Latency-associated Gene Expression Research Articles

Co-treatment with Clofazimine and Rapamycin eliminates drug-resistant tuberculosis by inducing polyfunctional central memory T cell responses.

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is acquiring drug resistance at a faster rate than the discovery of new antibiotics. Therefore, alternate therapies that can limit the drug resistance and disease recurrence are urgently needed. Emerging evidences indicate that combined treatment with antibiotics and an immunomodulator provides superior treatment efficacy. Clofazimine (CFZ) enhances the generation of T central memory (TCM) cells by blocking the Kv1.3+ potassium channels. Rapamycin (Rapa) facilitates M.tb clearance by inducing autophagy. In this study, we observed that the co-treatment with CFZ and Rapa potently eliminates both multiple and extensively drug-resistant (MDR and XDR) clinical isolates of M.tb in a mouse model by inducing robust T cell memory and polyfunctional TCM responses. Furthermore, co-treatment reduces the expression of latency-associated genes of M.tb in human macrophages. Therefore, CFZ and Rapa co-therapy holds promise for treating patients infected with MDR and XDR strains of M.tb.

A Single-Cell Approach to the Elusive Latent Human Cytomegalovirus Transcriptome.

ABSTRACTHerpesvirus latency has been difficult to understand molecularly due to low levels of viral genomes and gene expression. In the case of the betaherpesvirus human cytomegalovirus (HCMV), this is further complicated by the heterogeneity inherent to hematopoietic subpopulations harboring genomes and, as a consequence, the various patterns of infection that simultaneously exist in a host, ranging from latent to lytic. Single-cell RNA sequencing (scRNA-seq) provides tremendous potential in measuring the gene expression profiles of heterogeneous cell populations for a wide range of applications, including in studies of cancer, immunology, and infectious disease. A recent study by Shnayder et al. (mBio 9:e00013-18, 2018, https://doi.org/10.1128/mBio.00013-18) utilized scRNA-seq to define transcriptomal characteristics of HCMV latency. They conclude that latency-associated gene expression is similar to the late lytic viral program but at lower levels of expression. The study highlights the numerous challenges, from the definition of latency to the analysis of scRNA-seq, that exist in defining a latent transcriptome.

Defining the Transcriptional Landscape during Cytomegalovirus Latency with Single-Cell RNA Sequencing.

ABSTRACTPrimary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive transcriptome RNA sequencing (RNA-seq) atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence in vivo is prevalent in diverse tissues. Notably, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single-cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program, albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression.

Abstract 4794: Reduced Epstein-Barr virus replication in human papillomavirus-immortalized keratinocyte organotypic raft culture

Abstract An epidemic rise in oropharyngeal squamous cell carcinoma (OSCC) infected with human papillomavirus has been observed over the past decade. The majority of HPV+OSCC arise from the tonsils and base of tongue (BOT), which are lymphoid-rich regions that are known to harbor Epstein-Barr virus (EBV). Indeed, we previously reported HPV/EBV co-infection in 25% of tonsillar and 70% of BOT tumors. HPV is a DNA tumor virus that specifically infects proliferating basal cells in stratified squamous epithelia. HPV replication is facilitated by viral oncoproteins E6 and E7, which create an S-phase-like environment via degradation of cell cycle regulators p53 and Rb, respectively. EBV is also an established DNA tumor virus that replicates in differentiated epithelia in its normal lifecycle. However, EBV exhibits a latent infection characterized by oncogene expression and lack of viral replication in EBV-associated carcinomas. E6/E7-mediated alteration of the cell cycle results in an interruption in epithelial differentiation, which is required for EBV replication in epithelial cells. Thus, we hypothesize that HPV immortalization of keratinocytes shifts the EBV life cycle from replication to latency. To test our hypothesis, we developed an EBV/HPV co-infection model utilizing organotypic raft culture to produce stratified epithelia in vitro. Tonsillar keratinocytes immortalized by HPV were EBV-infected by co-culture with IgG-induced EBV-positive Akata Burkitt’s lymphoma cells four days after differentiation induction. Raft tissues were harvested 6 days post-EBV infection for analysis of viral life cycles. EBV infection did not alter HPV replication or gene expression. In HPV-negative tonsillar keratinocytes, robust EBV replication was observed that was blocked by treatment with acyclovir, a nucleoside analog that inhibits EBV lytic replication. In contrast, a dramatic decrease in EBV DNA levels was observed in HPV-positive keratinocytes, which was not further reduced by acyclovir. Human telomerase-immortalized normal oral keratinocytes supported robust EBV replication in organotypic raft culture suggesting a specific effect of HPV rather than immortalization on EBV replication. Expression of E7 alone was sufficient to decrease EBV DNA levels. Preliminary data suggest no change in EBV infectivity between HPV-negative and HPV-positive rafts, and an increase in EBV latency-associated gene expression in HPV-positive cells. The ability of EBV to establish persistent latent infection in HPV-infected epithelial cells would contribute additional viral oncogene expression to explain the rapid development and progression of HPV+OSCC. Citation Format: Joseph T. Guidry, William K. Songock, Xiaohui Ma, Cherie-Ann O. Nathan, Jason M. Bodily, Rona S. Scott. Reduced Epstein-Barr virus replication in human papillomavirus-immortalized keratinocyte organotypic raft culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2017-4794

Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained Levels of Vascular Endothelial Growth Factors A and C Early during In Vitro Infection of Human Microvascular Dermal Endothelial Cells: Biological Implications

Kaposi's sarcoma (KS), a vascular tumor associated with human immunodeficiency virus type 1 infection, is characterized by spindle-shaped endothelial cells, inflammatory cells, cytokines, growth and angiogenic factors, and angiogenesis. KS spindle cells are believed to be of the lymphatic endothelial cell (LEC) type. Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) is etiologically linked to KS, and in vitro KSHV infection of primary human dermal microvascular endothelial cells (HMVEC-d) is characterized by the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, sustained induction of NF-kappaB and several cytokines, and growth and angiogenic factors. KSHV induced robust vascular endothelial growth factor A (VEGF-A) and VEGF-C gene expression as early as 30 min postinfection (p.i.) in serum-starved HMVEC-d, which was sustained throughout the observation period of 72 h p.i. Significant amounts of VEGF-A and -C were also detected in the culture supernatant of infected cells. VEGF-A and -C were also induced by UV-inactivated KSHV and envelope glycoprotein gpK8.1A, thus suggesting a role for virus entry stages in the early induction of VEGF and requirement of KSHV viral gene expression for sustained induction. Exogenous addition of VEGF-A and -C increased KSHV DNA entry into target cells and moderately increased latent ORF73 and lytic ORF50 promoter activation and gene expression. KSHV infection also induced the expression of lymphatic markers Prox-1 and podoplanin as early as 8 h p.i., and a paracrine effect was seen in the neighboring uninfected cells. Similar observations were also made in the pure blood endothelial cell (BEC)-TIME cells, thus suggesting that commitment to the LEC phenotype is induced early during KSHV infection of blood endothelial cells. Treatment with VEGF-C alone also induced Prox-1 expression in the BEC-TIME cells. Collectively, these studies show that the in vitro microenvironments of KSHV-infected endothelial cells are enriched, with VEGF-A and -C molecules playing key roles in KSHV biology, such as increased infection and gene expression, as well as in angiogenesis and lymphangiogenesis, thus recapitulating the microenvironment of early KS lesions.

Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained NF-κB Activation during De Novo Infection of Primary Human Dermal Microvascular Endothelial Cells That Is Essential for Viral Gene Expression

In vitro Kaposi's sarcoma-associated herpesvirus (KSHV) infection of primary human dermal microvascular endothelial (HMVEC-d) cells and human foreskin fibroblast (HFF) cells is characterized by the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, and induction of several cytokines, growth factors, and angiogenic factors. Since NF-kappaB is a key molecule involved in the regulation of several of these factors, here, we examined NF-kappaB induction during de novo infection of HMVEC-d and HFF cells. Activation of NF-kappaB was observed as early as 5 to 15 min postinfection by KSHV, and translocation of p65-NF-kappaB into nuclei was detected by immunofluorescence assay, electrophoretic mobility shift assay, and p65 enzyme-linked immunosorbent assay. IkappaB phosphorylation inhibitor (Bay11-7082) reduced this activation significantly. A sustained moderate level of NF-kappaB induction was seen during the observed 72 h of in vitro KSHV latency. In contrast, high levels of ERK1/2 activation at earlier time points and a moderate level of activation at later times were observed. p38 mitogen-activated protein kinase was activated only at later time points, and AKT was activated in a cyclic manner. Studies with UV-inactivated KSHV suggested a role for virus entry stages in NF-kappaB induction and a requirement for KSHV viral gene expression in sustained induction. Inhibition of NF-kappaB did not affect target cell entry by KSHV but significantly reduced the expression of viral latent open reading frame 73 and lytic genes. KSHV infection induced the activation of several host transcription factors, including AP-1 family members, as well as several cytokines, growth factors, and angiogenic factors, which were significantly affected by NF-kappaB inhibition. These results suggest that during de novo infection, KSHV induces sustained levels of NF-kappaB to regulate viral and host cell genes and thus possibly regulates the establishment of latent infection.

Cell cycle analysis of Epstein-Barr virus-infected cells following treatment with lytic cycle-inducing agents.

While Epstein-Barr virus (EBV) latency-associated gene expression is associated with cell cycle progression, the relationship between the EBV lytic program and the cell cycle is less clear. Using four different EBV lytic induction systems, we address the relationship between lytic cycle activation and the cell cycle. In three of these systems, G0 or G1 cell growth arrest signaling is observed prior to detection of the EBV immediate-early gene product Zta. In tetradecanoyl phorbol acetate-treated P3HR1 cultures and in 5-iodo-2'-deoxyuridine-treated NPC-KT cultures, cell cycle analysis of Zta-expressing cell populations showed a significant G1 bias during the early stages of lytic cycle progression. In contrast, treatment of the cell line Akata with anti-immunoglobulin (Ig) results in rapid induction of immediate-early gene expression, and accordingly, activation of the immediate-early gene product Zta precedes significant anti-Ig-induced cell cycle effects. Nevertheless, cell cycle analysis of the Zta-expressing population following anti-Ig treatment shows a bias for cells in G1, indicating that anti-Ig-mediated induction of Zta occurs more efficiently in cells traversing G1. Last, although 5-azacytidine treatment of Rael cells results in a G1 arrest in the total cell population which precedes the induction of Zta, cell cycle analysis of the Zta-expressing population shows a significant bias for cells with an apparent G2/M DNA content. This bias may result, in part, from activation of Zta expression following demethylation of the Zta promoter during S-phase. Together, these studies indicate that induction of Zta occurs through several distinct mechanisms, some of which may involve checkpoint signaling.

EBV regulates c-MYC, apoptosis, and tumorigenicity in Burkitt's lymphoma.

Epstein-Barr Virus (EBV) latency-associated gene expression in Burkitt’s lymphoma (BL) cells is limited to a small set of gene products, none of which are known to overtly influence cell growth potential. Designated type I latency, this program of viral gene expression is characteristic of BL tumor cells in vivo and is restricted to expression of the EBNA-1 protein, the BamHI-A rightward transcripts (BARTs), and two small non-coding RNAs, EBER-1 and EBER-2 (Arrand and Rymo 1982; Rowe et al. 1986; Gregory et al. 1990; Brooks et al. 1993; Fries et al. 1997). Notably, with the exception of EBNA-1, which is required for viral genome maintenance, none of these latency-associated gene products is essential for EBV-mediated immortalization of B lymphocytes in vitro (Swaminathan et al. 1991; Robertson et al. 1994). Consequently, a direct contribution of EBV to tumorigenic potential in BL has not been established. Thus, the dominant factor presumed to be responsible for maintenance of tumorigenicity in BL is the deregulated expression of the c-MYC proto-oncogene, which occurs as a consequence of chromosomal translocation (reviewed in Magrath 1990). This assumption, however, was challenged by the work of Takada and colleagues (Shimizu et al. 1994) who found that spontaneous loss of the EBV genome in the BL cell line Akata is associated with a concomitant loss of tumorigenic potential. The main objectives of this study were to define the contribution of EBV and individual latent gene products to tumorigenicity in BL using the Akata BL cell systemKeywordsTumorigenic PotentialUnited States Public Health ServiceCell Growth CycleAkata CellAkata Cell LineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Differential methylation of Epstein-Barr virus latency promoters facilitates viral persistence in healthy seropositive individuals.

Epstein-Barr virus (EBV) establishes a life-long infection in humans, with distinct viral latency programs predominating during acute and chronic phases of infection. Only a subset of the EBV latency-associated antigens present during the acute phase of EBV infection are expressed in the latently infected memory B cells that serve as the long-term EBV reservoir. Since the EBV immortalization program elicits a potent cellular immune response, downregulation of viral gene expression in the long-term latency reservoir is likely to facilitate evasion of the immune response and persistence of EBV in the immunocompetent host. Tissue culture and tumor models of restricted EBV latency have consistently demonstrated a critical role for methylation of the viral genome in maintaining the restricted pattern of latency-associated gene expression. Here we extend these observations to demonstrate that the EBV genomes in the memory B-cell reservoir are also heavily and discretely methylated. This analysis reveals that methylation of the viral genome is a normal aspect of EBV infection in healthy immunocompetent individuals and is not restricted to the development of EBV-associated tumors. In addition, the pattern of methylation very likely accounts for the observed inhibition of the EBV immortalization program and the establishment and maintenance of a restricted latency program. Thus, EBV appears to be the first example of a parasite that usurps the host cell-directed methylation system to regulate pathogen gene expression and thereby establish a chronic infection.

The abundant latency-associated transcripts of herpes simplex virus type 1 are bound to polyribosomes in cultured neuronal cells and during latent infection in mouse trigeminal ganglia.

During herpes simplex virus type 1 (HSV-1) latency, limited viral transcription takes place. This transcription has been linked to the ability of the HSV-1 genome to reactivate and consists of abundant 2.0- and 1.5-kb collinear latency-associated transcripts (LATs), spanned by minor hybridizing RNA (mLAT). The 1.5-kb LAT is derived from the 2.0-kb LAT by splicing, and both transcripts contain two large overlapping open reading frames. The molecular action mechanisms of the latency-associated gene expression are unknown, and no HSV-1 latency-encoded proteins have been convincingly demonstrated. We have cloned the entire latency-associated transcriptionally active HSV-1 DNA fragment (10.4 kb) under control of a constitutive promoter and generated a neuronal cell line (NA4) stably transfected with the viral LAT's region. NA4 cells produced the 2.0- and the 1.5-kb LATs. Northern blotting and reverse transcription-PCR analysis of RNA from NA4 cells and from trigeminal ganglia of mice latently infected with HSV-1 revealed that the two abundant LAT species were present in the polyribosomal RNA fractions. After addition of EDTA, which causes dissociation of mRNA-ribosome complexes, both LATs could be detected only in subpolyribosomal, but not in polyribosomal fractions. These results show that (i) HSV-1 LATs are bound to polyribosomes during latency in vivo, as well as in neuronal cells in vitro, and therefore might be translated, and that (ii) the NA4 cell line is a suitable tool with which to look for HSV-1 latency-encoded gene products.

Molecular biology of herpes simplex virus type 1 latency in the nervous system.

Herpes simplex virus (HSV) is one of the best studied examples of viral ability to remain latent in the human nervous system and to cause recurrent disease by reactivation. Intensive effort was directed in recent years to unveil the molecular viral mechanisms and the virus-host interactions associated with latent HSV infection. The discovery of the state of the latent viral DNA in nervous tissues and of the presence of latency-associated gene expression during latent infection, both differing from the situation during viral replication, provided important clues relevant to the pathogenesis of latent HSV infection. This review summarizes the current state of knowledge on the site of latent infection, the molecular phenomena of latency, and the mechanisms of the various stages of latency: acute infection, establishment and maintenance of latency, and reactivation. This information paved the way to recent trials aiming to use herpes viruses as vectors to deliver genes into the nervous system, an issue that is also addressed in this review.