NF-κB p50 Plays Distinct Roles in the Establishment and Control of Murine Gammaherpesvirus 68 Latency

ABSTRACT NF-κB signaling is critical to the survival and transformation of cells infected by the human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. Here we have examined how elimination of the NF-κB transcription factor p50 from mice affects the life cycle of murine gammaherpesvirus 68 (MHV68). Notably, mice lacking p50 in every cell type were unable to establish a sufficiently robust immune response to control MHV68 infection, leading to high levels of latently infected B cells detected in the spleen and persistent virus replication in the lungs. The latter correlated with very low levels of virus-specific immunoglobulin G (IgG) in the infected p50−/− mice at day 48 postinfection. Because the confounding impact of the loss of p50 on the host response to MHV68 infection prevented a direct analysis of the role of this NF-κB family member on MHV68 latency in B cells, we generated and infected mixed p50+/+/p50−/− bone marrow chimeric mice. We show that the chimeric mice were able to control acute virus replication and exhibited normal levels of virus-specific IgG at 3 months postinfection, indicating the induction of a normal host immune response to MHV68 infection. However, in p50+/+/p50−/− chimeric mice the p50−/− B cells exhibited a significant defect compared to p50+/+ B cells in supporting MHV68 latency. In addition to identifying a role for p50 in the establishment of latency, we determined that the absence of p50 in a subset of the hematopoietic compartment led to persistent virus replication in the lungs of the chimeric mice, providing evidence that p50 is required for controlling virus reactivation. Taken together, these data demonstrate that p50 is required for immune control by the host and has distinct tissue-dependent roles in the regulation of murine gammaherpesvirus latency during chronic infection.

Download Full-text

Perforin and Fas in murine gammaherpesvirus-specific CD8+ T cell control and morbidity

Journal of General Virology ◽

10.1099/0022-1317-82-8-1971 ◽

2001 ◽

Vol 82 (8) ◽

pp. 1971-1981 ◽

Cited By ~ 38

Author(s):

David J. Topham ◽

Rhonda C. Cardin ◽

Jan P. Christensen ◽

James W. Brooks ◽

Gabrielle T. Belz ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Infectious Virus ◽

Chimeric Mice ◽

Dna Virus ◽

Murine Gammaherpesvirus ◽

Ifn Γ ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68 ◽

Perforin Expression

The immune system uses both virus-specific T cells and B cells to control the acute and latent phases of respiratory infection with the murine gammaherpesvirus 68 (γHV-68). We sought to further define the important effector mechanisms for CD8+ T cells. First, depletion of the CD4+ T cells resulted in a failure of most animals to drive the virus into latency, although lytic virus in the lung was reduced by approximately 1000-fold from its peak. Second, the absence of either perforin or Fas alone had no impact on the ability to reduce titres of lytic virus in the lung. Further neutralization of IFN-γ in CD4-depleted P+/+, P−/− or Fas−/− mice had no effect. To define the requirements for Fas or perforin more clearly, two sets of chimeric mice were constructed differing in perforin expression by the T cells, and Fas on infected epithelial cells or lymphocytes. Animals with P−/− T cells and a Fas−/− lung failed to limit the shedding of infectious virus, regardless of whether CD4 T cells were present. In addition, we noted that having P−/− T cells in irradiated Fas+/+ hosts caused a lethal disease that was not apparent in the non-chimeric (unirradiated) P−/− (Fas+/+) mice. In another set of chimeric mice, P−/− T cells were able to limit persistent infection of B cells that expressed Fas, but not B cells that were Fas-deficient. These studies demonstrate that some degree of cytotoxicity via either perforin or Fas is essential for CD8+ T cells to control this DNA virus.

Download Full-text

Establishment and Maintenance of Long-Term Murine Gammaherpesvirus 68 Latency in B Cells in the Absence of CD40

Journal of Virology ◽

10.1128/jvi.79.5.2891-2899.2005 ◽

2005 ◽

Vol 79 (5) ◽

pp. 2891-2899 ◽

Cited By ~ 19

Author(s):

David O. Willer ◽

Samuel H. Speck

Keyword(s):

B Cells ◽

B Cell ◽

Virus Replication ◽

Receptor Expression ◽

Cell Phenotype ◽

Memory B Cell ◽

Murine Gammaherpesvirus ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (γHV68), like Epstein-Barr virus (EBV), establishes a chronic infection in its host by gaining access to the memory B-cell reservoir, where it persists undetected by the host's immune system. EBV encodes a membrane protein, LMP1, that appears to function as a constitutively active CD40 receptor, and is hypothesized to play a central role in EBV-driven differentiation of infected naive B cells to a memory B-cell phenotype. However, it has recently been shown that there is a critical role for CD40-CD40L interaction in B-cell immortalization by EBV (K.-I. Imadome, M. Shirakata, N. Shimizu, S. Nonoyama, and Y. Yamanashi, Proc. Natl. Acad. Sci. USA 100:7836-7840, 2003), indicating that LMP1 does not adequately recapitulate all of the necessary functions of CD40. The role of CD40 receptor expression on B cells for the establishment and maintenance of γHV68 latency is unclear. Data previously obtained with a competition model, demonstrated that in the face of CD40-sufficient B cells, γHV68 latency in CD40-deficient B cells waned over time in chimeric mice (I.-J. Kim, E. Flano, D. L. Woodland, F. E. Lund, T. D. Randall, and M. A. Blackman, J. Immunol. 171:886-892, 2003). To further investigate the role of CD40 in γHV68 latency in vivo, we have characterized the infection of CD40 knockout (CD40−/−) mice. Here we report that, consistent with previous observations, γHV68 efficiently established a latent infection in B cells of CD40−/− mice. Notably, unlike the infection of normal C57BL/6 mice, significant ex vivo reactivation from splenocytes harvested from infected CD40−/− mice 42 days postinfection was observed. In addition, in contrast to γHV68 infection of C57BL/6 mice, the frequency of infected naive B cells remained fairly stable over a 3-month period postinfection. Furthermore, a slightly higher frequency of γHV68 infection was observed in immunoglobulin D (IgD)-negative B cells, which was stably maintained over a period of 3 months postinfection. The presence of virus in IgD-negative B cells indicates that γHV68 may either directly infect memory B cells present in CD40−/− mice or be capable of driving differentiation of naive CD40−/− B cells. A possible explanation for the apparent discrepancy between the failure of γHV68 latency to be maintained in CD40-deficient B cells in the presence of CD40-sufficient B cells and the stable maintenance of γHV68 B-cell latency in CD40−/− mice came from examining virus replication in the lungs of infected CD40−/− mice, where we observed significantly higher levels of virus replication at late times postinfection compared to those in infected C57BL/6 mice. Taken together, these findings are consistent with a model in which chronic virus infection of CD40−/− mice is maintained through virus reactivation in the lungs and reseeding of latency reservoirs.

Download Full-text

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

Download Full-text

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.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.00001-07 ◽

2007 ◽

Vol 81 (18) ◽

pp. 9870-9877 ◽

Cited By ~ 18

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.

Download Full-text

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.

Download Full-text

The Murine Gammaherpesvirus 68 M2 Gene Is Required for Efficient Reactivation from Latently Infected B Cells

Journal of Virology ◽

10.1128/jvi.00066-11 ◽

2011 ◽

Vol 85 (6) ◽

pp. 3041-3041

Author(s):

J. H. Herskowitz ◽

M. A. Jacoby ◽

S. H. Speck

Keyword(s):

B Cells ◽

Murine Gammaherpesvirus ◽

Latently Infected ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.80.3.1592-1598.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1592-1598 ◽

Cited By ~ 36

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.

Download Full-text