scholarly journals The M2 gene product of murine gammaherpesvirus 68 is required for efficient colonization of splenic follicles but is not necessary for expansion of latently infected germinal centre B cells

2004 ◽  
Vol 85 (10) ◽  
pp. 2789-2797 ◽  
Author(s):  
J. Pedro Simas ◽  
Sofia Marques ◽  
Anne Bridgeman ◽  
Stacey Efstathiou ◽  
Heiko Adler
Keyword(s):  
B Cells ◽  
Gene Product ◽  
Memory B Cells ◽  
Germinal Centre ◽  
Wild Type Virus ◽  
Wild Type ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

Infection of mice with murine gammaherpesvirus 68 is characterized by a marked transient expansion of latently infected splenic germinal centre (GC) B cells, which is followed by lower levels of persistent infection in GC and memory B cells. Virus transcription within GC B cells is restricted to a number of latency-associated open reading frames, including M2. This gene encodes a structurally unique protein of unknown function, which has been shown to be essential for the transient peak of virus latency during the establishment of latent infection in the spleen. This study shows that upon infection of mice with M2-defective viruses, at 14 days post-infection during the establishment of latency in the spleen, there was a reduction in the number of latently infected follicles when compared with wild-type virus. However, the mean number of latently infected cells within each follicle was equivalent between wild-type and M2-defective viruses. Late in infection, disruption of M2 resulted in sustained and abnormally high levels of virus persistence in splenic GC B cells but not memory B cells. These data indicate that during the establishment of latency in the spleen, the M2 gene product is required for efficient colonization of splenic follicles but is dispensable for the expansion of latently infected GC B cells and that M2 might be a critical modulator of B-cell function.

scholarly journals The ORF49 Protein of Murine Gammaherpesvirus 68 Cooperates with RTA in Regulating Virus Replication

2007 ◽  
Vol 81 (18) ◽  
pp. 9870-9877 ◽  
Author(s):  
Sangmi Lee ◽  
Hye-Jeong Cho ◽  
Jung-Jin Park ◽  
Yong-Sun Kim ◽  
Seungmin Hwang ◽  
...  
Keyword(s):  
Virus Replication ◽  
Wild Type Virus ◽  
Wild Type ◽  
Mapping Study ◽  
Murine Gammaherpesvirus ◽  
Downstream Target ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Target Promoters

ABSTRACT Our functional mapping study of murine gammaherpesvirus 68 (MHV-68, or γHV-68) revealed that a mutant harboring a transposon at the ORF49 locus (ORF49null) evidenced a highly attenuated in vitro growth. ORF49 resides adjacent to and in an opposite direction from RTA, the primary switch of the gammaherpesvirus life cycle. A FLAG-tagged ORF49 protein was able to transcomplement ORF49null, and a revertant of ORF49null restored its attenuated growth to a level comparable to that of the wild type. The FLAG-tagged ORF49 protein promoted the ability of RTA to activate downstream target promoters and enhanced virus replication from the ORF50null virus in the presence of RTA. Furthermore, ORF49 enhanced wild-type virus replication by increasing the RTA transcript levels. Our data indicate that ORF49 may perform an important function in MHV-68 replication in cooperation with RTA.

scholarly journals A Gammaherpesvirus 68 Gene 50 Null Mutant Establishes Long-Term Latency in the Lung but Fails To Vaccinate against a Wild-Type Virus Challenge

2006 ◽  
Vol 80 (3) ◽  
pp. 1592-1598 ◽  
Author(s):  
Janice M. Moser ◽  
Michael L. Farrell ◽  
Laurie T. Krug ◽  
Jason W. Upton ◽  
Samuel H. Speck
Keyword(s):  
B Cells ◽  
Virus Replication ◽  
Antiviral Drug ◽  
Lytic Replication ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68

ABSTRACT The gammaherpesvirus immediate-early genes are critical regulators of virus replication and reactivation from latency. Rta, encoded by gene 50, serves as the major transactivator of the lytic program and is highly conserved among all the gammaherpesviruses, including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (γHV68). Introduction of a translation stop codon in γHV68 gene 50 (gene 50.stop γHV68) demonstrated that Rta is essential for virus replication in vitro. To investigate the role that virus replication plays in the establishment and maintenance of latency, we infected mice with gene 50.stop γHV68. Notably, the gene 50.stop virus established a long-term infection in lung B cells following intranasal infection of mice but was unable to establish latency in the spleen. This complete block in the establishment of latency in the spleen was also seen when lytic virus production was inhibited by treating mice infected with wild-type virus with the antiviral drug cidofovir, implicating virus replication and not an independent function of Rta in the establishment of splenic latency. Furthermore, we showed that gene 50.stop γHV68 was unable to prime the immune system and was unable to protect against a challenge with wild-type γHV68, despite its ability to chronically infect lung B cells. These data indicate gammaherpesviruses that are unable to undergo lytic replication in vivo may not be viable vaccine candidates despite the detection of cells harboring viral genome at late times postinfection.

scholarly journals Identification of Infected B-Cell Populations by Using a Recombinant Murine Gammaherpesvirus 68 Expressing a Fluorescent Protein

2009 ◽  
Vol 83 (13) ◽  
pp. 6484-6493 ◽  
Author(s):  
Christopher M. Collins ◽  
Jeremy M. Boss ◽  
Samuel H. Speck
Keyword(s):  
B Cells ◽  
Virus Infection ◽  
Plasma Cells ◽  
Fluorescent Protein ◽  
Viral Latency ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Infection of inbred mice with murine gammaherpesvirus 68 (MHV68) has proven to be a powerful tool to study gammaherpesvirus pathogenesis. However, one of the limitations of this system has been the inability to directly detect infected cells harvested from infected animals. To address this issue, we generated a transgenic virus that expresses the enhanced yellow fluorescent protein (YFP), driven by the human cytomegalovirus immediate-early promoter and enhancer, from a neutral locus within the viral genome. This virus, MHV68-YFP, replicated and established latency as efficiently as did the wild-type virus. During the early phase of viral latency, MHV68-YFP efficiently marked latently infected cells in the spleen after intranasal inoculation. Staining splenocytes for expression of various surface markers demonstrated the presence of MHV68 in distinct populations of splenic B cells harboring MHV68. Notably, these analyses also revealed that markers used to discriminate between newly formed, follicular and marginal zone B cells may not be reliable for phenotyping B cells harboring MHV68 since virus infection appears to modulate cell surface expression levels of CD21 and CD23. However, as expected, we observed that the overwhelming majority of latently infected B cells at the peak of latency exhibited a germinal center phenotype. These analyses also demonstrated that a significant percentage of MHV68-infected splenocytes at the peak of viral latency are plasma cells (ca. 15% at day 14 and ca. 8% at day 18). Notably, the frequency of virus-infected plasma cells correlated well with the frequency of splenocytes that spontaneously reactivate virus upon explant. Finally, we observed that the efficiency of marking latently infected B cells with the MHV68-YFP recombinant virus declined at later times postinfection, likely due to shut down of transgene expression, and indicating that the utility of this marking strategy is currently limited to the early stages of virus infection.

scholarly journals Induction of Protective Immunity against Murine Gammaherpesvirus 68 Infection in the Absence of Viral Latency

2009 ◽  
Vol 84 (5) ◽  
pp. 2453-2465 ◽  
Author(s):  
Qingmei Jia ◽  
Michael L. Freeman ◽  
Eric J. Yager ◽  
Ian McHardy ◽  
Leming Tong ◽  
...  
Keyword(s):  
Human Herpesvirus ◽  
Lytic Replication ◽  
Wild Type Virus ◽  
Wild Type ◽  
Transcription Activator ◽  
Vaccine Strategy ◽  
Murine Gammaherpesvirus ◽  
Barr Virus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

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.

scholarly journals B cells latently infected with murine gammaherpesvirus 68 (MHV‐68) are present in the mouse thymus–A step toward immune evasion?

2018 ◽  
Vol 49 (2) ◽  
pp. 351-352
Author(s):  
Tomoyoshi Yamano ◽  
Madlen Steinert ◽  
Beatrix Steer ◽  
Ludger Klein ◽  
Wolfgang Hammerschmidt ◽  
...  
Keyword(s):  
B Cells ◽  
Immune Evasion ◽  
Mouse Thymus ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

scholarly journals Role for MyD88 Signaling in Murine Gammaherpesvirus 68 Latency

2008 ◽  
Vol 82 (8) ◽  
pp. 3853-3863 ◽  
Author(s):  
Lisa M. Gargano ◽  
Janice M. Moser ◽  
Samuel H. Speck
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Response ◽  
Wild Type ◽  
Tlr Signaling ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
B Cell Response

ABSTRACT Toll-like receptors (TLRs) are known predominantly for their role in activating the innate immune response. Recently, TLR signaling via MyD88 has been reported to play an important function in development of a B-cell response. Since B cells are a major latency reservoir for murine gammaherpesvirus 68 (MHV68), we investigated the role of TLR signaling in the establishment and maintenance of MHV68 latency in vivo. Mice deficient in MyD88 (MyD88−/−) or TLR3 (TLR3−/−) were infected with MHV68. Analysis of splenocytes recovered at day 16 postinfection from MyD88−/− mice compared to those from wild-type control mice revealed a lower frequency of (i) activated B cells, (ii) germinal-center B cells, and (iii) class-switched B cells. Accompanying this substantial defect in the B-cell response was an approximately 10-fold decrease in the establishment of splenic latency. In contrast, no defect in viral latency was observed in TLR3−/− mice. Analysis of MHV68-specific antibody responses also demonstrated a substantial decrease in the kinetics of the response in MyD88−/− mice. Analysis of wild-type × MyD88−/− mixed-bone-marrow chimeric mice demonstrated that there is a selective failure of MyD88−/− B cells to participate in germinal-center reactions as well as to become activated and undergo class switching. In addition, while MHV68 established latency efficiently in the MyD88-sufficient B cells, there was again a ca. 10-fold reduction in the frequency of MyD88−/− B cells harboring latent MHV68. This phenotype indicates that MyD88 is important for the establishment of MHV68 latency and is directly related to the role of MyD88 in the generation of a B-cell response. Furthermore, the generation of a B-cell response to MHV68 was intrinsic to B cells and was independent of the interleukin-1 receptor, a cytokine receptor that also signals through MyD88. These data provide evidence for a unique role for MyD88 in the establishment of MHV68 latency.

scholarly journals Lung Epithelial Cells Are a Major Site of Murine Gammaherpesvirus Persistence

1998 ◽  
Vol 187 (12) ◽  
pp. 1941-1951 ◽  
Author(s):  
James P. Stewart ◽  
Edward J. Usherwood ◽  
Alan Ross ◽  
Heather Dyson ◽  
Tony Nash
Keyword(s):  
B Cells ◽  
Epithelial Cells ◽  
B Cell ◽  
Virus Genome ◽  
Lung Epithelial Cells ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Deficient Mice

It is currently believed that latently infected, resting B lymphocytes are central to gammaherpesvirus persistence, whereas mucosal epithelial cells are considered nonessential. We have readdressed the question of nonlymphoid persistence using murine gammaherpesvirus 68 (MHV-68). To dissect lymphoid from nonlymphoid persistence, we used μMT transgenic mice that are defective in B cells. MHV-68 DNA persisted in the lungs of intact and B cell–deficient mice. Both episomal and linear forms of the virus genome were present in lungs, implying the presence of both latency and productive replication. In situ hybridization for virus tRNA transcripts revealed latent MHV-68 in pulmonary epithelial cells. Infectious virus was recovered from the lungs of μMT mice after T cell depletion, showing that the persisting virus DNA was reactivatable. Finally, using adoptive transfer of B cells into B cell–deficient mice, it was shown that virus persisting in lungs seeded splenic B cells, and virus resident in the spleen seeded the lungs. These results show that mucosal epithelia can act as a nonlymphoid reservoir for gammaherpesvirus persistence, and that there is a two-way movement of virus between lymphoid and nonlymphoid compartments during persistence.

scholarly journals Murine Gammaherpesvirus 68 Cyclin D Homologue Is Required for Efficient Reactivation from Latency

2000 ◽  
Vol 74 (15) ◽  
pp. 7016-7023 ◽  
Author(s):  
Ann T. Hoge ◽  
Sara B. Hendrickson ◽  
William H. Burns
Keyword(s):  
Acute Infection ◽  
Human Herpesvirus ◽  
Wild Type Virus ◽  
Cyclin D ◽  
Type Virus ◽  
Wild Type ◽  
Virus Growth ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (MHV68) is a gammaherpesvirus that was first isolated from murid rodents. MHV68 establishes a latent infection in the spleen and other lymphoid organs. Several gammaherpesviruses, including herpesvirus saimiri, human herpesvirus 8, and MHV68, encode proteins with extensive homology to the D-type cyclins. To study the function of the cyclin homologue, a recombinant MHV68 has been constructed that lacks the cyclin homologue and expresses β-galactosidase as a marker (MHV68cy−). MHV68cy− grows in vitro with kinetics and to titers similar to those of the wild type. BALB/c mice infected with mixtures of equivalent amounts of the wild type and MHV68cy− show deficient growth of the MHV68cy− in an acute infection. Infection of SCID mice with virus mixtures also showed decreased MHV68cy−virus growth, indicating that the deficiency is not mediated by T or B cells. Although mice infected with mixtures containing 100 times as much MHV68cy− had greater splenic titers of the mutant virus than wild-type virus in acute infection, at 28 days postinfection splenocytes from these mice reactivated primarily wild-type virus. Quantitative PCR data indicate that equivalent genomes were present in the latent state. Reinsertion of the cyclin homologue into the cyclin-deleted virus restored the wild-type phenotype. These results indicate that the MHV68 cyclin D homologue mediates important functions in the acute infection and is required for efficient reactivation from latency.

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