Leming Tong

University of California, Los Angeles, Los Angeles, CA, United States

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Publications (16)68.77 Total impact

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    ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infecting two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the function of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early, early gene expression, or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA-sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that co-regulates the viral late genes as a group.
    Journal of Virology 07/2014; · 5.08 Impact Factor
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    ABSTRACT: Gammaherpesviruses such as KSHV and EBV establish lifelong persistent infections through latency in lymphocytes. These viruses have evolved several strategies to counteract the various components of the innate and adaptive immune systems. We conducted an unbiased screen using the genetically and biologically related virus, MHV-68, to find viral ORFs involved in the inhibition of type I interferon signaling and identified a conserved viral dUTPase, ORF54. Here we define the contribution of ORF54 in type I interferon inhibition by ectopic expression and through the use of genetically modified MHV-68. ORF54 and an ORF54 lacking dUTPase enzymatic activity efficiently inhibit type I interferon signaling by inducing the degradation of the type I interferon receptor protein IFNAR1. Subsequently, we show in vitro that the lack of ORF54 causes a reduction in lytic replication in the presence of type I interferon signaling. Investigation of the physiological consequence of IFNAR1 degradation and importance of ORF54 during MHV-68 in vivo infection demonstrates that ORF54 has an even greater impact on persistent infection than on lytic replication. MHV-68 lacking ORF54 expression is unable to efficiently establish latent infection in lymphocytes, although it replicates relatively normally in lung tissues. However, infection of IFNAR-/- mice alleviates this phenotype, emphasizing the specific role of ORF54 in type I interferon inhibition. Infection of mice and cells by a recombinant MHV-68 virus harboring a site specific mutation in ORF54 rendering the dUTPase inactive demonstrates that dUTPase enzymatic activity is not required for anti-interferon function of ORF54. Moreover, we find that dUTPase activity is dispensable at all stages of MHV-68 infection analyzed. Overall, our data suggest that ORF54 has evolved anti-interferon activity in addition to its dUTPase enzymatic activity, and that it is actually the anti-interferon role that renders ORF54 critical for establishing an effective persistent infection of MHV-68.
    PLoS Pathogens 10/2011; 7(10):e1002292. · 8.14 Impact Factor
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    ABSTRACT: Here we describe the cloning of a sequenced WUMS isolate of murine gammaherpesvirus-68 (MHV-68, ╬│HV-68, also known as MuHV-4) as a bacterial artificial chromosome (BAC). We engineered the insertion of the BAC sequence flanked by loxP sites into the left end of the viral genome before the M1 open reading frame. The infectious viruses were reconstituted following transfection of the MHV-68 BAC DNA into cells. The MHV-68 BAC-derived virus replicated indistinguishably from the wild-type virus in cultured cells. Excision of the BAC insert was efficiently achieved by coexpressing the Cre recombinase. Although the BAC insertion did not significantly affect acute productive infection in the lung, it severely compromised the ability of MHV-68 to establish splenic latency. Removal of the BAC sequence restored the wild-type level of latency. Site-specific mutagenesis was carried out by RecA-mediated recombination to demonstrate that this infectious BAC clone can be used for genetic studies of MHV-68.
    BioMed Research International 01/2011; 2011:926258. · 2.88 Impact Factor
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    ABSTRACT: Human gammaherpesviruses, Epstein-Barr virus, and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus are important pathogens associated with diseases, including lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) is used as an experimental model system to study the host immune control of infection and explore novel vaccine strategies based on latency-deficient live viruses. We studied the properties and the potential of a recombinant MHV-68 (AC-RTA) in which the genes required for persistent infection were replaced by a constitutively expressed viral transcription activator, RTA, which dictates the virus to lytic replication. After intranasal infection of mice, replication of AC-RTA in the lung was attenuated, and no AC-RTA virus or viral DNA was detected in the isolated splenocytes, indicating a lack of latency in the spleen. Infection of the AC-RTA virus elicited both cellular immune responses and virus-specific IgG at a level comparable to that elicited by infection of the wild-type virus. Importantly, vaccination of AC-RTA was able to protect mice against subsequent challenge by the wild-type MHV-68. AC-RTA provides a vaccine strategy for preventing infection of human gammaherpesviruses. Furthermore, our results suggest that immunity to the major latent antigens is not required for protection.
    Journal of Virology 12/2009; 84(5):2453-65. · 5.08 Impact Factor
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    ABSTRACT: A conserved herpesviral kinase, designated ORF36 in murine gamma-herpesvirus 68 (MHV-68), plays multiple vital roles in the viral life cycle. Here, we show by screening mutant viruses that ORF36 counteracts the antiviral type I interferon (IFN) response. ORF36 specifically binds to the activated form of interferon regulatory factor 3 (IRF-3) in the nucleus, inhibiting IRF-3 interaction with the cotranscriptional activator CBP and thereby suppressing the recruitment of RNA polymerase II to the interferon beta promoter. The anti-IFN function of ORF36 is conserved among herpesvirus subfamilies, although the conserved kinase activity is not absolutely required for this function. MHV-68 lacking ORF36 induces a greater interferon response and is attenuated in vitro and in vivo, where acute viral infection in the lung and latency in the spleen are compromised. Our data suggest that herpesviruses have evolved within their conserved kinase an anti-IFN activity critical for evasion of host immunity and for persistence.
    Cell host & microbe 03/2009; 5(2):166-78. · 13.02 Impact Factor
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    ABSTRACT: A hallmark of productive infection by DNA viruses is the coupling of viral late gene expression to genome replication. Here we report the identification of open reading frame 30 (ORF30) and ORF34 as viral trans factors crucial for activating late gene transcription following viral DNA replication during lytic infection of murine gammaherpesvirus 68 (MHV-68). The mutant virus lacking either ORF30 or ORF34 underwent normal DNA replication but failed to express viral late gene transcripts, leading to nonproductive infection. In a reporter assay system, ORF30 and ORF34 were required for MHV-68 to activate the viral late gene promoters. Furthermore, studies using chromatin immunoprecipitation assays showed that the recruitment of RNA polymerase II to the viral late promoters during lytic infection was significantly reduced in the absence of ORF30 or ORF34. Together, the results suggest that ORF30 and ORF34 may play an important role in the assembly of the transcription initiation complex at the late gene promoters. Our discovery of the viral mutants that uncouple late gene transcription from DNA replication lays an important foundation to dissect the mechanism of this critical step of gene expression regulation.
    Journal of Virology 01/2009; 83(5):2265-73. · 5.08 Impact Factor
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    ABSTRACT: Gammaherpesviruses establish life-long persistency inside the host and cause various diseases during their persistent infection. However, the systemic interaction between the virus and host in vivo has not been studied in individual hosts continuously, although such information can be crucial to control the persistent infection of the gammaherpesviruses. For the noninvasive and continuous monitoring of the interaction between gammaherpesvirus and the host, a recombinant murine gammaherpesvirus 68 (MHV-68, a gammaherpesvirus 68) was constructed to express a firefly luciferase gene driven by the viral M3 promoter (M3FL). Real-time monitoring of M3FL infection revealed novel sites of viral replication, such as salivary glands, as well as acute replication in the nose and the lung and progression to the spleen. Continuous monitoring of M3FL infection in individual mice demonstrated the various kinetics of transition to different organs and local clearance, rather than systemically synchronized clearance. Moreover, in vivo spontaneous reactivation of M3FL from latency was detected after the initial clearance of acute infection and can be induced upon treatment with either a proteasome inhibitor Velcade or an immunosuppressant cyclosporine A. Taken together, our results demonstrate that the in vivo replication and reactivation of gammaherpesvirus are dynamically controlled by the locally defined interaction between the virus and the host immune system and that bioluminescence imaging can be successfully used for the real-time monitoring of this dynamic interaction of MHV-68 with its host in vivo.
    Journal of Virology 11/2008; 82(24):12498-509. · 5.08 Impact Factor
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    ABSTRACT: Kaposi's sarcoma-associated herpesvirus and murine gammaherpesvirus-68 (MHV-68) establish latent infections and are associated with various types of malignancies. They are members of the gamma-2 herpesvirus subfamily and encode a replication and transcriptional activator, RTA, which is necessary and sufficient to disrupt latency and initiate the viral lytic cycle in vitro. We have constructed a recombinant MHV-68 virus that overexpresses RTA. This virus has faster replication kinetics in vitro and in vivo, is deficient in establishing latency, exhibits a reduction in the development of a mononucleosis-like disease in mice, and can protect mice against challenge by wild-type MHV-68. The present study, by using MHV-68 as an in vivo model system, demonstrated that RTA plays a critical role in the control of viral latency and suggests that latency is a determinant of viral pathogenesis in vivo.
    Journal of Virology 10/2004; 78(17):9215-23. · 5.08 Impact Factor
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    ABSTRACT: Murine gammaherpesvirus 68 (MHV-68 [also referred to as gammaHV68]) is phylogenetically related to Kaposi's sarcoma-associated herpesvirus (KSHV [also referred to as HHV-8]) and Epstein-Barr virus (EBV). However, unlike KSHV or EBV, MHV-68 readily infects fibroblast and epithelial cell lines derived from several mammalian species, providing a system to study productive and latent infections as well as reactivation of gammaherpesviruses in vivo and in vitro. To carry out rapid genome-wide analysis of MHV-68 gene expression, we made DNA arrays containing nearly all of the known and predicted open reading frames (ORFs) of the virus. RNA obtained from an MHV-68 latently infected cell line, from cells lytically infected with MHV-68 in culture, and from the lung tissue of infected mice was used to probe the MHV-68 arrays. Using a tightly latent B-cell line (S11E), the MHV-68 latent transcription program was quantitatively described. Using BHK-21 cells and infected mice, we demonstrated that latent genes are transcribed during lytic replication and are relatively independent of de novo protein synthesis. We determined that the transcription profiles at the peak of lytic gene expression are similar in cultured fibroblast and in the lung of infected mice. Finally, the MHV-68 DNA arrays were used to examine the gene expression profile of a recombinant virus that overexpresses replication and transcription activator (RTA), C-RTA/MHV-68, during lytic replication in cell culture. The recombinant virus replicates faster then the parental strain and the DNA arrays revealed that nearly every MHV-68 ORF examined was activated by RTA overexpression. Examination of the gene expression patterns of C-RTA/MHV-68 over a time course led to the finding that the M3 promoter is RTA responsive in the absence of other viral factors.
    Journal of Virology 11/2003; 77(19):10488-503. · 5.08 Impact Factor
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    ABSTRACT: Rta, encoded primarily by open reading frame 50, is well conserved among gammaherpesviruses. It has been shown that the Rta proteins of Epstein Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV-68; also referred to as gamma HV68) play an important role in viral reactivation from latency. However, the role of Rta during productive de novo infection has not been characterized in gammaherpesviruses. Since there are cell lines that can support efficient productive de novo infection by MHV-68 but not EBV or KSHV, we examined whether MHV-68 Rta plays a role in initiating viral lytic replication in productively infected cells. Rta, functioning as a transcriptional activator, can activate the viral promoter of early lytic genes. The amino acid sequence alignments of the Rta homologues suggest that the organizations of their functional domains are similar, with the DNA binding and dimerization domains at the N terminus and the trans-activation domain at the C terminus. We constructed two mutants of MHV-68 Rta, Rd1 and Rd2, with deletions of 112 and 243 amino acids from the C terminus, respectively. Rd1 and Rd2 could no longer trans-activate the promoter of MHV-68 gene 57, consistent with the deletions of their trans-activation domains at the C terminus. Furthermore, Rd1 and Rd2 were able to function as dominant-negative mutants, inhibiting trans-activation of wild-type Rta. To study whether Rd1 and Rd2 blocked viral lytic replication, purified virion DNA was cotransfected with Rd1 or Rd2 into fibroblasts. Expression of viral lytic proteins was greatly suppressed, and the yield of infectious viruses was reduced up to 10(4)-fold. Stable cell lines constitutively expressing Rd2 were established and infected with MHV-68. Transcription of the immediate-early gene, rta, and the early gene, tk, of the virus was reduced in these cell lines. The presence of Rd2 also led to attenuation of viral lytic protein expression and virion production. The ability of Rta dominant-negative mutants to inhibit productive infection suggests that the trans-activation function of Rta is essential for MHV-68 lytic replication. We propose that a single viral protein, Rta, governs the initiation of MHV-68 lytic replication during both reactivation and productive de novo infection.
    Journal of Virology 11/2001; 75(19):9262-73. · 5.08 Impact Factor
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    ABSTRACT: Myxococcus xanthus is a gram-negative soil bacterium that initiates a complex developmental program in response to starvation. A transposon insertion (Tn5-lac omega109) mutant with developmental deficiencies was isolated and characterized in this study. A strain containing this insertion mutation in an otherwise wild-type background showed delayed developmental aggregation for about 12 h and sporulated at 1-2% of the wild-type level. Tn5-lac omega109 was found to have disrupted the M. xanthus wbgB gene, which is located 2.1 kb downstream of the M. xanthus lipopolysacharide (LPS) O-antigen biosynthesis genes wzm wzt wbgA. The deduced polypeptide sequence of WbgB shares significant similarity with bacterial glycosyltransferases including M. xanthus WbgA. The wbgB::Tn5-lac omega109 mutant was found to be defective in LPS O-antigen synthesis by immunochemical analysis. Further mutational analysis indicated that the defects of the wbgB::Tn5-lac omega109 mutant were not the result of polar effects on downstream genes. Various motility assays demonstrated that the Tn5-lac omega109 mutation affected both social (S) and adventurous (A) gliding motility of M. xanthus cells. The pleiotrophic effects of wbgB mutations indicate the importance of LPS O-antigen biosynthesis for various cellular functions in M. xanthus.
    Archives of Microbiology 01/2001; 174(6):399-405. · 1.91 Impact Factor
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    ABSTRACT: Myxococcus xanthus is a Gram-negative gliding bacterium that aggregates and develops into multicellular fruiting bodies in response to starvation. Two chemosensory systems (frz and dif), both of which are homologous to known chemotaxis proteins, were previously identified through characterization of various developmental mutants. This study aims to examine the interaction between these two systems since both of them are required for fruiting body formation of M. xanthus. Through detailed phenotypic analyses of frz and dif double mutants, we found that both frz and dif are involved in cellular reversal and social motility; however, the frz genes are epistatic in controlling cellular reversal, whereas the dif genes are epistatic in controlling social motility. The study suggests that the integration of these two chemotaxis systems may play a central role in controlling the complicated social behaviors of M. xanthus.
    FEMS Microbiology Letters 12/2000; 192(2):211-5. · 2.05 Impact Factor
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    ABSTRACT: Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M. xanthus S motility. We have demonstrated here that M. xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacterium Pseudomonas aeruginosa, the M. xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the dif genes are linked to the M. xanthus dsp region, a locus known to be crucial for M. xanthus fibril biogenesis and S gliding.
    Journal of Bacteriology 11/2000; 182(20):5793-8. · 3.19 Impact Factor
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    ABSTRACT: Myxococcus xanthus is a Gram-negative gliding bacterium that aggregates and develops into multicellular fruiting bodies in response to starvation. Two chemosensory systems (frz and dif), both of which are homologous to known chemotaxis proteins, were previously identified through characterization of various developmental mutants. This study aims to examine the interaction between these two systems since both of them are required for fruiting body formation of M. xanthus. Through detailed phenotypic analyses of frz and dif double mutants, we found that both frz and dif are involved in cellular reversal and social motility; however, the frz genes are epistatic in controlling cellular reversal, whereas the dif genes are epistatic in controlling social motility. The study suggests that the integration of these two chemotaxis systems may play a central role in controlling the complicated social behaviors of M. xanthus.
    FEMS Microbiology Letters 10/2000; 192(2):211 - 215. · 2.05 Impact Factor
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    Infectious Agents and Cancer 7(1).
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    ABSTRACT: All cells were propagated in Dulbeco's Modified Eagle Medium containing antibiotics and 10% serum (Fetal Bovine Serum for 293T, Vero and MEF cells; Bovine Calf Serum for NIH3T3 and NIH3T12 cells). MEF cells with passage 5~6 were used to investigate the growth property of viruses by multistep growth curve. NIH3T12 cells were used to propagate the wild-type MHV-68 and its mutant derivatives for in vitro and in vivo study. In addition, it was also used for chromatin immunoprecipitation assay after viral infection. Vero cells were used to titer infectious viruses and latent load by plaque forming assay and infectious center assay, respectively. 293T cells were mainly used to transfect plasmid DNA for dual luciferase assay, immunofluorescence, co-immunoprecipitation, and chromatin immunoprecipitation assay. NIH3T3 cells were mainly used to transfect plasmid DNA for dual luciferase assay, ELISA, and IFN transcripts quantitation. The stable cell lines constitutively expressing IRF-3(dDBD) or IRF-3(5D) were generated by transfecting linearized plasmids expressing pEBB-IRF-3(dDBD)/pHB-IRF-3-C227(5D) or pHB- IRF-3-F(5D) with the linearized pBabe-puro into 293T or NIH3T3. The transfected cells were selected for by the puromycin-resistance (1 ug/ml for 293T; 6 ug/ml for NIH3T3), and the stable expression of the transfected genes were confirmed by immunoblotting.

Publication Stats

389 Citations
68.77 Total Impact Points

Institutions

  • 2000–2011
    • University of California, Los Angeles
      • • Department of Medicine
      • • Department of Molecular and Medical Pharmacology
      • • Molecular Biology Institute
      Los Angeles, CA, United States
    • University of Texas Medical School
      • Department of Microbiology and Molecular Genetics
      Houston, Texas, United States
  • 2001
    • Washington University in St. Louis
      San Luis, Missouri, United States