASSRs in schizophrenic patients was significantly lower than that in healthy subjects. However, the correlation between ASSRs phase index and clinical symptoms and neurocognitive deficits has yet to be systematically studied. The purpose of this study was to investigate the dysfunction of neural activity of gamma rhythmdys function and its association with clinical symptoms and neurocognition in patients with schizophrenia.

Aims: To compare and verify the difference in energy and phase coherence of 20 Hz and 40 Hz ASSRs between schizophrenia and healthy participants, and to explore the correlation between schizophrenia ASSRs and neurocognitive deficits.

Method: Auditory steady-state evoked potentials by repeated auditory stimuli in 24 patients with schizophrenia and 30 healthy controls were recorded. The Positive and Negative Syndrome Scale (PANSS) was used to assess the clinical symptoms of the patients. MATRICS Consensus Cognitive Battery (MCCB) was used for the assessment of neurocognitive function. The correlation between indices, such as ASSRs energy, phase locking factor and phase coherence, and clinical and cognitive assessment was also systematically compared between two groups.

Results: Compared with the control group, the patient group had differences in cognitive domains including information processing speed (=-2.39, =0.021), attention/vigilance (=-2.36, =0.023), verbal learning (=-3.11, =0.003), and reasoning and problem solving (=-2.60, =0.012). The energy of 40 Hz ASSRs in the patient group was significantly lower than that in the control group (=-2.291, =0.032), and their phase locking factor and inter-trial phase coherence index were lower than control group (=-3.017, =0.004 and =3.131, =0.003), which was also significantly correlated to reasoning and problem solving function deficits.

Conclusion: Patients with schizophrenia had defects in multiple cognitive domains, and their 40 Hz ASSRs energy was low. Specifically, their phase locking characteristics and phase coherence were poor, which was to some extent related to reasoning ability and thinking disorder.
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http://dx.doi.org/10.11919/j.issn.1002-0829.217078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925596PMC
February 2018

Activation of Kaposi's sarcoma-associated herpesvirus (KSHV) by inhibitors of class III histone deacetylases: identification of sirtuin 1 as a regulator of the KSHV life cycle.

J Virol 2014 Jun 26;88(11):6355-67. Epub 2014 Mar 26.

Department of Pediatrics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

Unlabelled: Kaposi's sarcoma-associated herpesvirus (KSHV) establishes persistent latent infection in immunocompetent hosts. Disruption of KSHV latency results in viral lytic replication, which promotes the development of KSHV-related malignancies in immunocompromised individuals. While inhibitors of classes I and II histone deacetylases (HDACs) potently reactivate KSHV from latency, the role of class III HDAC sirtuins (SIRTs) in KSHV latency remains unclear. Here, we examined the effects of inhibitors of SIRTs, nicotinamide (NAM) and sirtinol, on KSHV reactivation from latency. Treatment of latently KSHV-infected cells with NAM or sirtinol induced transcripts and proteins of the master lytic transactivator RTA (ORF50), early lytic genes ORF57 and ORF59, and late lytic gene ORF65 and increased the production of infectious virions. NAM increased the acetylation of histones H3 and H4 as well as the level of the active histone H3 trimethyl Lys4 (H3K4me3) mark but decreased the level of the repressive histone H3 trimethyl Lys27 (H3K27me3) mark in the RTA promoter. Consistent with these results, we detected SIRT1 binding to the RTA promoter. Importantly, knockdown of SIRT1 was sufficient to increase the expression of KSHV lytic genes. Accordingly, the level of the H3K4me3 mark in the RTA promoter was increased following SIRT1 knockdown, while that of the H3K27me3 mark was decreased. Furthermore, SIRT1 interacted with RTA and inhibited RTA transactivation of its own promoter and that of its downstream target, the viral interleukin-6 gene. These results indicate that SIRT1 regulates KSHV latency by inhibiting different stages of viral lytic replication and link the cellular metabolic state with the KSHV life cycle.

Importance: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent of several malignancies, including Kaposi's sarcoma, commonly found in immunocompromised patients. While latent infection is required for the development of KSHV-induced malignancies, viral lytic replication also promotes disease progression. However, the mechanism controlling KSHV latent versus lytic replication remains unclear. In this study, we found that class III histone deacetylases (HDACs), also known as SIRTs, whose activities are linked to the cellular metabolic state, mediate KSHV replication. Inhibitors of SIRTs can reactivate KSHV from latency. SIRTs mediate KSHV latency by epigenetically silencing a key KSHV lytic replication activator, RTA. We found that one of the SIRTs, SIRT1, binds to the RTA promoter to mediate KSHV latency. Knockdown of SIRT1 is sufficient to induce epigenetic remodeling and KSHV lytic replication. SIRT1 also interacts with RTA and inhibits RTA's transactivation function, preventing the expression of its downstream genes. Our results indicate that SIRTs regulate KSHV latency by inhibiting different stages of viral lytic replication and link the cellular metabolic state with the KSHV life cycle.
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http://dx.doi.org/10.1128/JVI.00219-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4093851PMC
June 2014

Viral cyclin promotes KSHV-induced cellular transformation and tumorigenesis by overriding contact inhibition.

Cell Cycle 2014 13;13(5):845-58. Epub 2014 Jan 13.

Department of Molecular Medicine; University of Texas Health Science Center at San Antonio; San Antonio, TX USA; Department of Molecular Microbiology and Immunology; Keck School of Medicine; University of Southern California; Los Angeles, CA USA; Department of Pediatrics; University of Texas Health Science Center at San Antonio; San Antonio, TX USA.

Kaposi sarcoma-associated herpesvirus (KSHV) is a tumor virus encoding several proto-oncogenes. However, the roles of these viral genes in KSHV-induced tumorigenesis have not been defined. In this study, we used a recently developed model of KSHV-induced cellular transformation and tumorigenesis combining with a reverse genetic system to examine the role of a KSHV latent gene vCyclin (ORF72), a cellular Cyclin D2 homolog, in KSHV-induced oncogenesis. Deletion of vCyclin did not affect cell proliferation and cell cycle progression at a low-density condition, when cells were at an active proliferation state. However, vCyclin mutant cells were contact-inhibited and arrested at G 1 phase at a high-density condition. As a result, vCyclin mutant cells formed less and smaller colonies in soft agar assay. Nude mice inoculated with vCyclin mutant cells had reduced tumor incidence and extended tumor latency and survival compared with mice inoculated with wild-type (WT) virus-infected cells. WT but not mutant virus effectively induced Cyclin-dependent kinase inhibitor p27/Kip1 Ser10 phosphorylation and cytoplasmic relocalization. shRNA knockdown of p27 released the blockage of the mutant cells from cell cycle arrest at G 1 phase at a high-density condition. Together, these results indicate that vCyclin primarily functions to enhance cellular transformation and tumorigenesis by promoting cell cycle progression and cell proliferation at a contact-inhibited condition.
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http://dx.doi.org/10.4161/cc.27758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979920PMC
May 2015

p53 binding prevents phosphatase-mediated inactivation of diphosphorylated c-Jun N-terminal kinase.

J Biol Chem 2012 May 30;287(21):17554-17567. Epub 2012 Mar 30.

Departments of Biochemistry and The University of Texas Health Science Center, San Antonio, Texas 78229; Greehey Children's Cancer Research Institute, The University of Texas Health Science Center, San Antonio, Texas 78229. Electronic address:

c-Jun N-terminal kinase (JNK) is a serine/threonine phosphotransferase whose sustained activation in response to genotoxic stress promotes apoptosis. In Drosophila, the normally rapid JNK-dependent apoptotic response to genotoxic stress is significantly delayed in Dmp53 (Drosophila p53) mutants. Likewise, the extent of JNK activity after UV irradiation is dependent on p53 in murine embryonic fibroblasts with loss of p53 resulting in diminished JNK activity. Together, these results suggest that p53 potentiates the JNK-dependent response to genotoxic stress; however, the mechanism whereby p53 stimulates JNK activity remains undefined. Here, we demonstrate that both Drosophila and human p53 can directly stimulate JNK activity independently of p53-dependent gene transcription. Furthermore, we demonstrate that both the Drosophila and human p53 orthologs form a physical complex with diphosphorylated JNK ((DP)JNK) both in vivo and in vitro, suggesting that the interaction is evolutionarily conserved. Focusing on human p53, we demonstrate that the interaction maps to the DNA binding domain (hp53(DBD)). Intriguingly, binding of p53(DBD) alone to (DP)JNK prevented its inactivation by MAPK phosphatase (MKP)-5; however, JNK was still able to phosphorylate c-Jun while in a complex with the p53(DBD). Apparent dissociation constants for the p53(DBD)·(DP)JNK (274 ± 14 nm) and MKP-5·(DP)JNK (55 ± 8 nm) complexes were established; however, binding of MKP-5 and p53 to JNK was not mutually exclusive. Together, these results suggest that stress-dependent increases in p53 levels potentiate JNK activation by preventing its rapid dephosphorylation by MKPs and that the simultaneous activation of p53 and JNK may constitute a "fail-safe" switch for the JNK-dependent apoptotic response.
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http://dx.doi.org/10.1074/jbc.M111.319277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366850PMC
May 2012

Direct and efficient cellular transformation of primary rat mesenchymal precursor cells by KSHV.

J Clin Invest 2012 Mar 1;122(3):1076-81. Epub 2012 Feb 1.

Tumor Virology Program, Greehey Children’s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

Infections by viruses are associated with approximately 12% of human cancer. Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several malignancies commonly found in AIDS patients. The mechanism of KSHV-induced oncogenesis remains elusive, due in part to the lack of an adequate experimental system for cellular transformation of primary cells. Here, we report efficient infection and cellular transformation of primary rat embryonic metanephric mesenchymal precursor cells (MM cells) by KSHV. Cellular transformation occurred at as early as day 4 after infection and in nearly all infected cells. Transformed cells expressed hallmark vascular endothelial, lymphatic endothelial, and mesenchymal markers and efficiently induced tumors in nude mice. KSHV established latent infection in MM cells, and lytic induction resulted in low levels of detectable infectious virions despite robust expression of lytic genes. Most KSHV-induced tumor cells were in a latent state, although a few showed heterogeneous expression of lytic genes. This efficient system for KSHV cellular transformation of primary cells might facilitate the study of growth deregulation mechanisms resulting from KSHV infections.
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http://dx.doi.org/10.1172/JCI58530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3287217PMC
March 2012

A cluster of transcripts encoded by KSHV ORF30-33 gene locus.

Virus Genes 2012 Apr 17;44(2):225-36. Epub 2011 Dec 17.

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, People's Republic of China.

Kaposi's sarcoma-associated herpesvirus ORF30-33 locus encodes four genes with unknown functions. We performed transcriptional mapping of these genes. Northern-hybridization, 5'- and 3'-rapid amplification of cDNA ends, and DNA sequencing identified four transcripts of 3.7, 3.6, 2.7, and 1.4 kb, none of which has alternative splicing. While all transcripts have the same termination site, their start sites vary. All transcripts are not expressed or only weakly expressed in latent cells but can be chemically induced. The 3.7 and 3.6 kb transcripts contain all four genes and are sensitive to cycloheximide (CH) but resistant to phosphonoacetic acid (PAA), indicating that they are early lytic transcripts. The 2.7 kb transcript contains ORF32 and ORF33 genes while the 1.4 kb transcript contains the ORF33 gene. Both transcripts are sensitive to CH and PAA, indicating that they are late lytic transcripts. Furthermore, we identified four promoters with functional TATA boxes, none of which is directly transactivated by RTA. Examination of the 5' untranslated region of ORF31 failed to identify any functional internal ribosome entry sites. These results define the transcriptional patterns of the ORF30-33 locus, which should help the delineation of its function.
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http://dx.doi.org/10.1007/s11262-011-0698-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3290719PMC
April 2012

Masking of speech in people with first-episode schizophrenia and people with chronic schizophrenia.

Schizophr Res 2012 Jan 21;134(1):33-41. Epub 2011 Oct 21.

Department of Psychology, Speech and Hearing Research Center, Key Laboratory on Machine Perception (Ministry of Education), Peking University, Beijing 100871, China.

In "cocktail-party" environments, although listeners feel it difficult to recognize attended speech due to both energetic masking and informational masking, they can use various perceptual/cognitive cues, such as content and voice primes, to facilitate their attention to target speech. In patients with schizophrenia, both speech-perception deficits and increased vulnerability to masking stimuli generally occur. This study investigated whether speech recognition in first-episode patients (FEPs) and chronic patients (CPs) of schizophrenia is more vulnerable to noise masking and/or speech masking than that in demographics-matched-healthy controls, and whether patients with schizophrenia can use primes to unmask speech. In a trial under the priming condition, before the target sentence containing three keywords was co-presented with a noise or speech masker, the prime (early part of the sentence including the first two keywords) was recited in quiet with the target-speaker's voice. The results show that in patients, target-speech recognition was more impaired under speech-masking conditions than noise-masking conditions, and the impairment in CPs (n=22) was larger than that in FEPs (n=12). Although working memory for holding prime-content information in patients, especially CPs, was more vulnerable to masking, especially speech masking, than that in healthy controls, patients were still able to use the prime to unmask the last keyword. Thus, in "cocktail-party" environments, speech recognition in people with schizophrenia is more vulnerable to masking, particularly informational masking, and the speech-recognition impairment augments as the illness progresses. However, people with schizophrenia can use the content/voice prime to reduce energetic masking and informational masking of target speech.
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http://dx.doi.org/10.1016/j.schres.2011.09.019DOI Listing
January 2012

Reactive oxygen species hydrogen peroxide mediates Kaposi's sarcoma-associated herpesvirus reactivation from latency.

PLoS Pathog 2011 May 19;7(5):e1002054. Epub 2011 May 19.

Tumor Virology Program, Greehey Children's Cancer Research Institute, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the host following an acute infection. Reactivation from latency contributes to the development of KSHV-induced malignancies, which include Kaposi's sarcoma (KS), the most common cancer in untreated AIDS patients, primary effusion lymphoma and multicentric Castleman's disease. However, the physiological cues that trigger KSHV reactivation remain unclear. Here, we show that the reactive oxygen species (ROS) hydrogen peroxide (H₂O₂) induces KSHV reactivation from latency through both autocrine and paracrine signaling. Furthermore, KSHV spontaneous lytic replication, and KSHV reactivation from latency induced by oxidative stress, hypoxia, and proinflammatory and proangiogenic cytokines are mediated by H₂O₂. Mechanistically, H₂O₂ induction of KSHV reactivation depends on the activation of mitogen-activated protein kinase ERK1/2, JNK, and p38 pathways. Significantly, H₂O₂ scavengers N-acetyl-L-cysteine (NAC), catalase and glutathione inhibit KSHV lytic replication in culture. In a mouse model of KSHV-induced lymphoma, NAC effectively inhibits KSHV lytic replication and significantly prolongs the lifespan of the mice. These results directly relate KSHV reactivation to oxidative stress and inflammation, which are physiological hallmarks of KS patients. The discovery of this novel mechanism of KSHV reactivation indicates that antioxidants and anti-inflammation drugs could be promising preventive and therapeutic agents for effectively targeting KSHV replication and KSHV-related malignancies.
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http://dx.doi.org/10.1371/journal.ppat.1002054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098240PMC
May 2011

Cloning the simian varicella virus genome in E. coli as an infectious bacterial artificial chromosome.

Arch Virol 2011 May 13;156(5):739-46. Epub 2011 Apr 13.

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, 72205, USA.

Simian varicella virus (SVV) is closely related to human varicella-zoster virus and causes varicella and zoster-like disease in nonhuman primates. In this study, a mini-F replicon was inserted into a SVV cosmid, and infectious SVV was generated by co-transfection of Vero cells with overlapping SVV cosmids. The entire SVV genome, cloned as a bacterial artificial chromosome (BAC), was stably propagated upon serial passage in E. coli. Transfection of pSVV-BAC DNA into Vero cells yielded infectious SVV (rSVV-BAC). The mini-F vector sequences flanked by loxP sites were removed by co-infection of Vero cells with rSVV-BAC and adenovirus expressing Cre-recombinase. Recombinant SVV generated using the SVV-BAC genetic system has similar molecular and in vitro replication properties as wild-type SVV. To demonstrate the utility of this approach, a SVV ORF 10 deletion mutant was created using two-step Red-mediated recombination. The results indicate that SVV ORF 10, which encodes a homolog of the HSV-1 virion VP-16 transactivator protein, is not essential for in vitro replication but is required for optimal replication in cell culture.
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http://dx.doi.org/10.1007/s00705-010-0889-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3269341PMC
May 2011

Simian varicella virus open reading frame 63/70 expression is required for efficient virus replication in culture.

J Neurovirol 2011 Jun 9;17(3):274-80. Epub 2011 Apr 9.

Department of Neurology, University of Colorado School of Medicine, 12700 E. 19th Avenue, Box B182, Aurora, CO 80045, USA.

Simian varicella virus (SVV) open reading frame (ORF) 63, duplicated in the virus genome as ORF 70, is homologous to varicella zoster virus ORF 63/70. Transfection of bacterial artificial chromosome clones containing the wild-type SVV genome and mutants with stop codons in ORF 70, in both ORFs 63 and 70 and the repaired virus DNA sequences into Vero cells produced a cytopathic effect (CPE). The onset of CPE was much slower with the double-mutant transfectants (10 days vs. 3 days) and plaques were smaller. While SVV ORF 63 is not required for replication in culture, its expression leads to robust virus replication.
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http://dx.doi.org/10.1007/s13365-011-0025-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3204661PMC
June 2011

Recent advances in cloning herpesviral genomes as infectious bacterial artificial chromosomes.

Cell Cycle 2011 Feb 1;10(3):434-40. Epub 2011 Feb 1.

Tumor virology Program, Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

Herpesviruses are common but important pathogens in humans and animals. These viruses have large complex genomes encoding genes with diverse functions in different phases of their life cycle and associated diseases. In the last decade, genomes of herpesviruses cloned as infectious bacterial artificial chromosomes (BACs) have become powerful tools for delineating the functions of viral genes and understanding the pathogenesis of their associated diseases. Here we review the history of herpesviral genetics and recent advances in methods for cloning herpesviral genomes as infectious BACs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3115017PMC
http://dx.doi.org/10.4161/cc.10.3.14708DOI Listing
February 2011

Rhesus rhadinovirus infection of rhesus fibroblasts occurs through clathrin-mediated endocytosis.

J Virol 2010 Nov 8;84(22):11709-17. Epub 2010 Sep 8.

The University of Texas Health Science Center, San Antonio, TX 78229, USA.

Rhesus rhadinovirus (RRV) is a gammaherpesvirus closely related to Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus linked to the development of Kaposi's sarcoma and several other lymphoproliferative diseases, including primary effusion lymphoma and multicentric Castleman's disease. RRV naturally infects rhesus macaques and induces lymphoproliferative diseases under experimental conditions, making it an excellent model for the study of KSHV. Unlike KSHV, which grows poorly in cell culture, RRV replicates efficiently in rhesus fibroblasts (RFs). In this study, we have characterized the entry pathway of RRV in RFs. Using a luciferase-expressing recombinant RRV (RRV-luciferase), we show that the infectivity of RRV is reduced by inhibitors of endosomal acidification. RRV infectivity is also reduced by inhibitors of clathrin-mediated but not caveola-mediated endocytosis, indicating that RRV enters into RFs via clathrin-mediated endocytosis. Using a red fluorescent protein (RFP)-expressing recombinant RRV (RRV-RFP), we show that RRV particles are colocalized with markers of endocytosis (early endosome antigen 1) and clathrin-mediated endocytosis (clathrin heavy chain) during entry into RFs. RRV particles are also colocalized with transferrin, which enters cells by clathrin-mediated endocytosis, but not with cholera toxin B, which enters cells by caveola-mediated endocytosis. Inhibition of clathrin-mediated endocytosis with a dominant-negative construct of EPS15, an essential component of clathrin-coated pits, blocked the entry of RRV into RFs. Together, these results indicate that RRV entry into RFs is mediated by clathrin-mediated endocytosis.
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http://dx.doi.org/10.1128/JVI.01429-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2977882PMC
November 2010

Autoexcision of bacterial artificial chromosome facilitated by terminal repeat-mediated homologous recombination: a novel approach for generating traceless genetic mutants of herpesviruses.

J Virol 2010 Mar 13;84(6):2871-80. Epub 2010 Jan 13.

Tumor Virology Program, Greehey Children's Cancer Research Institute, Departments of Pediatrics, Microbiology and Immunology, Molecular Medicine, and Medicine, and Cancer Therapy and Research Center, the University of Texas Health Science Center, San Antonio, Texas 78229, USA.

Infectious bacterial artificial chromosomes (BACs) of herpesviruses are powerful tools for genetic manipulation. However, the presence of BAC vector sequence in the viral genomes often causes genetic and phenotypic alterations. While the excision of the BAC vector cassette can be achieved by homologous recombination between extra duplicate viral sequences or loxP site-mediated recombination, these methods either are inefficient or leave a loxP site mark in the viral genome. Here we describe the use of viral intrinsic repeat sequences, which are commonly present in herpesviral genomes, to excise the BAC vector cassette. Using a newly developed in vitro transposon-based cloning approach, we obtained an infectious BAC of rhesus rhadinovirus (RRV) strain RRV26-95 with the BAC vector cassette inserted in the terminal repeat (TR) region. We showed that the BAC vector cassette was rapidly excised upon reconstitution in cells predominantly through TR-mediated homologous recombination. Genetic and phenotypic analysis showed that the BAC-excised virus was reversed to wild-type RRV. Using this autoexcisable BAC clone, we successfully generated an RRV mutant with a deletion of Orf50, which encodes a replication and transcription activator (RTA) protein. Together, these results illustrate the usefulness of TR for genetic manipulation of herpesviruses when combined with the novel transposon-based cloning approach.
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http://dx.doi.org/10.1128/JVI.01734-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2826046PMC
March 2010

A sequence-independent in vitro transposon-based strategy for efficient cloning of genomes of large DNA viruses as bacterial artificial chromosomes.

Nucleic Acids Res 2009 Jan 6;37(1):e2. Epub 2008 Nov 6.

Greehey Children's Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78229, USA.

Bacterial artificial chromosomes (BACs) derived from genomes of large DNA viruses are powerful tools for functional delineation of viral genes. Current methods for cloning the genomes of large DNA viruses as BACs require prior knowledge of the viral sequences or the cloning of viral DNA fragments, and are tedious because of the laborious process of multiple plaque purifications, which is not feasible for some fastidious viruses. Here, we describe a novel method for cloning the genomes of large DNA viruses as BACs, which entails direct in vitro transposition of viral genomes with a BAC cassette, and subsequent recovery in Escherichia coli. Determination of insertion sites and adjacent viral sequences identify the BAC clones for genetic manipulation and functional characterization. Compared to existing methods, this new approach is highly efficient, and does not require any information on viral sequences or cloning of viral DNA fragments, and plaque purifications. This method could potentially be used for discovering previously unidentified viruses.
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http://dx.doi.org/10.1093/nar/gkn890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615602PMC
January 2009

Genetic disruption of KSHV major latent nuclear antigen LANA enhances viral lytic transcriptional program.

Virology 2008 Sep 5;379(2):234-44. Epub 2008 Aug 5.

Tumor Virology Program, Greehey Children's Cancer Research Institute, Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, 8403 Floyd Curl Drive, San Antonio, TX 78229, USA.

Following primary infection, KSHV establishes a lifelong persistent latent infection in the host. The mechanism of KSHV latency is not fully understood. The latent nuclear antigen (LANA or LNA) encoded by ORF73 is one of a few viral genes expressed during KSHV latency, and is consistently detected in all KSHV-related malignancies. LANA is essential for KSHV episome persistence, and regulates the expression of viral lytic genes through epigenetic silencing, and inhibition of the expression and transactivation function of the key KSHV lytic replication initiator RTA (ORF50). In this study, we used a genetic approach to examine the role of LANA in regulating KSHV lytic replication program. Deletion of LANA did not affect the expression of its adjacent genes vCyclin (ORF72) and vFLIP (ORF71). In contrast, the expression levels of viral lytic genes including immediate-early gene RTA, early genes MTA (ORF57), vIL-6 (ORF-K2) and ORF59, and late gene ORF-K8.1 were increased before and after viral lytic induction with 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. This enhanced expression of viral lytic genes was also observed following overexpression of RTA with or without simultaneous chemical induction. Consistent with these results, the LANA mutant cells produced more infectious virions than the wild-type virus cells did. Furthermore, genetic repair of the mutant virus reverted the phenotypes to those of wild-type virus. Together, these results have demonstrated that, in the context of viral genome, LANA contributes to KSHV latency by regulating the expression of RTA and its downstream genes.
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http://dx.doi.org/10.1016/j.virol.2008.06.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2626151PMC
September 2008

Molecular biology of KSHV in relation to AIDS-associated oncogenesis.

Cancer Treat Res 2007 ;133:69-127

Tiumor Virology Program, Children's Cancer Research Institute, Department of Pediatrics, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

KSHV has been established as the causative agent of KS, PEL, and MCD, malignancies occurring more frequently in AIDS patients. The aggressive nature of KSHV in the context of HIV infection suggests that interactions between the two viruses enhance pathogenesis. KSHV latent infection and lytic reactivation are characterized by distinct gene expression profiles, and both latency and lytic reactivation seem to be required for malignant progression. As a sophisticated oncogenic virus, KSHV has evolved to possess a formidable repertoire of potent mechanisms that enable it to target and manipulate host cell pathways, leading to increased cell proliferation, increased cell survival, dysregulated angiogenesis, evasion of immunity, and malignant progression in the immunocompromised host. Worldwide, approximately 40.3 million people are currently living with HIV infection. Of these, a significant number are coinfected with KSHV. The complex interplay between the two viruses dramatically elevates the risk for development of KSHV-induced malignancies, KS, PEL, and MCD. Although HAART significantly reduces HIV viral load, the entire T-cell repertoire and immune function may not be completely restored. In fact, clinically significant immune deficiency is not necessary for the induction of KSHV-related malignancy. Because of variables such as lack of access to therapy noncompliance with prescribed treatment, failure to respond to treatment and the development of drug-resistant strains of HIV, KSHV-induced malignancies will continue to present as major health concerns.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798888PMC
http://dx.doi.org/10.1007/978-0-387-46816-7_3DOI Listing
September 2007

Functional characterization of Kaposi's sarcoma-associated herpesvirus ORF45 by bacterial artificial chromosome-based mutagenesis.

J Virol 2006 Dec 11;80(24):12187-96. Epub 2006 Oct 11.

Department of Microbiology, School of Dental Medicine, University of Pennsylvania, 240 S. 40th Street, Philadelphia, PA 19104, USA.

Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an immediate-early protein. This protein is also present in virions as a tegument protein. ORF45 protein interacts with interferon regulatory factor 7 (IRF-7) and inhibits virus-induced type I interferon production by blocking activation of IRF-7. To define further the function of ORF45 and the mechanism underlying its action, we constructed an ORF45-null recombinant virus genome (BAC-stop45) by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BAC-stop45 genomes were generated. When monolayers of 293T BAC36 and 293T BAC-stop45 cells were induced with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate, no significant difference was found between them in overall viral gene expression and lytic DNA replication, but induced 293T BAC-stop45 cells released 10-fold fewer virions to the medium than did 293T BAC36 cells. When ORF45-null virus was used to infect cells, lower infectivity was observed than for wild-type BAC36. These results suggest that KSHV ORF45 plays roles in both early and late stages of viral infection, probably in viral ingress and egress.
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http://dx.doi.org/10.1128/JVI.01275-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1676278PMC
December 2006

Kaposi's sarcoma-associated herpesvirus induction of chromosome instability in primary human endothelial cells.

Cancer Res 2004 Jun;64(12):4064-8

Tumor Virology Program, Children Cancer Research Institute and Department of Pediatrics, The University of Texas Health Science Center at San Antonio, 78229, USA.

Chromosome instability contributes to the multistep oncogenesis of cancer cells. Kaposi's sarcoma (KS), an angiogenic vascular spindle cancer of endothelial cells, displays stage advancement with lesions at early stage being hyperproliferative, whereas lesions at late stage are clonal or multiclonal and can exhibit a neoplastic nature and chromosome instability. Although infection with KS-associated herpesvirus (KSHV) has been associated with the initiation and promotion of KS, the mechanism of KS neoplastic transformation remains unclear. We show that KSHV infection of primary human umbilical vein endothelial cells induces abnormal mitotic spindles and centrosome duplication. As a result, KSHV-infected cells manifest chromosome instability, including chromosomal misalignments and laggings, mitotic bridges, and formation of micronuclei and multinucleation. Our results indicate that KSHV infection could predispose cells to malignant transformation through induction of genomic instability and contributes to the development of KS.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-0657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5257260PMC
June 2004

Kaposi's sarcoma-associated herpesvirus glycoprotein K8.1 is dispensable for virus entry.

J Virol 2004 Jun;78(12):6389-98

BIOMMED, School of Veterinary Medicine, Louisiana State University, Skip Bertman Drive, Baton Rouge, LA 70803, USA.

Kaposi's sarcoma-associated herpesvirus (KSHV) is considered the etiologic agent of Kaposi's sarcoma and several lymphoproliferative disorders. Recently, the KSHV genome was cloned into a bacterial artificial chromosome and used to construct a recombinant KSHV carrying a deletion of the viral interferon regulatory factor gene (F. C. Zhou, Y. J. Zhang, J. H. Deng, X. P. Wang, H. Y. Pan, E. Hettler, and S. J. Gao, J. Virol. 76:6185-6196, 2002). The K8.1 glycoprotein is a structural component of the KSHV particle and is thought to facilitate virus entry by binding to heparan sulfate moieties on cell surfaces. To further address the role of K8.1 in virus infectivity, a K8.1-null recombinant virus (BAC36DeltaK8.1) was constructed by deletion of most of the K8.1 open reading frame and insertion of a kanamycin resistance gene cassette within the K8.1 gene. Southern blotting and diagnostic PCR confirmed the presence of the engineered K8.1 gene deletion. Transfection of the wild-type genome (BAC36) and mutant genome (BAC36DeltaK8.1) DNAs into 293 cells in the presence or absence of the complementing plasmid (pCDNAK8.1A), transiently expressing the K8.1A gene, produced infectious virions in the supernatants of transfected cells. These results demonstrated that the K8.1 glycoprotein is not required for KSHV entry into 293 cells.
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http://dx.doi.org/10.1128/JVI.78.12.6389-6398.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC416545PMC
June 2004

[Construction of pseudorabies virus Ea TK-/gE-/gp63- mutant strain and the study on its biological property].

Wei Sheng Wu Xue Bao 2002 Jun;42(3):370-4

Key Laboratory of Agricultural Microbiology, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China.

Using pseudorabies virus Ea strain as material, we inserted LacZ gene expression cassette into gE gene. After blue plaque and plaque purification, a recombinant virus PRVEa TK-/gE-/LacZ+ generated. Utilizing EcoR I site in LacZ gene, digested PRVEa TK-/gE-/LacZ+ genome DNA was cotransfected into PK-15 cells with plasmid pFBBS, then PRVEa TK-/gE-/gp63- generated after plaque purification. Four pairs of primers amplification demonstrated the virus was pure TK-/gE-/gp63- mutant virus. PCR product sequence indicates there were 205 bp deletion in TK gene; 1247 bp deletion in gE,gp63 and intergenic region of PRVEa TK-/gE-/gp63- mutant virus genome DNA. Inoculation to Balb/C mice with PRVEa TK-/gE-/gp63- indicates the virulence is reduced greatly.
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June 2002

Protection induced by intramuscular immunization with DNA vaccines of pseudorabies in mice, rabbits and piglets.

Vaccine 2002 Jan;20(7-8):1205-14

Laboratory of Animal Virology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Hubei Province 430070, Wuhan, China.

Glycoprotein gene gB, gC and gD of pseudorabies virus (PrV) strain Ea, which was isolated locally in Wuhan, were cloned from the viral genome DNA and expressed in vitro controlled by the major immediately-early promotor/enhancer of HCMV. In the presented paper, Balb/c mice, rabbits and piglets were vaccinated intramuscularly two times at 2-week interval with those eukaryotic expression plasmid pcDB, pcDC and pcDD, respectively. The animals injected with pcDB, pcDC, pcDD or mix DNA developed anti-PrV antibodies. Neutralizing antibody titers obtained 2-5log(2), 2 weeks after the second vaccination. Cellular immune responses were also detected by lymphoproliferation assay and cytotoxic T lymphocyte (CTL) activity assay in all groups vaccinated with DNA. Immune responses elicited by DNA vaccines provided protections with different degrees against lethal dose PrV challenge. In mice, protections induced by pcDC, pcDD or mix DNA were 100%, similar to that by inactivated vaccine. Protections were more than 50% induced by pcDC, pcDD or mix DNA in rabbits. Protections induced by pcDB were the lowest among DNA immunization in mice or rabbits. However, pcDB could elicit the higher cellular responses in rabbits or piglets. In piglets, body temperatures of animals injected with pcDB, pcDC, pcDD or mix DNA did not change significantly after challenge with 2x10(5) pfu of PrV strain Ea, and the means daily growth post-challenge of those animals were higher than those injected with inactivated vaccine or parental plasmid. Neither DNA vaccines nor inactivated vaccine could prevent or delay virus excretion after challenge. Our experiments in experimental animals and natural hosts suggested the efficiency and potential application of DNA vaccines for pseudorabies in pigs.
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http://dx.doi.org/10.1016/s0264-410x(01)00416-9DOI Listing
January 2002