No abstract

DNA methylation of retroviral promoters and enhancers localized in the provirus 5′ long terminal repeat (LTR) is considered to be a mechanism of transcriptional suppression that allows retroviruses to evade host immune responses and antiretroviral drugs. However, the role of DNA methylation in the control of HIV-1 latency has never been unambiguously demonstrated, in contrast to the apparent importance of transcriptional interference and chromatin structure, and has never been studied in HIV-1-infected patients. Here, we show in an in vitro model of reactivable latency and in a latent reservoir of HIV-1-infected patients that CpG methylation of the HIV-1 5′ LTR is an additional epigenetic restriction mechanism, which controls resistance of latent HIV-1 to reactivation signals and thus determines the stability of the HIV-1 latency. CpG methylation acts as a late event during establishment of HIV-1 latency and is not required for the initial provirus silencing. Indeed, the latent reservoir of some aviremic patients contained high proportions of the non-methylated 5′ LTR. The latency controlled solely by transcriptional interference and by chromatin-dependent mechanisms in the absence of significant promoter DNA methylation tends to be leaky and easily reactivable. In the latent reservoir of HIV-1-infected individuals without detectable plasma viremia, we found HIV-1 promoters and enhancers to be hypermethylated and resistant to reactivation, as opposed to the hypomethylated 5′ LTR in viremic patients. However, even dense methylation of the HIV-1 5′LTR did not confer complete resistance to reactivation of latent HIV-1 with some histone deacetylase inhibitors, protein kinase C agonists, TNF-α, and their combinations with 5-aza-2deoxycytidine: the densely methylated HIV-1 promoter was most efficiently reactivated in virtual absence of T cell activation by suberoylanilide hydroxamic acid. Tight but incomplete control of HIV-1 latency by CpG methylation might have important implications for strategies aimed at eradicating HIV-1 infection.

Natural killer (NK) cells are affected by infection with human cytomegalovirus (HCMV) manifested by increased expression of the HLA-E binding activating receptor NKG2C. We here show that HCMV seropositivity was associated with a profound expansion of NKG2C killer cell immunoglobulin-like receptors (KIRs) specific for self-HLA class I molecules, with predominant usage of KIR2DL2/3. KIR engagement dampened NKG2C-mediated activation suggesting that such biased expression of self-specific KIRs may preserve self-tolerance and limit immune-pathology during viral infection. Together, these findings shed new light on how the human NK-cell compartment adjusts to HCMV infection resulting in clonal expansion and differentiation of educated and polyfunctional NK cells. IntroductionNatural killer (NK) cells have the ability to kill targets without prior sensitization and their involvement in antiviral and antitumor immunity is well established [1,2]. Recent studies have demonstrated a high degree of functional heterogeneity in the NK-cell compartment attributable to a vast network of inhibitory or activating receptors that allow these cells to recognize target cells [3,4]. Killer cell immunoglobulin-like receptors (KIR) and CD94/NKG2 heterodimers are two major types of HLA class I binding receptors that regulate NK cell function [5,6]. Both these receptor-families exist in activating and inhibitory forms and contribute to the functional education of human NK cells by interactions with their cognate ligands [7], whereas KIR are expressed in a stochastic manner with a variegated distribution in the NK cell population [8,9], NKG2A is expressed on all CD56bright NK cells and disappears gradually during differentiation of CD56 dim NK cells [10,11]. NKG2C and NKG2A are covalently associated with CD94 [12]. Both NKG2A and NKG2C specifically interact with the non-classical MHC class-Ib molecule HLA-E, which is expressed at low levels on almost all nucleated cells, and presents peptides derived from signal sequences of other HLA class-I molecules [13]. The affinity of their interaction depends on the sequence of the HLA-E-bound nonamers and is higher for NKG2A than for NKG2C [14,15]. In the CD56 dim subset, NKG2C expression largely excludes NKG2A expression [10,16]. Expression of NKG2C is induced by co-culture with HCMV-infected fibroblasts and correlates with HCMV seropositivity in healthy donors [16,17]. Recently, NKG2C 1 NK cells were shown to expand during HIV and hantavirus infections in HCMV-seropositive patients, suggesting that HCMV may prime the NK-cell compartment for specific expansion of the NKG2C 1 subset upon additional viral encounters [18,19]. Two recent papers have demonstrated increased expression of NKG2C on NK cells in patients with chronic HBV and HCV infection [20,21]. Therefore, we choose this clinical setting to perform an in-depth characterization of the NKG2C 1 NK-cell subset. We show that NKG2C 1 CD56 dim NK cells are terminally differentiated, highly polyfunctional and display a clonal expression of inhib...

Recent advances in molecular biology have led to the development of novel small molecules that target specific viral proteins of the hepatitis C virus (HCV) life cycle. These drugs, collectively termed directly acting antivirals (DAA) against HCV, include a range of non-structural (NS) 3/NS4A protease, NS5B polymerase, and NS5A inhibitors at various stages of clinical development. The rapid replication rate of HCV, along with the low fidelity of its polymerase, gives rise to generations of mutations throughout the viral genome resulting in remarkable sequence variation in the HCV population, known as a quasispecies. The efficacy of DAAs is limited by the presence of those mutations that give rise to amino-acid substitutions within the targeted protein, and that affect the viral sensitivity to these compounds. Thus, due to the high genetic variability of HCV, variants with reduced susceptibility to DAA can occur naturally even before treatment begins, but usually at low levels. Not surprisingly then, these changes are selected in patients either breaking through or not responding to potent DAA treatment. In vitro or in vivo, six major position mutations in the NS3 HCV protease (36, 54, 155, 156, 168, and 170) have now been reported associated with different levels of resistance. The amino acid composition at several of the drug resistance sites can vary between the HCV genotypes/subtypes, resulting in different consensus amino acids leading to a reduction in replicative fitness as well as reduced DAA sensitivity. Different amino acid diversity profiles for HCV genotypes/subtypes suggest differences in the position/type of immune escape and drug resistance mutations. Also, different pathways of resistance profiles based on the chemical scaffold (linear or macrocyclic) of the protease inhibitors have been described. This review first describes how resistance to a protease inhibitor can develop and then provides an overview of the mechanism of how particular mutations confer varying levels of resistance to protease inhibitor, which have been identified and characterized using both genotypic and phenotypic tools. Future potential therapeutic strategies to assist patients who do develop resistance to protease inhibitors are also outlined. The challenge developing new HCV protease inhibitors should take into consideration not only the antiviral potency of the drugs, the occurrence and importance of side effects, the frequency of oral administration, but also the resistance profiles of these agents.