A Genome-wide CRISPR Screen Identifies ZCCHC14 as a Host Factor Required for Hepatitis B Surface Antigen Production.

A hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.

Physician-Scientist Careers in the Biotechnology and Pharmaceutical Industries.

Many careers are open to physician-scientists in the biotechnology and pharmaceutical sectors. However, research is structured very differently in these environments compared to academic medicine. This article highlights these differences and the reasons for them, then outlines the different career paths available to physician-scientists in the variegated worlds of biotechnology and pharmaceutical companies.

JC polyoma viruria associates with protection from chronic kidney disease independently from apolipoprotein L1 genotype in African Americans.

Background: Viral infections can trigger chronic kidney disease (CKD) and the urine virome may inform risk. The Natural History of APOL1-Associated Nephropathy Study (NHAANS) reported that urine JC polyomavirus (JCPyV) associated with a lower risk of APOL1-associated nephropathy in African Americans. Herein, association was assessed between urine JCPyV with CKD in African Americans independent from the APOL1 genotype. Methods: Quantitative polymerase chain reaction was performed for urinary detection of JCPyV and BK polyoma virus (BKPyV) in 200 newly recruited nondiabetic African Americans. A combined analysis was performed in these individuals plus 300 NHAANS participants. Results: In the 200 new participants, urine JCPyV was present in 8.8% of CKD cases and 45.8% of nonnephropathy controls (P = 3.0 × 10-8). In those with APOL1 renal-risk genotypes, JCPyV was detected in 5.1% of cases and 40.0% of controls (P = 0.0002). In those lacking APOL1 renal-risk genotypes, JCPyV was detected in 12.2% of cases and 48.8% of controls (P = 8.5 × 10-5). BKPyV was detected in 1.3% of cases and 0.8% of controls (P = 0.77). In a combined analysis with 300 NHAANS participants (n = 500), individuals with urine JCPyV had a 63% lower risk of CKD compared with those without urine JCPyV (odds ratio 0.37; P = 4.6 × 10-6). RNA fluorescence in situ hybridization confirmed the presence of JCPyV genomic DNA and JCPyV messenger RNA (mRNA) in nondiseased kidney. Conclusions: Inverse relationships exist between JCPyV viruria and non-diabetic CKD. Future studies should determine whether renal inflammation associated with CKD is less permissive for JCPyV reactivation/replication or whether JCPyV is a marker of reduced host immune responsiveness that diminishes immune pathologic contributions to CKD.

Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation.

Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection.

Inefficient Codon Usage Impairs mRNA Accumulation: the Case of the v-FLIP Gene of Kaposi's Sarcoma-Associated Herpesvirus.

UNLABELLED: Latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes encode a homolog of cellular FLICE-inhibitory proteins (termed v-FLIP) that activates NF-κB and can trigger important proinflammatory and antiapoptotic changes in latently infected cells. The protein is present at very low levels in infection and has generally been difficult to efficiently express in recombinant vectors. Here we show that codon usage in the v-FLIP gene is strikingly suboptimal. Optimization of codon use in expression vectors, as expected, restores efficient protein expression. Surprisingly, however, it also dramatically increases the steady-state level of v-FLIP mRNA, at least in part by increasing mRNA stability. When codon-optimized v-FLIP sequences are reintroduced into intact KSHV genomes, the resulting virus expresses readily detectable monocistronic v-FLIP mRNAs that are undetectable in wild-type (WT) infection by blot hybridization, suggesting that such RNAs are in fact transcribed in WT infection but fail to accumulate. The overexpression of v-FLIP by codon-optimized latent genomes results in a 5- to 7-fold decrement in virus production following lytic induction, indicating that maximizing NF-κB signaling is deleterious to induction. These studies provide a clear explanation for the evolution of inefficient codon usage in this gene and point to a strong connection between translational efficiency and RNA accumulation in mammalian cells. IMPORTANCE: This study reports that inefficient codon usage in a herpesviral gene is strikingly correlated with the inability of its mRNA to accumulate in cells; correction of efficient translatability restores RNA abundance. A similar correlation has been reported in yeast species, but the mechanisms operating in mammalian cells appear substantially different.

Site-specific association with host and viral chromatin by Kaposi's sarcoma-associated herpesvirus LANA and its reversal during lytic reactivation.

UNLABELLED: Latency-associated nuclear antigen (LANA), a multifunctional protein expressed by the Kaposi sarcoma-associated herpesvirus (KSHV) in latently infected cells, is required for stable maintenance of the viral episome. This is mediated by two interactions: LANA binds to specific sequences (LBS1 and LBS2) on viral DNA and also engages host histones, tethering the viral genome to host chromosomes in mitosis. LANA has also been suggested to affect host gene expression, but both the mechanism(s) and role of this dysregulation in KSHV biology remain unclear. Here, we have examined LANA interactions with host chromatin on a genome-wide scale using chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) and show that LANA predominantly targets human genes near their transcriptional start sites (TSSs). These host LANA-binding sites are generally found within transcriptionally active promoters and display striking overrepresentation of a consensus DNA sequence virtually identical to the LANA-binding site 1 (LBS1) motif in KSHV DNA. Comparison of the ChIP-seq profile with whole-transcriptome (high-throughput sequencing of RNA transcripts [RNA-seq]) data reveals that few of the genes that are differentially regulated in latent infection are occupied by LANA at their promoters. This suggests that direct LANA binding to promoters is not the prime determinant of altered host transcription in KSHV-infected cells. Most surprisingly, the association of LANA to both host and viral DNA is strongly disrupted during the lytic cycle of KSHV. This disruption can be prevented by the inhibition of viral DNA synthesis, suggesting the existence of novel and potent regulatory mechanisms linked to either viral DNA replication or late gene expression. IMPORTANCE: Here, we employ complementary genome-wide analyses to evaluate the distribution of the highly abundant latency-associated nuclear antigen, LANA, on the host genome and its impact on host gene expression during KSHV latent infection. Combined, ChIP-seq and RNA-seq reveal that LANA accumulates at active gene promoters that harbor specific short DNA sequences that are highly reminiscent of its cognate binding sites in the virus genome. Unexpectedly, we found that such association does not lead to remodeling of global host transcription during latency. We also report for the first time that LANA's ability to bind host and viral chromatin is highly dynamic and is disrupted in cells undergoing an extensive lytic reactivation. This therefore suggests that the association of LANA to chromatin during a productive infection cycle is controlled by a new regulatory mechanism.

KSHV 2.0: a comprehensive annotation of the Kaposi's sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features.

Productive herpesvirus infection requires a profound, time-controlled remodeling of the viral transcriptome and proteome. To gain insights into the genomic architecture and gene expression control in Kaposi's sarcoma-associated herpesvirus (KSHV), we performed a systematic genome-wide survey of viral transcriptional and translational activity throughout the lytic cycle. Using mRNA-sequencing and ribosome profiling, we found that transcripts encoding lytic genes are promptly bound by ribosomes upon lytic reactivation, suggesting their regulation is mainly transcriptional. Our approach also uncovered new genomic features such as ribosome occupancy of viral non-coding RNAs, numerous upstream and small open reading frames (ORFs), and unusual strategies to expand the virus coding repertoire that include alternative splicing, dynamic viral mRNA editing, and the use of alternative translation initiation codons. Furthermore, we provide a refined and expanded annotation of transcription start sites, polyadenylation sites, splice junctions, and initiation/termination codons of known and new viral features in the KSHV genomic space which we have termed KSHV 2.0. Our results represent a comprehensive genome-scale image of gene regulation during lytic KSHV infection that substantially expands our understanding of the genomic architecture and coding capacity of the virus.

A unique herpesviral transcriptional program in KSHV-infected lymphatic endothelial cells leads to mTORC1 activation and rapamycin sensitivity.

Immunosuppression therapy following organ transplantation is a significant factor in the development and progression of Kaposi's sarcoma-associated herpesvirus (KSHV)-induced posttransplant Kaposi's sarcoma (KS). Switching from cyclosporine to the mTOR inhibitor rapamycin is reported to promote KS regression without allograft rejection. Examining the underlying molecular basis for this clinical observation, we find that KSHV infection selectively upregulates mTOR signaling in primary human lymphatic endothelial cells (LECs), but not blood endothelial cells (BECs), and sensitizes LECs to rapamycin-induced apoptosis. Viral transcriptome analysis revealed that while infected BECs display conventional latency, KSHV-infected LECs support a radically different program involving widespread deregulation of both latent and lytic genes. ORF45, a lytic gene selectively expressed in infected LECs, is required for mTOR activation and critical for rapamycin sensitivity. These studies reveal the existence of a unique herpesviral gene expression program corresponding to neither canonical latency nor lytic replication, with important pathogenetic and therapeutic consequences.

Kaposi's sarcoma-derived cell line SLK is not of endothelial origin, but is a contaminant from a known renal carcinoma cell line.

Kaposi's sarcoma (KS) is an endothelial cell-derived tumor. Investigations of the molecular mechanisms of KS pathogenesis and the identification of drugs for treatment of KS depend critically on valid cell-culture models. Two major immortalized cell lines are available for KS research. Recently, the KS cell line KS Y-1 has been shown to be cross-contaminated with the T24 urinary bladder cancer cell line (ATCC HTB-4). Here, we show by short tandem repeat profiling that the second KS cell line, SLK, is indistinguishable from the clear-cell renal-cell carcinoma cell line Caki-1. Immunocytochemical detection of cytokeratin expression confirmed the epithelial-cell origin of SLK cells. Our findings indicate that SLK cells are not of endothelial origin and should not be used in future studies as a model for KS-derived endothelial tumor cells. We suggest that in the future, more attention needs to be paid to the authenticity of cells in lines derived from human tissues.

In-depth investigation of archival and prospectively collected samples reveals no evidence for XMRV infection in prostate cancer.

XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.

Construction and manipulation of a new Kaposi's sarcoma-associated herpesvirus bacterial artificial chromosome clone.

Efficient genetic modification of herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) has come to rely on bacterial artificial chromosome (BAC) technology. In order to facilitate this approach, we generated a new KSHV BAC clone, called BAC16, derived from the rKSHV.219 virus, which stems from KSHV and Epstein-Barr virus-coinfected JSC1 primary effusion lymphoma (PEL) cells. Restriction enzyme and complete sequencing data demonstrate that the KSHV of JSC1 PEL cells showed a minimal level of sequence variation across the entire viral genome compared to the complete genomic sequence of other KSHV strains. BAC16 not only stably propagated in both Escherichia coli and mammalian cells without apparent genetic rearrangements, but also was capable of robustly producing infectious virions (∼5 × 10(7)/ml). We also demonstrated the utility of BAC16 by generating deletion mutants of either the K3 or K5 genes, whose products are E3 ligases of the membrane-associated RING-CH (MARCH) family. While previous studies have shown that individual expression of either K3 or K5 results in efficient downregulation of the surface expression of major histocompatibility complex class I (MHC-I) molecules, we found that K5, but not K3, was the primary factor critical for the downregulation of MHC-I surface expression during KSHV lytic reactivation or following de novo infection. The data presented here demonstrate the utility of BAC16 for the generation and characterization of KSHV knockout and mutant recombinants and further emphasize the importance of functional analysis of viral genes in the context of the KSHV genome besides the study of individual gene expression.

Kaposi's sarcoma-associated herpesvirus ORF54/dUTPase downregulates a ligand for the NK activating receptor NKp44.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes long-term latent infection in humans and can cause cancers in endothelial and B cells. A functioning immune system is vital for restricting viral proliferation and preventing KSHV-dependent neoplasms. While natural killer (NK) lymphocytes are known to target virus-infected cells for destruction, their importance in the anti-KSHV immune response is not currently understood. Activating receptors on NK cells recognize ligands on target cells, including the uncharacterized ligand(s) for NKp44, termed NKp44L. Here we demonstrate that several NK ligands are affected when KSHV-infected cells are induced to enter the lytic program. We performed a screen of most of the known KSHV genes and found that the product of the ORF54 gene could downregulate NKp44L. The ORF54-encoded protein is a dUTPase; however, dUTPase activity is neither necessary nor sufficient for the downregulation of NKp44L. In addition, we find that ORF54 can also target proteins of the cytokine receptor family and the mechanism of downregulation involves perturbation of membrane protein trafficking. The ORF54-related proteins of other human herpesviruses do not possess this activity, suggesting that the KSHV homolog has evolved a novel immunoregulatory function and that the NKp44-NKp44L signaling pathway contributes to antiviral immunity.

Multiple defects, including premature apoptosis, prevent Kaposi's sarcoma-associated herpesvirus replication in murine cells.

The development of a mouse model for Kaposi's sarcoma-associated herpesvirus (KSHV) infection has been impeded by the limited host range of the virus. Here, we have examined the molecular basis of this host range restriction. KSHV efficiently enters murine cells and establishes latency. However, ectopic expression of the lytic switch protein RTA (replication and transcription activator) in these cells induces little viral gene expression and no virus production. Upon treatment with histone deacetylase inhibitors, KSHV-infected murine cells display more extensive but aberrant viral transcription and do not support either viral DNA synthesis or the production of infectious virions. These aberrantly infected cells also display markedly enhanced apoptosis. Genetic ablation of the mitochondrial apoptotic pathway in these cells prolongs their survival and permits viral DNA replication but does not rescue the generation of virions. We conclude that multiple defects, both prior to and following DNA synthesis, restrict lytic KSHV infection in murine cells.

Infection of lymphoblastoid cell lines by Kaposi's sarcoma-associated herpesvirus: critical role of cell-associated virus.

Kaposi's sarcoma-associated herpesvirus (KSHV) displays strong lymphotropism in vivo, but paradoxically, established B cell lines have largely been refractory to infection by soluble KSHV virions. Here we show that this block can be overcome by exposure to cell-associated virus. Doxycycline-inducible recombinant KSHV.219 (rKSHV.219)-harboring SLK (iSLK.219) cells were employed as KSHV donors. Cocultivation of lymphoid cell lines with reactivated iSLK.219 cells resulted in readily demonstrable viral entry into each cell line; similar observations were made in primary tonsillar B cell cultures. Moreover, infected lymphoid cells were able to outgrow upon puromycin selection, indicating development of persistent infection. Infected BJAB cells display signatures of latent infection, including classical latency-associated transcripts, a punctate pattern of LANA expression, and episomal maintenance of the KSHV genome. However, when lytically activated by various chemical stimuli, infected BJAB cells were able to produce only low levels of infectious virions. These data demonstrate that (i) cell-associated viruses can bypass viral entry blocks in most lymphoid cell lines, (ii) the determinants of cell-associated virus entry differ from those of soluble virion infection, and (iii) immortalized lymphoblastoid lines have partial postentry blocks to efficient lytic reactivation.

Viral infection in acute exacerbation of idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis is a progressive, uniformly fatal interstitial lung disease. An acute exacerbation of idiopathic pulmonary fibrosis is an episode of acute respiratory worsening without an identifiable etiology. Occult viral infection has been proposed as a possible cause of acute exacerbation. OBJECTIVES: To use unbiased genomics-based discovery methods to define the role of viruses in acute exacerbation of idiopathic pulmonary fibrosis. METHODS: Bronchoalveolar lavage and serum from patients with acute exacerbation of idiopathic pulmonary fibrosis, stable disease, and acute lung injury were tested for viral nucleic acid using multiplex polymerase chain reaction, pan-viral microarray, and high-throughput cDNA sequencing. MEASUREMENTS AND MAIN RESULTS: Four of forty-three patients with acute exacerbation of idiopathic pulmonary fibrosis had evidence of common respiratory viral infection (parainfluenza [n = 1], rhinovirus [n = 2], coronavirus [n = 1]); no viruses were detected in the bronchoalveolar lavage from stable patients. Pan-viral microarrays revealed additional evidence of viral infection (herpes simplex virus [n = 1], Epstein-Barr virus [n = 2], and torque teno virus [TTV] [n = 12]) in patients with acute exacerbation. TTV infection was significantly more common in patients with acute exacerbation than stable controls (P = 0.0003), but present in a similar percentage of acute lung injury controls. Deep sequencing of a subset of acute exacerbation cases confirmed the presence of TTV but did not identify additional viruses. CONCLUSIONS: Viral infection was not detected in most cases of acute exacerbation of idiopathic pulmonary fibrosis. TTV was present in a significant minority of cases, and cases of acute lung injury; the clinical significance of this finding remains to be determined.

Transcriptome sequencing demonstrates that human papillomavirus is not active in cutaneous squamous cell carcinoma.

ß-Human papillomavirus (ß-HPV) DNA is present in some cutaneous squamous cell carcinomas (cuSCCs), but no mechanism of carcinogenesis has been determined. We used ultra-high-throughput sequencing of the cancer transcriptome to assess whether papillomavirus transcripts are present in these cancers. In all, 67 cuSCC samples were assayed for ß-HPV DNA by PCR, and viral loads were measured with type-specific quantitative PCR. A total of 31 SCCs were selected for whole transcriptome sequencing. Transcriptome libraries were prepared in parallel from the HPV18-positive HeLa cervical cancer cell line and HPV16-positive primary cervical and periungual SCCs. Of the tumors, 30% (20/67) were positive for ß-HPV DNA, but there was no difference in ß-HPV viral load between tumor and normal tissue (P=0.310). Immunosuppression and age were significantly associated with higher viral load (P=0.016 for immunosuppression; P=0.0004 for age). Transcriptome sequencing failed to identify papillomavirus expression in any of the skin tumors. In contrast, HPV16 and HPV18 mRNA transcripts were readily identified in primary cervical and periungual cancers and HeLa cells. These data demonstrate that papillomavirus mRNA expression is not a factor in the maintenance of cuSCCs.

Viral microRNA target allows insight into the role of translation in governing microRNA target accessibility.

It is widely believed that functional mammalian microRNA (miRNA) recognition sequences are located preferentially in the 3' untranslated region (3'UTR) of target mRNAs. Nonetheless, putative miRNA target sites within coding regions have been found at lower frequency in genome-wide studies, and several have been genetically validated. To account for these findings, it has been proposed that translation may inhibit miRNA access to target sites. Here we identify a naturally occurring viral miRNA target that, owing to the compact nature of the viral transcriptome, is situated naturally in the coding region of one transcript and in the 3'UTR of an overlapping mRNA. Examination of the expression of these mRNAs reveals that the cognate miRNA can inhibit expression in both contexts, but inhibition is more potent when the target site is in the UTR. Similarly, forced translation of the target site in the UTR diminished, but did not abolish, its down-regulation by the miRNA. These data affirm that miRNAs can exert regulatory effects on targets within coding regions; however, the dampening of these effects by translation likely accounts for the observed selection for target sites in the 3'UTRs.

Infection of primary human tonsillar lymphoid cells by KSHV reveals frequent but abortive infection of T cells.

The lymphotropic herpesvirus KSHV principally infects B cells in vivo and is linked to several human B cell lymphoproliferative syndromes. Here we examine the susceptibility of primary tonsillar lymphocytes to infection by a recombinant KSHV (rKSHV.219) that constitutively expresses GFP. At an MOI of ~1, ca. 5-10% of CD19+ B cells became GFP-positive. Surprisingly, in the same culture many more T cells became infected. However, in contrast to isolated B cells, isolated infected T cells did not support correct viral transcription and did not produce infectious virus, indicating the presence of one or more post-entry blocks to lytic KSHV replication in T cells. No immortalization or transformation has yet been observed in either B or T cells. These results affirm the feasibility of studying KSHV infection in primary lymphoid cells, and help to rationalize the detection of KSHV DNA in rare human T cell lymphomas in vivo.

Generation of a doxycycline-inducible KSHV producer cell line of endothelial origin: maintenance of tight latency with efficient reactivation upon induction.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and at least two B cell lymphoproliferative diseases: primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). B cells derived from PEL are latently infected, and can be induced to lytic replication by treatment with chemical agents like TPA or butyrate, which have pleiotropic effects on host cell signaling and chromatin structure. Most of these lines also display moderate levels of spontaneous lytic induction, which complicates analysis of latency. Here we describe the creation of latently infected cell lines derived from SLK endothelial cells that (i) display tight control of KSHV latency, with little spontaneous reactivation and (ii) are efficiently inducible by doxycycline, avoiding the need for pleiotropic inducing agents. These cells produce substantial quantities of infectious KSHV, and should be useful for studies of the latent-lytic switch and the impact of lytic replication on host cell biology.