Lytic Replication and Reactivation from B Cells Is Not Required for Establishing or Maintaining Gammaherpesvirus Latency In Vivo.

Gammaherpesviruses (GHVs) are lymphotropic tumor viruses with a biphasic infectious cycle. Lytic replication at the primary site of infection is necessary for GHVs to spread throughout the host and establish latency in distal sites. Dissemination is mediated by infected B cells that traffic hematogenously from draining lymph nodes to peripheral lymphoid organs, such as the spleen. B cells serve as the major reservoir for viral latency, and it is hypothesized that periodic reactivation from latently infected B cells contributes to maintaining long-term chronic infection. While fundamentally important to an understanding of GHV biology, aspects of B cell infection in latency establishment and maintenance are incompletely defined, especially roles for lytic replication and reactivation in this cell type. To address this knowledge gap and overcome limitations of replication-defective viruses, we generated a recombinant murine gammaherpesvirus 68 (MHV68) in which ORF50, the gene that encodes the essential immediate-early replication and transcription activator protein (RTA), was flanked by loxP sites to enable conditional ablation of lytic replication by ORF50 deletion in cells that express Cre recombinase. Following infection of mice that encode Cre in B cells with this virus, splenomegaly and viral reactivation from splenocytes were significantly reduced; however, the number of latently infected splenocytes was equivalent to WT MHV68. Despite ORF50 deletion, MHV68 latency was maintained over time in spleens of mice at levels approximating WT, reactivation-competent MHV68. Treatment of infected mice with lipopolysaccharide (LPS), which promotes B cell activation and MHV68 reactivation ex vivo, yielded equivalent increases in the number of latently infected cells for both ORF50-deleted and WT MHV68, even when mice were simultaneously treated with the antiviral drug cidofovir to prevent reactivation. Together, these data demonstrate that productive viral replication in B cells is not required for MHV68 latency establishment and support the hypothesis that B cell proliferation facilitates latency maintenance in vivo in the absence of reactivation. IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system and place infected hosts at risk for developing lymphomas and other diseases. It is hypothesized that gammaherpesviruses must initiate acute infection in these cells to establish and maintain long-term infection, but this has not been directly tested. We report here the use of a viral genetic system that allows for cell-type-specific deletion of a viral gene that is essential for replication and reactivation. We employ this system in an in vivo model to reveal that viral replication is not required to initiate or maintain infection within B cells.

Rheumatology Clinic Staff Needs: Barriers and Strategies to Addressing High Blood Pressure and Smoking Risk.

OBJECTIVE: Patients with rheumatologic conditions are at elevated risk of cardiovascular disease (CVD) due to inflammatory and traditional risk factors, such as high blood pressure (BP) and smoking. However, rheumatology clinics rarely address traditional risk factors, although they are routinely assessed and modifiable in primary care. The present study sought to (1) characterize rheumatology clinic staff's work process for addressing high BP and smoking and (2) identify barriers and strategies for effective management of these risk factors. METHODS: We conducted 7 focus groups with medical assistants, nurses, and scheduling staff from 4 adult rheumatology clinics across 2 health systems (BP focus groups, n = 23; smoking, n = 20). Transcripts were analyzed using thematic analysis to elucidate barriers and strategies. RESULTS: We found 3 clinic work processes for the management of high BP and smoking risk: (1) risk identification, (2) follow-up within the clinic, and (3) follow-up with primary care and community resources. Within these processes, we identified barriers and strategies grouped into themes: (1) time, (2) clinic workflows, (3) technology and resources, (4) staff's attitudes and knowledge, and (5) staff's perceptions of patients. The most pervasive barriers were (1) no structured system for follow-up and (2) staff confidence and skill in initiating conversations about health-related behavior change. CONCLUSIONS: Our study identified generalizable gaps in rheumatology staff's work processes and competencies for addressing high BP and smoking in patients. Future efforts to support staff needs should target (1) systems for follow-up within and outside the clinic and (2) conversation support tools.

Deletion of Murine Gammaherpesvirus Gene M2 in Activation-Induced Cytidine Deaminase-Expressing B Cells Impairs Host Colonization and Viral Reactivation.

Gammaherpesviruses (GHVs) are DNA tumor viruses that establish lifelong, chronic infections in lymphocytes of humans and other mammals. GHV infections are associated with numerous cancers, especially in immunocompromised hosts. While it is known that GHVs utilize host germinal center (GC) B cell responses during latency establishment, an understanding of how viral gene products function in specific B cell subsets to regulate this process is incomplete. Using murine gammaherpesvirus 68 (MHV68) as a small-animal model to define mechanisms of GHV pathogenesis in vivo, we generated a virus in which the M2 gene was flanked by loxP sites (M2.loxP), enabling the use of Cre-lox technology to define M2 function in specific cell types in infection and disease. The M2 gene encodes a protein that is highly expressed in GC B cells that promotes plasma cell differentiation and viral reactivation. M2 was efficiently deleted in Cre-expressing cells, and the presence of loxP sites flanking M2 did not alter viral replication or latency in mice that do not express Cre. In contrast, M2.loxP MHV68 exhibited a deficit in latency establishment and reactivation that resembled M2-null virus, following intranasal (IN) infection of mice that express Cre in all B cells (CD19-Cre). Nearly identical phenotypes were observed for M2.loxP MHV68 in mice that express Cre in germinal center (GC) B cells (AID-Cre). However, colonization of neither draining lymph nodes after IN infection nor the spleen after intraperitoneal (IP) infection required M2, although the reactivation defect was retained. Together, these data confirm that M2 function is B cell-specific and demonstrate that M2 primarily functions in AID-expressing cells to facilitate MHV68 dissemination to distal latency reservoirs within the host and reactivation from latency. Our study reveals that a viral latency gene functions within a distinct subset of cells to facilitate host colonization.IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system that can lead to lymphomas and other diseases. To facilitate colonization of a host, gammaherpesviruses encode gene products that manipulate processes involved in cellular proliferation and differentiation. Whether and how these viral gene products function in specific cells of the immune system is poorly defined. We report here the use of a viral genetic system that allows for deletion of specific viral genes in discrete populations of cells. We employ this system in an in vivo model to demonstrate cell-type-specific requirements for a particular viral gene. Our findings reveal that a viral gene product can function in distinct cellular subsets to direct gammaherpesvirus pathogenesis.

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice.

Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.

Host Tumor Suppressor p18INK4c Functions as a Potent Cell-Intrinsic Inhibitor of Murine Gammaherpesvirus 68 Reactivation and Pathogenesis.

Gammaherpesviruses are common viruses associated with lifelong infection and increased disease risk. Reactivation from latency aids the virus in maintaining infection throughout the life of the host and is responsible for a wide array of disease outcomes. Previously, we demonstrated that the virus-encoded cyclin (v-cyclin) of murine gammaherpesvirus 68 (γHV68) is essential for optimal reactivation from latency in normal mice but not in mice lacking the host tumor suppressor p18INK4c (p18). Whether p18 plays a cell-intrinsic or -extrinsic role in constraining reactivation remains unclear. Here, we generated recombinant viruses in which we replaced the viral cyclin with the cellular p18INK4c gene (p18KI) for targeted expression of p18, specifically within infected cells. We find that the p18KI virus is similar to the cyclin-deficient virus (cycKO) in lytic infection, establishment of latency, and infected cell reservoirs. While the cycKO virus is capable of reactivation in p18-deficient mice, expression of p18 from the p18KI virus results in a profound reactivation defect. These data demonstrate that p18 limits reactivation within latently infected cells, functioning in a cell-intrinsic manner. Further, the p18KI virus showed greater attenuation of virus-induced lethal pneumonia than the cycKO virus, indicating that p18 could further restrict γHV68 pathogenesis even in p18-sufficient mice. These studies demonstrate that host p18 imposes the requirement for the viral cyclin to reactivate from latency by functioning in latently infected cells and that p18 expression is associated with decreased disease, thereby identifying p18 as a compelling host target to limit chronic gammaherpesvirus pathogenesis.IMPORTANCE Gammaherpesviruses are ubiquitous viruses associated with multiple malignancies. The propensity to cycle between latency and reactivation results in an infection that is never cleared and often difficult to treat. Understanding the balance between latency and reactivation is integral to treating gammaherpesvirus infection and associated disease outcomes. This work characterizes the role of a novel inhibitor of reactivation, host p18INK4c, thereby bringing more clarity to a complex process with significant outcomes for infected individuals.

Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection.

Gammaherpesvirus (GHV) pathogenesis is a complex process that involves productive viral replication, dissemination to tissues that harbor lifelong latent infection, and reactivation from latency back into a productive replication cycle. Traditional loss-of-function mutagenesis approaches in mice using murine gammaherpesvirus 68 (MHV68), a model that allows for examination of GHV pathogenesis in vivo, have been invaluable for defining requirements for specific viral gene products in GHV infection. But these approaches are insufficient to fully reveal how viral gene products contribute when the encoded protein facilitates multiple processes in the infectious cycle and when these functions vary over time and from one host tissue to another. To address this complexity, we developed an MHV68 genetic platform that enables cell-type-specific and inducible viral gene deletion in vivo. We employed this system to re-evaluate functions of the MHV68 latency-associated nuclear antigen (mLANA), a protein with roles in both viral replication and latency. Cre-mediated deletion in mice of loxP-flanked ORF73 demonstrated the necessity of mLANA in B cells for MHV68 latency establishment. Impaired latency during the transition from draining lymph nodes to blood following mLANA deletion also was observed, supporting the hypothesis that B cells are a major conduit for viral dissemination. Ablation of mLANA in infected germinal center (GC) B cells severely impaired viral latency, indicating the importance of viral passage through the GC for latency establishment. Finally, induced ablation of mLANA during latency resulted in complete loss of affected viral genomes, indicating that mLANA is critically important for maintenance of viral genomes during stable latency. Collectively, these experiments provide new insights into LANA homolog functions in GHV colonization of the host and highlight the potential of a new MHV68 genetic platform to foster a more complete understanding of viral gene functions at discrete stages of GHV pathogenesis.

Murine Gammaherpesvirus 68 Expressing Kaposi Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen (LANA) Reveals both Functional Conservation and Divergence in LANA Homologs.

Latency-associated nuclear antigen (LANA) is a multifunctional protein encoded by members of the Rhadinovirus genus of gammaherpesviruses. Studies using murine gammaherpesvirus 68 (MHV68) demonstrated that LANA is important for acute replication, latency establishment, and reactivation in vivo Despite structural similarities in their DNA-binding domains (DBDs), LANA homologs from Kaposi sarcoma-associated herpesvirus (KSHV) and MHV68 exhibit considerable sequence divergence. We sought to determine if KSHV and MHV68 LANA homologs are functionally interchangeable. We generated an MHV68 virus that encodes KSHV LANA (kLANA) in place of MHV68 LANA (mLANA) and evaluated the virus's capacity to replicate, establish and maintain latency, and reactivate. kLANA knock-in (KLKI) MHV68 was replication competent in vitro and in vivo but exhibited slower growth kinetics and lower titers than wild-type (WT) MHV68. Following inoculation of mice, KLKI MHV68 established and maintained latency in splenocytes and peritoneal cells but did not reactivate efficiently ex vivo kLANA repressed the MHV68 promoter for ORF50, the gene that encodes the major lytic transactivator protein RTA, while mLANA did not, suggesting a likely mechanism for the KLKI MHV68 phenotypes. Bypassing this repression by providing MHV68 RTA in trans rescued KLKI MHV68 replication in tissue culture and enabled detection of KLKI MHV68 reactivation ex vivo These data demonstrate that kLANA and mLANA are functionally interchangeable for establishment and maintenance of latency and suggest that repression of lytic replication by kLANA, as previously shown with KSHV, is a kLANA-specific function that is transferable to MHV68.IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are members of the Rhadinovirus genus of gammaherpesviruses. These viruses establish lifelong infections that place their respective human and murine hosts at risk for cancer. Latency-associated nuclear antigen (LANA) is a conserved Rhadinovirus protein that is necessary for long-term chronic infection by these viruses. To better understand the conserved functions performed by LANA homologs, we generated a recombinant MHV68 virus that encodes the KSHV LANA protein in place of the MHV68 LANA homolog. We determined that the KSHV LANA protein is capable of supporting MHV68 latency in a mouse model of chronic infection but also functions to repress viral replication. This work describes an in vivo model system for defining evolutionarily conserved and divergent functions of LANA homologs in Rhadinovirus infection and disease.

A gammaherpesvirus cooperates with interferon-alpha/beta-induced IRF2 to halt viral replication, control reactivation, and minimize host lethality.

The gammaherpesviruses, including Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish latency in memory B lymphocytes and promote lymphoproliferative disease in immunocompromised individuals. The precise immune mechanisms that prevent gammaherpesvirus reactivation and tumorigenesis are poorly defined. Murine gammaherpesvirus 68 (MHV68) is closely related to EBV and KSHV, and type I (alpha/beta) interferons (IFNαß) regulate MHV68 reactivation from both B cells and macrophages by unknown mechanisms. Here we demonstrate that IFNß is highly upregulated during latent infection, in the absence of detectable MHV68 replication. We identify an interferon-stimulated response element (ISRE) in the MHV68 M2 gene promoter that is bound by the IFNαß-induced transcriptional repressor IRF2 during latency in vivo. The M2 protein regulates B cell signaling to promote establishment of latency and reactivation. Virus lacking the M2 ISRE (ISREΔ) overexpresses M2 mRNA and displays uncontrolled acute replication in vivo, higher latent viral load, and aberrantly high reactivation from latency. These phenotypes of the ISREΔ mutant are B-cell-specific, require IRF2, and correlate with a significant increase in virulence in a model of acute viral pneumonia. We therefore identify a mechanism by which a gammaherpesvirus subverts host IFNαß signaling in a surprisingly cooperative manner, to directly repress viral replication and reactivation and enforce latency, thereby minimizing acute host disease. Since we find ISREs 5' to the major lymphocyte latency genes of multiple rodent, primate, and human gammaherpesviruses, we propose that cooperative subversion of IFNαß-induced IRFs to promote latent infection is an ancient strategy that ensures a stable, minimally-pathogenic virus-host relationship.

Latent herpesvirus infection arms NK cells.

Natural killer (NK) cells were identified by their ability to kill target cells without previous sensitization. However, without an antecedent "arming" event, NK cells can recognize, but are not equipped to kill, target cells. How NK cells become armed in vivo in healthy hosts is unclear. Because latent herpesviruses are highly prevalent and alter multiple aspects of host immunity, we hypothesized that latent herpesvirus infection would arm NK cells. Here we show that NK cells from mice latently infected with Murid herpesvirus 4 (MuHV-4) were armed as evidenced by increased granzyme B protein expression, cytotoxicity, and interferon-gamma production. NK-cell arming occurred rapidly in the latently infected host and did not require acute viral infection. Furthermore, NK cells armed by latent infection protected the host against a lethal lymphoma challenge. Thus, the immune environment created by latent herpesvirus infection provides a mechanism whereby host NK-cell function is enhanced in vivo.

CXCR4 is a key regulator of neutrophil release from the bone marrow under basal and stress granulopoiesis conditions.

The number of neutrophils in the blood is tightly regulated to ensure adequate protection against microbial pathogens while minimizing damage to host tissue. Neutrophil homeostasis in the blood is achieved through a balance of neutrophil production, release from the bone marrow, and clearance from the circulation. Accumulating evidence suggests that signaling by CXCL12, through its major receptor CXCR4, plays a key role in maintaining neutrophil homeostasis. Herein, we generated mice with a myeloid lineage-restricted deletion of CXCR4 to define the mechanisms by which CXCR4 signals regulate this process. We show that CXCR4 negatively regulates neutrophil release from the bone marrow in a cell-autonomous fashion. However, CXCR4 is dispensable for neutrophil clearance from the circulation. Neutrophil mobilization responses to granulocyte colony-stimulating factor (G-CSF), CXCL2, or Listeria monocytogenes infection are absent or impaired, suggesting that disruption of CXCR4 signaling may be a common step mediating neutrophil release. Collectively, these data suggest that CXCR4 signaling maintains neutrophil homeostasis in the blood under both basal and stress granulopoiesis conditions primarily by regulating neutrophil release from the bone marrow.

Murine gammaherpesvirus 68 genes both induce and suppress lymphoproliferative disease.

Gammaherpesvirus infection is associated with an increased incidence of lymphoproliferative disease in immunocompromised hosts. Murine gammaherpesvirus 68 (gammaHV68) infection of BALB beta(2)-microglobulin-deficient (BALB beta(2)m(-/-)) mice provides an animal model for analysis of the mechanisms responsible for the induction of a lymphoproliferative disease, atypical lymphoid hyperplasia (ALH), that is pathologically similar to posttransplant lymphoproliferative disease associated with Epstein-Barr virus infection. Here we report that the gammaHV68 v-cyclin and v-bcl-2 genes are required for the efficient induction of gammaHV68-associated ALH in BALB beta(2)m(-/-) mice, while the v-GPCR gene is dispensable for ALH induction. In contrast to these findings, deletion of the viral M1 gene enhanced ALH. Thus, gammaHV68 genes can either inhibit or enhance the induction of lymphoproliferative disease in immunocompromised mice.

Herpesvirus latency confers symbiotic protection from bacterial infection.

All humans become infected with multiple herpesviruses during childhood. After clearance of acute infection, herpesviruses enter a dormant state known as latency. Latency persists for the life of the host and is presumed to be parasitic, as it leaves the individual at risk for subsequent viral reactivation and disease. Here we show that herpesvirus latency also confers a surprising benefit to the host. Mice latently infected with either murine gammaherpesvirus 68 or murine cytomegalovirus, which are genetically highly similar to the human pathogens Epstein-Barr virus and human cytomegalovirus, respectively, are resistant to infection with the bacterial pathogens Listeria monocytogenes and Yersinia pestis. Latency-induced protection is not antigen specific but involves prolonged production of the antiviral cytokine interferon-gamma and systemic activation of macrophages. Latency thereby upregulates the basal activation state of innate immunity against subsequent infections. We speculate that herpesvirus latency may also sculpt the immune response to self and environmental antigens through establishment of a polarized cytokine environment. Thus, whereas the immune evasion capabilities and lifelong persistence of herpesviruses are commonly viewed as solely pathogenic, our data suggest that latency is a symbiotic relationship with immune benefits for the host.

Extraskeletal mesenchymal chondrosarcoma: case report.

This is a case report of an extraskeletal mesenchymal chondrosarcoma (ESMC) that originally occurred in the retroperitoneum of a 24-year-old female and later metastasized to the left proximal humerus. Mesenchymal chondrosarcomas are very rare in comparison to conventional chondrosarcomas and even more so when arising from an extraskeletal location. In this report, we discuss the major characteristics of ESMC and offer a review of the current knowledge regarding this rare disease entity.