Immune modulation during latent herpesvirus infection.

Nearly all human beings, by the time they reach adolescence, are infected with multiple herpesviruses. At any given time, this family of viruses accounts for 35-40 billion human infections worldwide, making herpesviruses among the most prevalent pathogens known to exist. Compared to most other viruses, herpesviruses are also unique in that infection lasts the life of the host. Remarkably, despite their prevalence and persistence, little is known about how these viruses interact with their hosts, especially during the clinically asymptomatic phase of infection referred to as latency. This review explores data in human and animal systems that reveal the ability of latent herpesviruses to modulate the immune response to self and environmental antigens. From the perspective of the host, there are both potentially detrimental and surprisingly beneficial effects of this lifelong interaction. The realization that latent herpesvirus infection modulates immune responses in asymptomatic hosts forces us to reconsider what constitutes a 'normal' immune system in a healthy individual.

Type I interferon signaling enhances CD8+ T cell effector function and differentiation during murine gammaherpesvirus 68 infection.

UNLABELLED: CD8(+) T cell responses are critical to the control of replication and reactivation associated with gammaherpesvirus infection. Type I interferons (IFNs) have been shown to have direct and indirect roles in supporting CD8(+) T cell development and function during viral infection; however, the role of type I interferons during latent viral infection has not been examined. Mice deficient in type I IFN signaling (IFNAR1(-/-) mice) have high levels of reactivation during infection with murine gammaherpesvirus 68 (MHV68), a murine gammaherpesvirus model for Epstein-Barr virus. We hypothesized that type I IFNs function to enhance the anti-gammaherpesvirus CD8(+) T cell response. To test this, IFNAR1(-/-) mice were infected with MHV68 and the CD8(+) T cell response was analyzed. In the absence of type I IFN signaling, there was a marked increase in short-lived effector CD8(+) T cells, and MHV68-specific CD8(+) T cells had upregulated expression of PD-1 and reduced tumor necrosis factor alpha (TNF-α), gamma IFN (IFN-γ), and interleukin-2 (IL-2) production. Suppressing MHV68 replication early in infection using the antiviral cidofovir rescued CD8(+) T cell cytokine production and reduced PD-1 expression. However, suppressing high levels of reactivation in IFNAR1(-/-) mice failed to improve CD8(+) T cell cytokine production during latency. T cell-specific abrogation of type I IFN signaling showed that the effects of type I IFNs on the CD8(+) T cell response during MHV68 infection are independent of direct type I IFN signaling on T cells. Our findings support a model in which type I IFNs likely suppress MHV68 replication, thus limiting viral antigen and facilitating an effective gammaherpesvirus-directed CD8(+) T cell response. IMPORTANCE: The murine gammaherpesvirus MHV68 has both genetic and biologic homology to the human gammaherpesvirus Epstein-Barr virus (EBV), which infects over 90% of humans. Latent EBV infection and reactivation are associated with various life-threatening diseases and malignancies. Host suppression of gammaherpesvirus latency and reactivation requires both CD8(+) T cells as well as type I interferon signaling. Type I IFNs have been shown to critically support the antiviral CD8(+) T cell response in other virus models. Here, we identify an indirect role for type I IFN signaling in enhancing gammaherpesvirus-specific CD8(+) T cell cytokine production. Further, this function of type I IFN signaling can be partially rescued by suppressing viral replication during early MHV68 infection. Our data suggest that type I IFN signaling on non-T cells can enhance CD8(+) T cell function during gammaherpesvirus infection, potentially through suppression of MHV68 replication.

Gammaherpesvirus latency differentially impacts the generation of primary versus secondary memory CD8+ T cells during subsequent infection.

UNLABELLED: Unlike laboratory animals, humans are infected with multiple pathogens, including the highly prevalent herpesviruses. The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. Mice were first infected with murine gammaherpesvirus 68 (MHV68), a model of Epstein-Barr virus (EBV) infection, and then after latency was established, they were challenged with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). The initial replication of LCMV was lower in latently infected mice, and the maturation of dendritic cells was abated. Although the number of LCMV-specific effector CD8(+) T cells was not altered, they were skewed to a memory phenotype. In contrast, LCMV-specific effector CD4(+) T cells were increased in latently infected mice compared to those in mice infected solely with LCMV. When the memory phase was reached, latently infected mice had an LCMV-specific memory T cell pool that was increased relative to that found in singly infected mice. Importantly, LCMV-specific memory CD8(+) T cells had decreased CD27 and increased killer cell lectin-like receptor G1 (KLRG1) expression. Upon secondary challenge, LCMV-specific secondary effector CD8(+) T cells expanded and cleared the infection. However, the LCMV-specific secondary memory CD8(+) T cell pool was decreased in latently infected animals, abrogating the boosting effect normally observed following rechallenge. Taken together, these results demonstrate that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8(+) T cells during acute infection. IMPORTANCE: CD8(+) T cells are critical for the clearance of intracellular pathogens, including viruses, certain bacteria, and tumors. However, current models for memory CD8(+) T cell differentiation are derived from pathogen-free laboratory mice challenged with a single pathogen or vaccine vector. Unlike laboratory animals, all humans are infected with multiple acute and chronic pathogens, including the highly prevalent herpesviruses Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses (HSV), and varicella-zoster virus (VZV). The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. We observed that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8(+) T cells during acute infection. These results suggest that unlike pathogen-free laboratory mice, infection or immunization of latently infected humans may result in the generation of T cells with limited potential for long-term protection.

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.

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.

Organ-specific roles for transcription factor NF-kappaB in reovirus-induced apoptosis and disease.

Reovirus induces apoptosis in cultured cells and in vivo. In cell culture models, apoptosis is contingent upon a mechanism involving reovirus-induced activation of transcription factor NF-kappaB complexes containing p50 and p65/RelA subunits. To explore the in vivo role of NF-kappaB in this process, we tested the capacity of reovirus to induce apoptosis in mice lacking a functional nfkb1/p50 gene. The genetic defect had no apparent effect on reovirus replication in the intestine or dissemination to secondary sites of infection. In comparison to what was observed in wild-type controls, apoptosis was significantly diminished in the CNS of p50-null mice following reovirus infection. In sharp contrast, the loss of p50 was associated with massive reovirus-induced apoptosis and uncontrolled reovirus replication in the heart. Levels of IFN-beta mRNA were markedly increased in the hearts of wild-type animals but not p50-null animals infected with reovirus. Treatment of p50-null mice with IFN-beta substantially diminished reovirus replication and apoptosis, which suggests that IFN-beta induction by NF-kappaB protects against reovirus-induced myocarditis. These findings reveal an organ-specific role for NF-kappaB in the regulation of reovirus-induced apoptosis, which modulates encephalitis and myocarditis associated with reovirus infection.

Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease.

Infection of neonatal mice with some reovirus strains produces a disease similar to infantile biliary atresia, but previous attempts to correlate reovirus infection with this disease have yielded conflicting results. We used isogenic reovirus strains T3SA- and T3SA+, which differ solely in the capacity to bind sialic acid as a coreceptor, to define the role of sialic acid in reovirus encephalitis and biliary tract infection in mice. Growth in the intestine was equivalent for both strains following peroral inoculation. However, T3SA+ spread more rapidly from the intestine to distant sites and replicated to higher titers in spleen, liver, and brain. Strikingly, mice infected with T3SA+ but not T3SA- developed steatorrhea and bilirubinemia. Liver tissue from mice infected with T3SA+ demonstrated intense inflammation focused at intrahepatic bile ducts, pathology analogous to that found in biliary atresia in humans, and high levels of T3SA+ antigen in bile duct epithelial cells. T3SA+ bound 100-fold more efficiently than T3SA- to human cholangiocarcinoma cells. These observations suggest that the carbohydrate-binding specificity of a virus can dramatically alter disease in the host and highlight the need for epidemiologic studies focusing on infection by sialic acid-binding reovirus strains as a possible contributor to the pathogenesis of neonatal biliary atresia.

Unsupervised learning techniques reveal heterogeneity in memory CD8+ T cell differentiation following acute, chronic and latent viral infections.

CD8+ T lymphocytes are critical for the control of gammaherpesvirus latency. To determine how memory CD8+ T cells generated during latency differ from those primed during acute or chronic viral infection, we adoptively transferred naive P14 CD8+ T cells into uninfected recipients, and examined surface proteins, cytokines and transcription factors following infection with the Armstrong (acute) or Clone 13 (chronic) strains of lymphocytic choriomeningitis virus (LCMV), or murine gammaherpesvirus 68 (MHV68) expressing the LCMV epitope DbGP33-41. By performing k-means clustering and generating self organizing maps (SOM), we observed increased short-lived effector-like, CD27lo CD62Llo and Bcl-6lo CD8+ T cells following latent infection. In addition, we found that memory CD8+ T cells from latent primed mice underwent less expansion following adoptive transfer and antigen rechallenge. Data from cluster models were combined and visualized by principal component analysis (PCA) demonstrating memory CD8+ T cells from latent infection occupy an intermediate differentiation space.

(C)Re-combining textbook models of virus spread within the host.

Classic viral pathogenesis models postulate that tissues supporting efficient virus replication promote virus dissemination, which culminates in clinical illness. In this issue of Cell Host & Microbe, Sacher and colleagues use Cre/loxP recombination to label murine cytomegalovirus during replication in distinct cell types in vivo. Strikingly, they demonstrate that the most productive cell type in the host-the hepatocyte-contributes no progeny to dissemination to other tissues.

Gamma interferon blocks gammaherpesvirus reactivation from latency.

Establishment of latent infection and reactivation from latency are critical aspects of herpesvirus infection and pathogenesis. Interfering with either of these steps in the herpesvirus life cycle may offer a novel strategy for controlling herpesvirus infection and associated disease pathogenesis. Prior studies show that mice deficient in gamma interferon (IFN-gamma) or the IFN-gamma receptor have elevated numbers of cells reactivating from murine gammaherpesvirus 68 (gammaHV68) latency, produce infectious virus after the establishment of latency, and develop large-vessel vasculitis. Here, we demonstrate that IFN-gamma is a powerful inhibitor of reactivation of gammaHV68 from latency in tissue culture. In vivo, IFN-gamma controls viral gene expression during latency. Importantly, depletion of IFN-gamma in latently infected mice results in an increased frequency of cells reactivating virus. This demonstrates that IFN-gamma is important for immune surveillance that limits reactivation of gammaHV68 from latency.

Reovirus delays diabetes onset but does not prevent insulitis in nonobese diabetic mice.

Mice infected with reovirus develop abnormalities in glucose homeostasis. Reovirus strain type 3 Abney (T3A) was capable of systemic infection of nonobese diabetic (NOD) mice, an experimental model of autoimmune diabetes. Reovirus antigen was detected in pancreatic islets of T3A-infected mice, and primary cultures of pancreatic islets from NOD mice supported T3A growth. Significantly fewer T3A-infected animals compared to uninfected controls developed diabetes. However, despite the alteration in diabetes penetrance, insulitis was evident in T3A-infected mice. These results suggest that viral infection of NOD mice alters autoimmune responses to beta-cell antigens and thereby delays development of diabetes.

Alpha/beta interferons regulate murine gammaherpesvirus latent gene expression and reactivation from latency.

Alpha/beta interferon (IFN-alpha/beta) protects the host from virus infection by inhibition of lytic virus replication in infected cells and modulation of the antiviral cell-mediated immune response. To determine whether IFN-alpha/beta also modulates the virus-host interaction during latent virus infection, we infected mice lacking the IFN-alpha/beta receptor (IFN-alpha/betaR(-/-)) and wild-type (wt; 129S2/SvPas) mice with murine gammaherpesvirus 68 (gammaHV68), a lymphotropic gamma-2-herpesvirus that establishes latent infection in B cells, macrophages, and dendritic cells. IFN-alpha/betaR(-/-) mice cleared low-dose intranasal gammaHV68 infection with wt kinetics and harbored essentially wt frequencies of latently infected cells in both peritoneum and spleen by 28 days postinfection. However, latent virus in peritoneal cells and splenocytes from IFN-alpha/betaR(-/-) mice reactivated ex vivo with >40-fold- and 5-fold-enhanced efficiency, respectively, compared to wt cells. Depletion of IFN-alpha/beta from wt mice during viral latency also significantly increased viral reactivation, demonstrating an antiviral function of IFN-alpha/beta during latency. Viral reactivation efficiency was temporally regulated in both wt and IFN-alpha/betaR(-/-) mice. The mechanism of IFN-alpha/betaR action was distinct from that of IFN-gammaR, since IFN-alpha/betaR(-/-) mice did not display persistent virus replication in vivo. Analysis of viral latent gene expression in vivo demonstrated specific upregulation of the latency-associated gene M2, which is required for efficient reactivation from latency, in IFN-alpha/betaR(-/-) splenocytes. These data demonstrate that an IFN-alpha/beta-induced pathway regulates gammaHV68 gene expression patterns during latent viral infection in vivo and that IFN-alpha/beta plays a critical role in inhibiting viral reactivation during latency.

Isolation and molecular characterization of a novel type 3 reovirus from a child with meningitis.

Mammalian reoviruses are non-enveloped viruses that contain a segmented, double-stranded RNA genome. Reoviruses infect most mammalian species, although infection with these viruses in humans is usually asymptomatic. We report the isolation of a novel reovirus strain from a 6.5-week-old child with meningitis. Hemagglutination and neutralization assays indicated that the isolate is a serotype 3 strain, leading to the designation T3/Human/Colorado/1996 (T3C/96). Sequence analysis of the T3C/96 S1 gene segment, which encodes the viral attachment protein, sigma 1, confirmed the serotype assignment for this strain and indicated that T3C/96 is a novel reovirus isolate. T3C/96 is capable of systemic spread in newborn mice after peroral inoculation and produces lethal encephalitis. These results suggest that serotype 3 reoviruses can cause meningitis in humans.