Morning Flowers : Gavotte

https://digitalcommons.library.umaine.edu/mmb-ps/1145/thumbnail.jp

The Old Cathedral Chimes : Nocturne

https://digitalcommons.library.umaine.edu/mmb-ps/1854/thumbnail.jp

Out On The Bounding Ocean Deep : Bass Song

https://digitalcommons.library.umaine.edu/mmb-vp/2348/thumbnail.jp

Tracking Murine Gammaherpesvirus 68 Infection of Germinal Center B Cells In Vivo

Infection of mice with murine gammaherpesvirus 68 (MHV68) provides a tractable small animal model to study various aspects of persistent gammaherpesvirus infection. We have previously utilized a transgenic MHV68 that expresses enhanced yellow fluorescent protein (EYFP) to identify infected cells. While this recombinant MHV68 has been useful for identifying infected cell populations by flow cytometry, it has been suboptimal for identification of infected cells in tissue sections due to the high solubility of EYFP. Efficient detection of EYFP expressed from the MHV68 genome in tissue sections requires fixation of whole organs prior to sectioning, which frequently leads to over-fixation of some cellular antigens precluding their detection. To circumvent this issue, we describe the generation and characterization of a transgenic MHV68 harboring a fusion gene composed of the EYFP coding sequence fused to the histone H2B open reading frame. Because the H2bYFP fusion protein is tightly bound in nucleosomes in the nucleus it does not freely diffuse out of unfixed tissue sections, and thus eliminates the need for tissue fixation. We have used the MHV68-H2bYFP recombinant virus to assess the location and distribution of virus infected B cells in germinal centers during the peak of MHV68 latency in vivo. These analyses show that the physical location of distinct populations of infected germinal center B cells correlates well with their surface phenotype. Furthermore, analysis of the distribution of virus infection within germinal center B cell populations revealed that ca. 70% of MHV68 infected GC B cells are rapidly dividing centroblasts, while ca. 20% have a clear centrocyte phenotype. Finally, we have shown that marking of infected cells with MHV68-H2bYFP is extended long after the onset of latency – which should facilitate studies to track MHV68 latently infected cells at late times post-infection

Antibody-Independent Control of γ-Herpesvirus Latency via B Cell Induction of Anti-Viral T Cell Responses

B cells can use antibody-dependent mechanisms to control latent viral infections. It is unknown whether this represents the sole function of B cells during chronic viral infection. We report here that hen egg lysozyme (HEL)-specific B cells can contribute to the control of murine γ-herpesvirus 68 (γHV68) latency without producing anti-viral antibody. HEL-specific B cells normalized defects in T cell numbers and proliferation observed in B cell−/− mice during the early phase of γHV68 latency. HEL-specific B cells also reversed defects in CD8 and CD4 T cell cytokine production observed in B cell−/− mice, generating CD8 and CD4 T cells necessary for control of latency. Furthermore, HEL-specific B cells were able to present virally encoded antigen to CD8 T cells. Therefore, B cells have antibody independent functions, including antigen presentation, that are important for control of γ-herpesvirus latency. Exploitation of this property of B cells may allow enhanced vaccine responses to chronic virus infection

Inhibition of NF-κB Activation In Vivo Impairs Establishment of Gammaherpesvirus Latency

A critical determinant in chronic gammaherpesvirus infections is the ability of these viruses to establish latency in a lymphocyte reservoir. The nuclear factor (NF)-κB family of transcription factors represent key players in B-cell biology and are targeted by gammaherpesviruses to promote host cell survival, proliferation, and transformation. However, the role of NF-κB signaling in the establishment of latency in vivo has not been addressed. Here we report the generation and in vivo characterization of a recombinant murine gammaherpesvirus 68 (γHV68) that expresses a constitutively active form of the NF-κB inhibitor, IκBαM. Inhibition of NF-κB signaling upon infection with γHV68-IκBαM did not affect lytic replication in cell culture or in the lung following intranasal inoculation. However, there was a substantial decrease in the frequency of latently infected lymphocytes in the lung (90% reduction) and spleens (97% reduction) 16 d post intranasal inoculation. Importantly, the defect in establishment of latency in lung B cells could not be overcome by increasing the dose of virus 100-fold. The observed decrease in establishment of viral latency correlated with a loss of activated, CD69(hi) B cells in both the lungs and spleen at day 16 postinfection, which was not apparent by 6 wk postinfection. Constitutive expression of Bcl-2 in B cells did not rescue the defect in the establishment of latency observed with γHV68-IκBαM, indicating that NF-κB–mediated functions apart from Bcl-2–mediated B-cell survival are critical for the efficient establishment of gammaherpesvirus latency in vivo. In contrast to the results obtained following intranasal inoculation, infection of mice with γHV68-IκBαM by the intraperitoneal route had only a modest impact on splenic latency, suggesting that route of inoculation may alter requirements for establishment of virus latency in B cells. Finally, analyses of the pathogenesis of γHV68-IκBαM provides evidence that NF-κB signaling plays an important role during multiple stages of γHV68 infection in vivo and, as such, represents a key host regulatory pathway that is likely manipulated by the virus to establish latency in B cells

Identification of the In Vivo Role of a Viral bcl-2

Many γ-herpesviruses encode candidate oncogenes including homologues of host bcl-2 and cyclin proteins (v-bcl-2, v-cyclin), but the physiologic roles of these genes during infection are not known. We show for the first time in any virus system the physiologic role of v-bcl-2. A γ-herpesvirus v-bcl-2 was essential for efficient ex vivo reactivation from latent infection, and for both persistent replication and virulence during chronic infection of immunocompromised (interferon [IFN]-γ−/−) mice. The v-cyclin was also critical for the same stages in pathogenesis. Strikingly, while the v-bcl-2 and v-cyclin were important for chronic infection, these genes were not essential for viral replication in cell culture, viral replication during acute infection in vivo, establishment of latent infection, or virulence during acute infection. We conclude that v-bcl-2 and v-cyclin have important roles during latent and persistent γ-herpesvirus infection and that herpesviruses encode genes with specific roles during chronic infection and disease, but not acute infection and disease. As γ-herpesviruses primarily cause human disease during chronic infection, these chronic disease genes may be important targets for therapeutic intervention

Identification of Alternative Transcripts Encoding the Essential Murine Gammaherpesvirus Lytic Transactivator RTA

The essential immediate early transcriptional activator RTA, encoded by gene 50, is conserved among all characterized gammaherpesviruses. Analyses of a recombinant murine gammaherpesvirus 68 (MHV68) lacking both of the known gene 50 promoters (G50DblKo) revealed that this mutant retained the ability to replicate in the simian kidney epithelial cell line Vero but not in permissive murine fibroblasts following low-multiplicity infection. However, G50DblKo replication in permissive fibroblasts was partially rescued by high-multiplicity infection. In addition, replication of the G50DblKo virus was rescued by growth on mouse embryonic fibroblasts (MEFs) isolated from IFN-α/βR(−/−) mice, while growth on Vero cells was suppressed by the addition of alpha interferon (IFN-α). 5′ rapid amplification of cDNA ends (RACE) analyses of RNAs prepared from G50DblKo and wild-type MHV68-infected murine macrophages identified three novel gene 50 transcripts initiating from 2 transcription initiation sites located upstream of the currently defined proximal and distal gene 50 promoters. In transient promoter assays, neither of the newly identified gene 50 promoters exhibited sensitivity to IFN-α treatment. Furthermore, in a single-step growth analysis RTA levels were higher at early times postinfection with the G50DblKo mutant than with wild-type virus but ultimately fell below the levels of RTA expressed by wild-type virus at later times in infection. Infection of mice with the MHV68 G50DblKo virus demonstrated that this mutant virus was able to establish latency in the spleen and peritoneal exudate cells (PECs) of C57BL/6 mice with about 1/10 the efficiency of wild-type virus or marker rescue virus. However, despite the ability to establish latency, the G50DblKo virus mutant was severely impaired in its ability to reactivate from either latently infected splenocytes or PECs. Consistent with the ability to rescue replication of the G50DblKo mutant by growth on type I interferon receptor null MEFs, infection of IFN-α/βR(−/−) mice with the G50DblKo mutant virus demonstrated partial rescue of (i) acute virus replication in the lungs, (ii) establishment of latency, and (iii) reactivation from latency. The identification of additional gene 50/RTA transcripts highlights the complex mechanisms involved in controlling expression of RTA, likely reflecting time-dependent and/or cell-specific roles of different gene 50 promoters in controlling virus replication. Furthermore, the newly identified gene 50 transcripts may also act as negative regulators that modulate RTA expression. IMPORTANCE The viral transcription factor RTA, encoded by open reading frame 50 (Orf50), is well conserved among all known gammaherpesviruses and is essential for both virus replication and reactivation from latently infected cells. Previous studies have shown that regulation of gene 50 transcription is complex. The studies reported here describe the presence of additional alternatively initiated, spliced transcripts that encode RTA. Understanding how expression of this essential viral gene product is regulated may identify new strategies for interfering with infection in the setting of gammaherpesvirus-induced diseases

A surface groove essential for viral Bcl-2 function during chronic infection in vivo

Antiapoptotic Bcl-2 family proteins inhibit apoptosis in cultured cells by binding BH3 domains of proapoptotic Bcl-2 family members via a hydrophobic BH3 binding groove on the protein surface. We investigated the physiological importance of the BH3 binding groove of an antiapoptotic Bcl-2 protein in mammals in vivo by analyzing a viral Bcl-2 family protein. We show that the gamma-herpesvirus 68 (gammaHV68) Bcl-2 family protein (gammaHV68 v-Bcl-2), which is known to inhibit apoptosis in cultured cells, inhibits both apoptosis in primary lymphocytes and Bax toxicity in yeast. Nuclear magnetic resonance determination of the gammaHV68 v-Bcl-2 structure revealed a BH3 binding groove that binds BH3 domain peptides from proapoptotic Bcl-2 family members Bax and Bak via a molecular mechanism shared with host Bcl-2 family proteins, involving a conserved arginine in the BH3 peptide binding groove. Mutations of this conserved arginine and two adjacent amino acids to alanine (SGR to AAA) within the BH3 binding groove resulted in a properly folded protein that lacked the capacity of the wild-type gammaHV68 v-Bcl-2 to bind Bax BH3 peptide and to block Bax toxicity in yeast. We tested the physiological importance of this v-Bcl-2 domain during viral infection by engineering viral mutants encoding a v-Bcl-2 containing the SGR to AAA mutation. This mutation resulted in a virus defective for both efficient reactivation of gammaHV68 from latency and efficient persistent gammaHV68 replication. These studies demonstrate an essential functional role for amino acids in the BH3 peptide binding groove of a viral Bcl-2 family member during chronic infection

Cyclosporin A and FK506 Block Induction of the Epstein-Barr Virus Lyric Cycle by Anti-Immunoglobulin

AbstractThe Epstein-Barr virus (EBV) BZLF1 gene is expressed early upon induction of the viral lytic cycle and its protein product is unique in its ability to disrupt viral latency in some latently infected cell lines. Anti-immunoglobulin (anti-Ig) treatment of the Burkitt's lymphoma cell line Akata, which bears surface IgG, has previously been shown to synchronously induce transcription of the BZLF1 gene (K. Takada and Y. Ono, 1989, J. Virol. 63, 445-449). We have previously shown that anti-Ig induction of Akata cells activates expression of the tumor necrosis factor alpha (TNF-α) gene via a calcineurin-dependent mechanism (Goldfeld et al., 1992, Proc. Natl. Acad. Sci. USA 89, 12198-12201). Here, we report that anti-Ig induction of the EBV lytic cycle in Akata cells can be blocked by the immunosuppressants cyclosporin A and FK506. Furthermore, we demonstrate that synergistic induction by phorbol ester and calcium ionophore of a BZLF1 promoter-driven reporter construct in an EBV-negative BL cell line can be inhibited by addition of cyclosporin A. Thus, analogous to activation of TNF-α gene in Akata cells, anti-Ig induction of the BZLF1 promoter is most likely mediated by calcineurin and probably involves translocation to the nucleus of a transcription factor sequestered in the cytoplasm. As such, immunosuppressants may be useful probes for dissecting B cell activation pathways involved in regulating EBV gene transcription