SUMMARY Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα) as required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.

SummaryHepatitis B viruses (HBVs), which are enveloped viruses with reverse-transcribed DNA genomes, constitute the family Hepadnaviridae. An outstanding feature of HBVs is their streamlined genome organization with extensive gene overlap. Remarkably, the ∼1,100 bp open reading frame (ORF) encoding the envelope proteins is fully nested within the ORF of the viral replicase P. Here, we report the discovery of a diversified family of fish viruses, designated nackednaviruses, which lack the envelope protein gene, but otherwise exhibit key characteristics of HBVs including genome replication via protein-primed reverse-transcription and utilization of structurally related capsids. Phylogenetic reconstruction indicates that these two virus families separated more than 400 million years ago before the rise of tetrapods. We show that HBVs are of ancient origin, descending from non-enveloped progenitors in fishes. Their envelope protein gene emerged de novo, leading to a major transition in viral lifestyle, followed by co-evolution with their hosts over geologic eras.

SUMMARY Virus infection-induced global protein synthesis suppression is linked to assembly of stress granules (SGs), cytosolic aggregates of stalled translation preinitiation complexes. To study long-term stress responses, we developed an imaging approach for extended observation and analysis of SG dynamics during persistent hepatitis C virus (HCV) infection. In combination with type 1 interferon, HCV infection induces highly dynamic assembly/disassembly of cytoplasmic SGs, concomitant with phases of active and stalled translation, delayed cell division, and prolonged cell survival. Double-stranded RNA (dsRNA), independent of viral replication, is sufficient to trigger these oscillations. Translation initiation factor eIF2α phosphorylation by protein kinase R mediates SG formation and translation arrest. This is antagonized by the upregulation of GADD34, the regulatory subunit of protein phosphatase 1 dephosphorylating eIF2α. Stress response oscillation is a general mechanism to prevent long-lasting translation repression and a conserved host cell reaction to multiple RNA viruses, which HCV may exploit to establish persistence.

decreasing the levels of intracellular dNTPs 14,15 , which apparently compete with the 47 thymidine analog triphosphates for incorporation into HIV-1 cDNA during reverse 48 transcription 16 . We postulated that SAMHD1 could have a similar effect on nucleoside 49analog-based therapy in leukemia 6 . 50To investigate whether SAMHD1 expression enhances Ara-C cytotoxicity in AML 51 cells, we tested whether Ara-C sensitivity in 13 AML cell lines, determined by the half 52 maximal inhibitory concentration (IC 50 ), is correlated with SAMHD1 protein and mRNA 53 levels. Both SAMHD1 expression (Fig. 1a and Supplementary Fig. 1) and Ara-C sensitivity 54 (Supplementary Table 1) varied considerably among these cell lines. Unexpectedly, 55 SAMHD1 levels inversely correlated with Ara-C cytotoxicity (p=0.0037, Fig. 1b and 56 Supplementary Fig. 2a,b), as well as with the levels of early (Caspase 3 and 7 activity, 57 p=0.02, Supplementary Fig. 3a,b) and late (sub-G1 cells, apoptotic DNA fragmentation, 58 p=0.029, Supplementary Fig. 3c,d) markers of apoptosis. In contrast, no significant 59 correlation could be established between Ara-C IC 50 values and the expression of cellular 60 4 proteins previously implicated in Ara-C uptake or its conversion to Ara-CTP 1 , including 61 equilibrative nucleoside transporter (ENT1/SLC29A1), deoxycytidine kinase (DCK), cytidine 62 deaminase (CDA), deoxycytidilate deaminase (DCTD), or 5'-nucleotidase (NT5C2) (Fig. 63 1a,c-g). 64To further investigate its role in Ara-C resistance, we tested the effects of SAMHD1 65 deficiency by a number of approaches: (i) depletion of SAMHD1 in AML cell lines 66 expressing high endogenous SAMHD1 levels using either lentiviral vectors encoding 67 SAMHD1-specific shRNA or transfection with SAMHD1-specific siRNA; (ii) CRISPR/Cas9-68 mediated disruption of the SAMHD1 gene; and (iii) targeted degradation of SAMHD1 using 69 virus-like particles (VLPs) which shuttle the SAMHD1-interacting lentiviral Vpx protein 70 (Vpx-VLPs) into cells 7,8,17 (Fig. 2a and Supplementary Fig. 4). Vpx recruits SAMHD1 to a 71 cullin4A-RING E3 ubiquitin ligase (CRL4 DCAF1 ), which targets the enzyme for proteasomal 72 degradation 7,8 . 73SAMHD1 depletion in AML cell lines by RNA interference (OCI-AML3, THP-1), 74 SAMHD1 knockout (THP-1 -/-), or transduction with Vpx-VLPs (MonoMac6 cells, THP-1) 75 markedly sensitized AML cell lines to Ara-C toxicity relative to the respective controls (Fig. 76 2a,b and Supplementary Fig. 4). In contrast, SAMHD1 siRNA had only a marginal effect on 77 Ara-C toxicity in low SAMHD1-expressing HEL cells (Fig. 2a,b). Interestingly, we observed 78 SAMHD1 dependency, although less pronounced, for the purine analog fludarabine 79 ( Supplementary Fig. 5a); however, the IC 50 values for the topoisomerase II inhibitors 80 etoposide and daunorubicin, as well as for dFdC (2',2'-difluorodeoxycytidine; gemcitabine), 81were not consistently affected by SAMHD1 down-modulation ( Supplementary Fig. 5b-d), 82 indicating a certain degree of drug specificity. 83 5In HEL...

Hepatitis C virus (HCV) infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. To understand how the virus establishes its tightly controlled intracellular RNA replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular HCV RNA replication. We therefore quantitatively measured viral RNA and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly validated the model using additional, independent experiments. Model analysis was used to predict the efficacy of different classes of inhibitors and identified sensitive substeps of replication that could be targeted by current and future therapeutics. A protective replication compartment proved to be essential for sustained RNA replication, balancing translation versus replication and thus effectively limiting RNA amplification. The model predicts that host factors involved in the formation of this compartment determine cellular permissiveness to HCV replication. In gene expression profiling, we identified several key processes potentially determining cellular HCV replication efficiency.

Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer1. Cell entry of HCV2 and other pathogens3-5 is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model6 we show that a monoclonal antibody specific for TJ protein claudin-17 eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection via host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy.

Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7.

Changes in the airway microbiome may be important in the pathophysiology of chronic lung disease in patients with cystic fibrosis. However, little is known about the microbiome in early cystic fibrosis lung disease and the relationship between the microbiomes from different niches in the upper and lower airways. Therefore, in this cross-sectional study, we examined the relationship between the microbiome in the upper (nose and throat) and lower (sputum) airways from children with cystic fibrosis using next generation sequencing. Our results demonstrate a significant difference in both α and β-diversity between the nose and the two other sampling sites. The nasal microbiome was characterized by a polymicrobial community while the throat and sputum communities were less diverse and dominated by a few operational taxonomic units. Moreover, sputum and throat microbiomes were closely related especially in patients with clinically stable lung disease. There was a high inter-individual variability in sputum samples primarily due to a decrease in evenness linked to increased abundance of potential respiratory pathogens such as Pseudomonas aeruginosa. Patients with chronic Pseudomonas aeruginosa infection exhibited a less diverse sputum microbiome. A high concordance was found between pediatric and adult sputum microbiomes except that Burkholderia was only observed in the adult cohort. These results indicate that an adult-like lower airways microbiome is established early in life and that throat swabs may be a good surrogate in clinically stable children with cystic fibrosis without chronic Pseudomonas aeruginosa infection in whom sputum sampling is often not feasible.