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

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.

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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

Journal of General Virology ◽

10.1099/vir.0.80138-0 ◽

2004 ◽

Vol 85 (10) ◽

pp. 2789-2797 ◽

Cited By ~ 30

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.

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Lung Epithelial Cells Are a Major Site of Murine Gammaherpesvirus Persistence

Journal of Experimental Medicine ◽

10.1084/jem.187.12.1941 ◽

1998 ◽

Vol 187 (12) ◽

pp. 1941-1951 ◽

Cited By ~ 206

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.

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Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68.

Journal of Virology ◽

10.1128/jvi.70.10.6775-6780.1996 ◽

1996 ◽

Vol 70 (10) ◽

pp. 6775-6780 ◽

Cited By ~ 194

Author(s):

K E Weck ◽

M L Barkon ◽

L I Yoo ◽

S H Speck ◽

I V Virgin HW

Keyword(s):

B Cells ◽

Murine Gammaherpesvirus ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

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Dendritic Cells Loaded with Tumor B Cells Elicit Broad Immunity against Murine Gammaherpesvirus 68 but Fail To Prevent Long-Term Latency

Journal of Virology ◽

10.1128/jvi.00571-10 ◽

2010 ◽

Vol 84 (17) ◽

pp. 8975-8979

Author(s):

Janet Weslow-Schmidt ◽

Fang Ye ◽

Stephanie S. Cush ◽

Kathleen A. Stuller ◽

Marcia A. Blackman ◽

...

Keyword(s):

Dendritic Cells ◽

B Cells ◽

Acute Infection ◽

Dendritic Cell Vaccination ◽

Murine Gammaherpesvirus ◽

Attenuated Viruses ◽

Gammaherpesvirus 68 ◽

Vaccine Approach ◽

Murine Gammaherpesvirus 68

ABSTRACT It is still unknown whether a noninfectious gammaherpesvirus vaccine is able to prevent or reduce virus persistence. This led us to use dendritic cells loaded with tumor B cells as a vaccine approach for the murine gammaherpesvirus 68 (γHV68) model of infection. Dendritic cells loaded with UV-irradiated latently infected tumor B cells induce broad, strong, and long-lasting immunity against γHV68. Dendritic cell vaccination prevents the enlargement of lymph nodes and severely limits acute infection and early latency but does not prevent γHV68 from establishing long-term latency. Our findings support the concept that attenuated viruses may be the best vaccine option for preventing gammaherpesvirus persistence.

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Evidence for a Multiprotein Gamma-2 Herpesvirus Entry Complex

Journal of Virology ◽

10.1128/jvi.01141-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 13082-13091 ◽

Cited By ~ 21

Author(s):

Laurent Gillet ◽

Philip G. Stevenson

Keyword(s):

B Cells ◽

Specific Antibody ◽

Cell Binding ◽

Murine Gammaherpesvirus ◽

Single Entry ◽

Single Complex ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68 ◽

Better Than

ABSTRACT Herpesviruses use multiple virion glycoproteins to enter cells. How these work together is not well understood: some may act separately or they may form a single complex. Murine gammaherpesvirus 68 (MHV-68) gB, gH, gL, and gp150 all participate in entry. gB and gL are involved in binding, gB and gH are conserved fusion proteins, and gp150 inhibits cell binding until glycosaminoglycans are engaged. Here we show that a gH-specific antibody coprecipitates gB and thus that gH and gB are associated in the virion membrane. A gH/gL-specific antibody also coprecipitated gB, implying a tripartite complex of gL/gH/gB, although the gH/gB association did not require gL. The association was also independent of gp150, and gp150 was not demonstrably bound to gB or gH. However, gp150 incorporation into virions was partly gL dependent, suggesting that it too contributes to a single entry complex. gp150− and gL− gp150− mutants bound better than the wild type to B cells and readily colonized B cells in vivo. Thus, gp150 and gL appear to be epithelial cell-adapted accessories of a core gB/gH entry complex. The cell binding revealed by gp150 disruption did not require gL and therefore seemed most likely to involve gB.

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To the Editor Murine gammaherpesvirus 68 (MHV-68) escapes from NK-cell-mediated immune surveillance by a CEACAM1-mediated immune evasion mechanism

European Journal of Immunology ◽

10.1002/eji.201444593 ◽

2014 ◽

Vol 44 (8) ◽

pp. 2521-2522 ◽

Cited By ~ 5

Author(s):

Heiko Adler ◽

Beatrix Steer ◽

Eric Juskewitz ◽

Robert Kammerer

Keyword(s):

Immune Evasion ◽

Nk Cell ◽

Immune Surveillance ◽

Murine Gammaherpesvirus ◽

Immune Evasion Mechanism ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

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Signaling through Toll-Like Receptors Induces Murine Gammaherpesvirus 68 Reactivation In Vivo

Journal of Virology ◽

10.1128/jvi.01717-08 ◽

2008 ◽

Vol 83 (3) ◽

pp. 1474-1482 ◽

Cited By ~ 35

Author(s):

Lisa M. Gargano ◽

J. Craig Forrest ◽

Samuel H. Speck

Keyword(s):

B Cell ◽

Cell Activation ◽

B Cell Activation ◽

Toll Like Receptors ◽

Murine Gammaherpesvirus ◽

Latently Infected ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68 ◽

Stimulation Of

ABSTRACT Murine gammaherpesvirus 68 (MHV68) establishes a lifelong infection in mice and is used as a model pathogen to study the role of viral and host factors in chronic infection. The maintenance of chronic MHV68 infection, at least in some latency reservoirs, appears to be dependent on the capacity of the virus to reactivate from latency in vivo. However, the signals that lead to MHV68 reactivation in vivo are not well characterized. Toll-like receptors (TLRs), by recognizing the specific patterns of microbial components, play an essential role in the activation of innate immunity. In the present study, we investigated the capacity of TLR ligands to induce MHV68 reactivation, both in vitro and in vivo. The stimulation of latently infected B cell lines with ligands for TLRs 3, 4, 5, and 9 enhanced MHV68 reactivation; the ex vivo stimulation of latently infected primary splenocytes, recovered from infected mice, with poly(I:C), lipopolysaccharide, flagellin, or CpG DNA led to early B-cell activation, B-cell proliferation, and a significant increase in the frequency of latently infected cells reactivating the virus. In vivo TLR stimulation also induced B-cell activation and MHV68 reactivation, resulting in heightened levels of virus replication in the lungs which correlated with an increase in MHV68-specific CD8+ T-cell responses. Importantly, TLR stimulation also led to an increase in MHV68 latency, as evidenced by an increase in viral genome-positive cells 2 weeks post-in vivo stimulation by specific TLR ligands. Thus, these data demonstrate that TLR stimulation can drive MHV68 reactivation from latency and suggests that periodic pathogen exposure may contribute to the homeostatic maintenance of chronic gammaherpesvirus infection through stimulating virus reactivation and reseeding latency reservoirs.

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