Air-Liquid Interface System To Understand Epstein-Barr Virus-Associated Nasopharyngeal Carcinoma

Epstein-Barr virus (EBV) infects epithelial cells and is associated with epithelial malignancies. Although EBV reactivation is induced by epithelial differentiation, the available methods for differentiation are not widely used.Epstein-Barr virus (EBV) infects epithelial cells and is associated with epithelial malignancies. Although EBV reactivation is induced by epithelial differentiation, the available methods for differentiation are not widely used. In a recent study, Caves et al. (mSphere 3:e00152-18, 2018, https://doi.org/10.1128/mSphere.00152-18) explored the use of a new transwell-based air-liquid interface (ALI) system to differentiate EBV-infected nasopharyngeal carcinoma cells. They found that cells cultured in the ALI system expressed markers of differentiation and supported complete EBV reactivation. This system offers an easy method for differentiation that could be widely adopted. This system could be extended to other epithelial cell types

Chromatin remodeling controls Kaposi's sarcoma-associated herpesvirus reactivation from latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of three human malignancies, the endothelial cell cancer Kaposi's sarcoma, and two B cell cancers, Primary Effusion Lymphoma and multicentric Castleman's disease. KSHV has latent and lytic phases of the viral life cycle, and while both contribute to viral pathogenesis, lytic proteins contribute to KSHV-mediated oncogenesis. Reactivation from latency is driven by the KSHV lytic gene transactivator RTA, and RTA transcription is controlled by epigenetic modifications. To identify host chromatin-modifying proteins that are involved in the latent to lytic transition, we screened a panel of inhibitors that target epigenetic regulatory proteins for their ability to stimulate KSHV reactivation. We found several novel regulators of viral reactivation: an inhibitor of Bmi1, PTC-209, two additional histone deacetylase inhibitors, Romidepsin and Panobinostat, and the bromodomain inhibitor (+)-JQ1. All of these compounds stimulate lytic gene expression, viral genome replication, and release of infectious virions. Treatment with Romidepsin, Panobinostat, and PTC-209 induces histone modifications at the RTA promoter, and results in nucleosome depletion at this locus. Finally, silencing Bmi1 induces KSHV reactivation, indicating that Bmi1, a member of the Polycomb repressive complex 1, is critical for maintaining KSHV latency

Species-Specific Regions of Occludin Required by Hepatitis C Virus for Cell Entry▿

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. As HCV infects only human and chimpanzee cells, antiviral therapy and vaccine development have been hampered by the lack of a convenient small-animal model. In this study we further investigate how the species tropism of HCV is modulated at the level of cell entry. It has been previously determined that the tight junction protein occludin (OCLN) is essential for HCV host cell entry and that human OCLN is more efficient than the mouse ortholog at mediating HCV cell entry. To further investigate the relationship between OCLN sequence and HCV species tropism, we compared OCLN proteins from a range of species for their ability to mediate infection of naturally OCLN-deficient 786-O cells with lentiviral pseudoparticles bearing the HCV glycoproteins. While primate sequences function equivalently to human OCLN, canine, hamster, and rat OCLN had intermediate activities, and guinea pig OCLN was completely nonfunctional. Through analysis of chimeras between these OCLN proteins and alanine scanning mutagenesis of the extracellular domains of OCLN, we identified the second half of the second extracellular loop (EC2) and specific amino acids within this domain to be critical for modulating the HCV cell entry factor activity of this protein. Furthermore, this critical region of EC2 is flanked by two conserved cysteine residues that are essential for HCV cell entry, suggesting that a subdomain of EC2 may be defined by a disulfide bond

Temporal analysis of hepatitis C virus cell entry with occludin directed blocking antibodies.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. A better understanding of its life cycle, including the process of host cell entry, is important for the development of HCV therapies and model systems. Based on the requirement for numerous host factors, including the two tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), HCV cell entry has been proposed to be a multi-step process. The lack of OCLN-specific inhibitors has prevented a comprehensive analysis of this process. To study the role of OCLN in HCV cell entry, we created OCLN mutants whose HCV cell entry activities could be inhibited by antibodies. These mutants were expressed in polarized HepG2 cells engineered to support the complete HCV life cycle by CD81 and miR-122 expression and synchronized infection assays were performed to define the kinetics of HCV cell entry. During these studies, OCLN utilization differences between HCV isolates were observed, supporting a model that HCV directly interacts with OCLN. In HepG2 cells, both HCV cell entry and tight junction formation were impaired by OCLN silencing and restored by expression of antibody regulatable OCLN mutant. Synchronized infection assays showed that glycosaminoglycans and SR-BI mediated host cell binding, while CD81, CLDN1 and OCLN all acted sequentially at a post-binding stage prior to endosomal acidification. These results fit a model where the tight junction region is the last to be encountered by the virion prior to internalization

Viral Determinants of miR-122-Independent Hepatitis C Virus Replication.

Hepatitis C virus (HCV) replication requires binding of the liver-specific microRNA (miRNA) miR-122 to two sites in the HCV 5' untranslated region (UTR). Although we and others have shown that viral genetics impact the amount of active miR-122 required for replication, it is unclear if HCV can replicate in the complete absence of this miRNA. To probe the absolute requirements for miR-122 and the genetic basis for those requirements, we used clustered regularly interspaced short palindromic repeat (CRISPR) technology to knock out miR-122 in Huh-7.5 cells and reconstituted these knockout (KO) cells with either wild-type miR-122 or a mutated version of this miRNA. We then characterized the replication of the wild-type virus, as well as a mutated HCV bearing 5' UTR substitutions to restore binding to the mutated miR-122, in miR-122 KO Huh-7.5 cells expressing no, wild-type, or mutated miR-122. We found that while replication was most efficient when wild-type or mutated HCV was provided with the matched miR-122, inefficient replication could be observed in cells expressing the mismatched miR-122 or no miR-122. We then selected viruses capable of replicating in cells expressing noncognate miR-122 RNAs. Unexpectedly, these viruses contained multiple mutations throughout their first 42 nucleotides that would not be predicted to enhance binding of the provided miR-122. These mutations increased HCV RNA replication in cells expressing either the mismatched miR-122 or no miR-122. These data provide new evidence that HCV replication can occur independently of miR-122 and provide unexpected insights into how HCV genetics influence miR-122 requirements. IMPORTANCE Hepatitis C virus (HCV) is the leading cause of liver cancer in the Western Hemisphere. HCV infection requires miR-122, which is expressed only in liver cells, and thus is one reason that replication of this virus occurs efficiently only in cells of hepatic origin. To understand how HCV genetics impact miR-122 usage, we knocked out miR-122 using clustered regularly interspaced short palindromic repeat (CRISPR) technology and adapted virus to replicate in the presence of noncognate miR-122 RNAs. In doing so, we identified viral mutations that allow replication in the complete absence of miR-122. This work provides new insights into how HCV genetics influence miR-122 requirements and proves that replication can occur without this miRNA, which has broad implications for how HCV tropism is maintained

NLRX1 negatively modulates type I IFN to facilitate KSHV reactivation from latency

<div><p>Kaposi’s sarcoma-associated herpesvirus (KSHV) is a herpesvirus that is linked to Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV establishes persistent latent infection in the human host. KSHV undergoes periods of spontaneous reactivation where it can enter the lytic replication phase of its lifecycle. During KSHV reactivation, host innate immune responses are activated to restrict viral replication. Here, we report that NLRX1, a negative regulator of the type I interferon response, is important for optimal KSHV reactivation from latency. Depletion of NLRX1 in either iSLK.219 or BCBL-1 cells significantly suppressed global viral transcription levels compared to the control group. Concomitantly, fewer viral particles were present in either cells or supernatant from NLRX1 depleted cells. Further analysis revealed that upon NLRX1 depletion, higher IFNβ transcription levels were observed, which was also associated with a transcriptional upregulation of JAK/STAT pathway related genes in both cell lines. To investigate whether IFNβ contributes to NLRX1’s role in KSHV reactivation, we treated control and NLRX1 depleted cells with a TBK1 inhibitor (BX795) or TBK1 siRNA to block IFNβ production. Upon BX795 or TBK1 siRNA treatment, NLRX1 depletion exhibited less inhibitory effects on reactivation and infectious virion production, suggesting that NLRX1 facilitates KSHV lytic replication by negatively regulating IFNβ responses. Our data suggests that NLRX1 plays a positive role in KSHV lytic replication by suppressing the IFNβ response during the process of KSHV reactivation, which might serve as a potential target for restricting KSHV replication and transmission.</p></div

NLRX1 knock down results in enhanced interferon responses upon KSHV reactivation in BCBL-1 cells.

<p>BCBL-1 cells were transfected with NS or NLRX1 siRNA for 48 hours and then treated with TPA and sodium butyrate for various time points. (A) IFNβ mRNA in BCBL-1 cells was monitored by qRT-PCR. (B-D) RNA was extracted from duplicate samples and various JAK/STAT related mRNA levels were analyzed using a JAK/STAT real-time qPCR-based gene array. mRNA levels of viral genes were normalized to the mRNA levels of human β-actin to yield dCT as a measure or relative expression without clustering. Scatter plot comparison of relative mRNA level at 0 (B), 24 (C) and 48 (D) hours between NLRX1 siRNA treated cells and NS siRNA treated cells. (E-H) Scatter plot of relative mRNA level at 24 and 48 hours. NS siRNA group at 0 hours were set as the normalization control. (I) Heat map of JAK/STAT microarray. Relative Z-scores of each gene level was calculated by subtracting mean values for each individual gene, and then dividing by each gene standard deviation. Higher Z-scores are indicated by red, lower levels by blue as shown in the key. Data are presented as mean ± s.d. from at least three independent experiments. *indicates p<0.05. ** indicates p<0.01 by Student’s t-test.</p

NLRX1 is required for optimal KSHV reactivation in iSLK.219 cells.

<p>(A) iSLK.219 cells were transfected with NS or NLRX1 siRNA for 48 hours and then treated with Dox for various time points. A representative image of a field of cells expressing GFP and RFP are shown at 0, 24, 48 and 72 hours post-Dox treatment. (B-C) Whole well GFP/RFP intensities were monitored and quantitated by a Clariostar plate reader. (D) KSHV genome copy numbers in the supernatants of reactivated iSLK.219 cells. (E) KSHV genome copy numbers in reactivated iSLK.219 cells. Data are presented as mean ± s.d. from at least three independent experiments. * indicates p<0.05. ** indicates p<0.01 by Student’s t-test.</p

TBK1 siRNA rescues KSHV reactivation.

<p>iSLK.219 cells were transfected with NS, NLRX1 siRNA or NLRX1+TBK1 siRNAs for 48 hours. iSLK.219 were then reactivated with Dox for 0, 24 and 48 hours. (A) A representative image of a field of cells expressing GFP and RFP are shown at 0, 24, and 48 and hours post-Dox treatment. (B-C) Whole well GFP/RFP intensities were monitored and quantitated by a Clariostar plate reader. (D) NLRX1 knockdown efficiency was monitored by qRT-PCR. (E-G) qRT-PCR of ORF57, K8.1 and vIRF1 in reactivated iSLK.219 cells. Data are presented as mean ± s.d. from at least three independent experiments. * indicates p<0.05. ** indicates p<0.01 by Student’s t-test.</p

NLRX1 negatively regulates MAVS-dependent type I interferon response.

<p>(A) iSLK.219 cells were transfected with NS or NLRX1 siRNA for 72 hours. Cells were then transfected with poly I:C at 2 μg/μl for 4 hours before being harvested. RNA was subjected to IFNβ qRT-PCR analysis. (B) NLRX1 knockdown efficiency was monitored by qRT-PCR. (C) iSLK.219 cells were transfected with NS, NLRX1 siRNA for 72 hours. 1 μM BX795 was then added to one set of NLRX1 siRNA transfected samples 6 hours before the cells were reactivated with Dox for 0, 24 and 48 hours. IFNβ mRNA was monitored by qRT-PCR. (D) NLRX1 knockdown efficiency was monitored by qRT-PCR.</p