Generation of a Latency-Deficient Gammaherpesvirus That Is Protective against Secondary Infection

Rickabaugh, Tammy M.; Brown, Helen; Martinez-Guzman, DeeAnn; Wu, Ting-Ting; Tong, Lili; Yu, Fuqu; Cole, Steven W.; Sun, Ren

doi:10.1128/jvi.78.17.9215-9223.2004

Cited by 38 publications

(44 citation statements)

References 31 publications

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“…These late viral proteins were absent from BHK-21 cells infected with 30S or 34S. We also examined the expression of two well characterized late genes, ORF26 and ORF65 (27,29,(33)(34)(35), which encode viral capsid proteins and were readily detected in cells infected with the WT virus (Fig. 3B).…”

Section: Generation Of Null Mutants Of Orf30 and Orf34mentioning

confidence: 99%

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ORF30 and ORF34 Are Essential for Expression of Late Genes in Murine Gammaherpesvirus 68

Park²,

Kim³

et al. 2009

J Virol

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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.

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Section: Generation Of Null Mutants Of Orf30 and Orf34mentioning

confidence: 99%

“…Total DNA was isolated from cells and analyzed by Southern blot as described previously (39). The viral copy numbers were determined by quantitative real-time PCR using primers for the ORF65 (M9) gene (29). Western blots were used for detecting viral proteins with rabbit polyclonal antibodies against ORF65, ORF26, or MHV-68-infected rabbit cells.…”

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ORF30 and ORF34 Are Essential for Expression of Late Genes in Murine Gammaherpesvirus 68

Park²,

Kim³

et al. 2009

J Virol

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“…Real-time PCR, using QIAGEN Quantitect SYBR Green PCR master mix and primers against MHV-68 M1, was performed on 40 ng of DNA per sample. The samples were quantitated as previously described (26). Only the M/RTA and M-H/RTA constructs were able to produce an increase in viral genome copy number in comparison to results with the BAC transfected with pFLAG-CMV alone (Fig.…”

Section: Fig 2 Transfection Of Homologous Rta Proteins Into Mhv-68-mentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 RTA Reactivates Murine Gammaherpesvirus 68 from Latency

Rickabaugh

Brown²,

Wu³

et al. 2005

J Virol

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Murine gammaherpesvirus 68 (MHV-68), Kaposi's sarcoma-associated herpesvirus (HHV-8), and EpsteinBarr virus (EBV) are all members of the gammaherpesvirus family, characterized by their ability to establish latency in lymphocytes. The RTA protein, conserved in all gammaherpesviruses, is known to play a critical role in reactivation from latency. Here we report that HHV-8 RTA, not EBV RTA, was able to induce MHV-68 lytic viral proteins and DNA replication and processing and produce viable MHV-68 virions from latently infected cells at levels similar to those for MHV-68 RTA. HHV-8 RTA was also able to activate two MHV-68 lytic promoters, whereas EBV RTA was not. In order to define the domains of RTA responsible for their functional differences in viral promoter activation and initiation of the MHV-68 lytic cycle, chimeric RTA proteins were constructed by exchanging the N-terminal and C-terminal domains of the RTA proteins. Our data suggest that the species specificity of MHV-68 RTA resides in the N-terminal DNA binding domain.Gammaherpesviruses are distinguished by their ability to establish latency in lymphocytes and are associated with malignancies, such as various B-cell lymphomas (1-3, 31, 33) and Kaposi's sarcoma (5,20,22,24). Murine gammaherpesvirus 68 (MHV-68) is classified as a type 2 gammaherpesvirus and is currently used as a mouse model for human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 (HHV-8) and Epstein-Barr virus (EBV) (11,21,27,29). The knowledge gained from the use of MHV-68 is instrumental in understanding human gammaherpesvirus pathogenesis.RTA is an immediate-early viral transactivator protein conserved among gammaherpesviruses (14,18,30,37). The RTA protein of HHV-8 has been shown to be necessary and sufficient for reactivation of HHV-8 in latently infected cells (18,30). Ectopic expression of MHV-68 RTA is sufficient and necessary for reactivation of MHV-68 from latency and for activation of the lytic cycle of MHV-68 during de novo infection (36,37). This is in contrast to EBV, a type 1 gammaherpesvirus, which generally requires the cooperativity of two viral proteins, Zebra and RTA, for lytic replication and reactivation (6-8, 12, 38). To define the functional similarity or difference among these RTA proteins, we tested whether the RTA protein of HHV-8 or EBV could activate MHV-68 lytic promoters and reactivate MHV-68 from latency. The comparison of these three RTAs has allowed us to determine which domains of RTA are necessary for reactivation of MHV-68 and transactivation of viral lytic promoters.Activation of heterologous promoters by the RTA proteins. All three RTAs are known to activate their respective autologous RTA promoters in addition to the promoters of downstream lytic viral genes (9, 10, 13, 15-17, 25, 28). In order to determine if the RTA proteins also have the ability to transactivate promoters from heterologous viruses, we tested the ability of HHV-8 RTA and EBV RTA to transactivate two MHV-68 lytic promoters in a reporter assay. A 1.2-kb ...

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“…To circumvent these problems, several labs have explored vaccination strategies involving live attenuated viruses, taking advantage of the availability of mutant viruses that are incapable of establishing latency or that are capable of establishing a latent infection with various defects in reactivation (15)(16)(17)(18). The new finding that viral replication is not absolutely required for the establishment of latency has led to renewed interest in replicationdeficient mutants (12)(13)(14)19).…”

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A Replication-Deficient Murine γ-Herpesvirus Blocked in Late Viral Gene Expression Can Establish Latency and Elicit Protective Cellular Immunity

Kayhan

Yager

Lanzer

et al. 2007

The Journal of Immunology

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The human γ-herpesviruses, EBV and Kaposi’s sarcoma-associated herpesvirus, are widely disseminated and are associated with the onset of a variety of malignancies. Thus, the development of prophylactic and therapeutic vaccination strategies is an important goal. The experimental mouse γ-herpesvirus, γHV68 (or MHV-68), has provided an in vivo model for studying immune control of these persistent viruses. In the current studies, we have examined infectivity, immunogenicity, and protective efficacy following infection with a replication-deficient γHV68 blocked in late viral gene expression, ORF31STOP. The data show that ORF31STOP was able to latently infect B cells. However, the anatomical site and persistence of the infection depended on the route of inoculation, implicating a role for viral replication in viral spread but not the infectivity per se. Furthermore, i.p. infection with ORF31STOP elicited strong cellular immunity but a non-neutralizing Ab response. In contrast, intranasal infection was poorly immunogenic. Consistent with this, mice infected i.p. had enhanced control of both the lytic and latent viral loads following challenge with wild-type γHV68, whereas intranasal infected mice were not protected. These data provide important insight into mechanisms of infection and protective immunity for the γ-herpesviruses and demonstrate the utility of replication-deficient mutant viruses in direct testing of “proof of principal” vaccination strategies.

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Generation of a Latency-Deficient Gammaherpesvirus That Is Protective against Secondary Infection

Cited by 38 publications

References 31 publications

ORF30 and ORF34 Are Essential for Expression of Late Genes in Murine Gammaherpesvirus 68

ORF30 and ORF34 Are Essential for Expression of Late Genes in Murine Gammaherpesvirus 68

Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 RTA Reactivates Murine Gammaherpesvirus 68 from Latency

A Replication-Deficient Murine γ-Herpesvirus Blocked in Late Viral Gene Expression Can Establish Latency and Elicit Protective Cellular Immunity

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