Clinical latency and reactivation of AIDS-related mycobacterial infections

The immune mechanisms associated with the evolution from latent to clinically active mycobacterial coinfection in human immunodeficiency virus type 1 (HIV-1)-infected humans remain poorly understood. Previous work has demonstrated that macaques infected with simian immunodeficiency virus (SIVmac) can develop persistent Mycobacterium bovis BCG coinfection and a fatal SIV-related tuberculosis-like disease by 4 months after BCG inoculation. In the present study, SIVmac-infected monkeys that developed clinically quiescent mycobacterial infection after BCG inoculation were followed prospectively for the reactivation of the BCG and the development of SIV-related tuberculosis-like disease. The development of clinically latent BCG coinfection in these SIVmac-infected monkeys was characterized by a change from high to undetectable levels of bacterial organisms, with or without measurable BCG mRNA expression in lymph node cells. The reactivation of clinically latent BCG coinfection and development of SIV-related tuberculosis-like disease were then observed in these SIVmac-BCG-coinfected monkeys during a 21-month period of follow-up. The reactivation of SIV-related tuberculosis-like disease in these animals coincided with a severe depletion of CD4 T cells and a loss of BCG-specific T-cell responses. Interestingly, bacterial superantigen challenge of the SIVmac-BCG-coinfected monkeys resulted in an up-regulation of clinically latent BCG coinfection, suggesting that infection with superantigen-producing microbes may increase the susceptibility of individuals to the reactivation of AIDS-related mycobacterial coinfection. Thus, reactivation of latent mycobacterial infections in HIV-1-infected individuals may result from a loss of T-cell immunity or from a superimposed further compromise of the immune system.     

Histone acetylation and reactivation of Epstein-Barr virus from latency

Induction of the viral BZLF1 gene has previously been shown to be one of the first steps in the reactivation of Epstein-Barr virus (EBV). Using an EBV oriP episomal vector system, we have reconstituted the regulation of the promoter for BZLF1 on stably transfected episomes, mapped promoter elements required for that regulation, and investigated mechanisms that may control the switch between latency and the lytic cycle. Changes in histone acetylation at the promoter for the BZLF1 gene appear to be a key part of the reactivation mechanism of this herpesvirus.     

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.     

CpG methylation controls reactivation of HIV from latency

What are the purposes of prizes and recognitions? Are they lagging indicators of past achievements or leading indicators of things to come?     

The role of microRNAs in Epstein-Barr virus latency and lytic reactivation

Oncogenic viruses reprogram host gene expression driving proliferation, ensuring survival, and evading the immune response. The recent appreciation of microRNAs (miRNAs) as small non-coding RNAs that broadly regulate gene expression has provided new insight into this complex scheme of host control. This review highlights the role of viral and cellular miRNAs during the latent and lytic phases of the EBV life cycle.     

Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.     

Rise and survival of bovine herpesvirus 1 recombinants after primary infection and reactivation from latency

Recombination is thought to be an important source of genetic variation in herpesviruses. Several studies, performed in vitro or in vivo, detected recombinant viruses after the coinoculation of two distinguishable strains of the same herpesvirus species. However, none of these studies investigated the evolution of the relative proportions of parental versus recombinant progeny populations after coinoculation of the natural host, both during the excretion and the reexcretion period. In the present study, we address this by studying the infection of cattle with bovine herpesvirus 1 (BoHV-1). The recombination of two BoHV-1 mutants lacking either glycoprotein C (gC(-)/gE(+)) or E (gC(+)/gE(-)) was investigated after inoculation of cattle by the natural route of infection. The results demonstrated that (i) recombination is a frequent event in vivo since recombinants (gC(+)/gE(+) and gC(-)/gE(-)) were detected in all coinoculated calves, (ii) relative proportions of progeny populations evolved during the excretion period toward a situation where two populations (gC(+)/gE(+) and gC(-)/gE(+)) predominated without fully outcompeting the presence of the two other detected populations (gC(+)/gE(-) and gC(-)/gE(-)), and (iii) after reactivation from latency, no gC(+)/gE(-) and gC(-)/gE(-) progeny viruses were detected, although gC(+)/gE(-) mutants, when inoculated alone, were detected after reactivation treatment. In view of these data, the importance of gE in the biology of BoHV-1 infection and the role of recombination in herpesvirus evolution are discussed.     

Reactive oxygen species hydrogen peroxide mediates Kaposi's sarcoma-associated herpesvirus reactivation from latency

Kaposi''s sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the host following an acute infection. Reactivation from latency contributes to the development of KSHV-induced malignancies, which include Kaposi''s sarcoma (KS), the most common cancer in untreated AIDS patients, primary effusion lymphoma and multicentric Castleman''s disease. However, the physiological cues that trigger KSHV reactivation remain unclear. Here, we show that the reactive oxygen species (ROS) hydrogen peroxide (H₂O₂) induces KSHV reactivation from latency through both autocrine and paracrine signaling. Furthermore, KSHV spontaneous lytic replication, and KSHV reactivation from latency induced by oxidative stress, hypoxia, and proinflammatory and proangiogenic cytokines are mediated by H₂O₂. Mechanistically, H₂O₂ induction of KSHV reactivation depends on the activation of mitogen-activated protein kinase ERK1/2, JNK, and p38 pathways. Significantly, H₂O₂ scavengers N-acetyl-L-cysteine (NAC), catalase and glutathione inhibit KSHV lytic replication in culture. In a mouse model of KSHV-induced lymphoma, NAC effectively inhibits KSHV lytic replication and significantly prolongs the lifespan of the mice. These results directly relate KSHV reactivation to oxidative stress and inflammation, which are physiological hallmarks of KS patients. The discovery of this novel mechanism of KSHV reactivation indicates that antioxidants and anti-inflammation drugs could be promising preventive and therapeutic agents for effectively targeting KSHV replication and KSHV-related malignancies.     

Gamma interferon blocks gammaherpesvirus reactivation from latency in a cell type-specific manner

Gammaherpesviruses are important pathogens whose lifelong survival in the host depends critically on their capacity to establish and reactivate from latency, processes regulated by both viral genes and the host immune response. Previous work has demonstrated that gamma interferon (IFN-gamma) is a key regulator of chronic infection with murine gammaherpesvirus 68 (gammaHV68), a virus that establishes latent infection in B lymphocytes, macrophages, and dendritic cells. In mice deficient in IFN-gamma or the IFN-gamma receptor, gammaHV68 gene expression is altered during chronic infection, and peritoneal cells explanted from these mice reactivate more efficiently ex vivo than cells derived from wild-type mice. Furthermore, treatment with IFN-gamma inhibits reactivation of gammaHV68 from latently infected wild-type peritoneal cells, and depletion of IFN-gamma from wild-type mice increases the efficiency of reactivation of explanted peritoneal cells. These profound effects of IFN-gamma on chronic gammaHV68 latency and reactivation raise the question of which cells respond to IFN-gamma to control chronic gammaHV68 infection. Here, we show that IFN-gamma inhibited reactivation of peritoneal cells and spleen cells harvested from mice lacking B lymphocytes, but not wild-type spleen cells, suggesting that IFN-gamma may inhibit reactivation in a cell type-specific manner. To directly test this hypothesis, we expressed the diphtheria toxin receptor specifically on either B lymphocytes or macrophages and used diphtheria toxin treatment to deplete these specific cells in vivo and in vitro after establishing latency. We demonstrate that macrophages, but not B cells, are responsive to IFN-gamma-mediated suppression of gammaHV68 reactivation. These data indicate that the regulation of gammaherpesvirus latency by IFN-gamma is cell type specific and raise the possibility that cell type-specific immune deficiency may alter latency in distinct and important ways.     

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.     

Murine gammaherpesvirus 68 cyclin D homologue is required for efficient reactivation from latency

Murine gammaherpesvirus 68 (MHV68) is a gammaherpesvirus that was first isolated from murid rodents. MHV68 establishes a latent infection in the spleen and other lymphoid organs. Several gammaherpesviruses, including herpesvirus saimiri, human herpesvirus 8, and MHV68, encode proteins with extensive homology to the D-type cyclins. To study the function of the cyclin homologue, a recombinant MHV68 has been constructed that lacks the cyclin homologue and expresses beta-galactosidase as a marker (MHV68(cy-)). MHV68(cy-) grows in vitro with kinetics and to titers similar to those of the wild type. BALB/c mice infected with mixtures of equivalent amounts of the wild type and MHV68(cy-) show deficient growth of the MHV68(cy-) in an acute infection. Infection of SCID mice with virus mixtures also showed decreased MHV68(cy-) virus growth, indicating that the deficiency is not mediated by T or B cells. Although mice infected with mixtures containing 100 times as much MHV68(cy-) had greater splenic titers of the mutant virus than wild-type virus in acute infection, at 28 days postinfection splenocytes from these mice reactivated primarily wild-type virus. Quantitative PCR data indicate that equivalent genomes were present in the latent state. Reinsertion of the cyclin homologue into the cyclin-deleted virus restored the wild-type phenotype. These results indicate that the MHV68 cyclin D homologue mediates important functions in the acute infection and is required for efficient reactivation from latency.     

Adaptive and innate transforming growth factor beta signaling impact herpes simplex virus 1 latency and reactivation

Innate and adaptive immunity play important protective roles by combating herpes simplex virus 1 (HSV-1) infection. Transforming growth factor β (TGF-β) is a key negative cytokine regulator of both innate and adaptive immune responses. Yet, it is unknown whether TGF-β signaling in either immune compartment impacts HSV-1 replication and latency. We undertook genetic approaches to address these issues by infecting two different dominant negative TGF-β receptor type II transgenic mouse lines. These mice have specific TGF-β signaling blockades in either T cells or innate cells. Mice were ocularly infected with HSV-1 to evaluate the effects of restricted innate or adaptive TGF-β signaling during acute and latent infections. Limiting innate cell but not T cell TGF-β signaling reduced virus replication in the eyes of infected mice. On the other hand, blocking TGF-β signaling in either innate cells or T cells resulted in decreased latency in the trigeminal ganglia of infected mice. Furthermore, inhibiting TGF-β signaling in T cells reduced cell lysis and leukocyte infiltration in corneas and trigeminal ganglia during primary HSV-1 infection of mice. These findings strongly suggest that TGF-β signaling, which generally functions to dampen immune responses, results in increased HSV-1 latency.