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.

Hepatitis C virus (HCV) has infected around 160 million individuals. Current therapies have limited efficacy and are fraught with side effects. To identify cellular HCV dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. Using this approach we identified the MAPK/ERK regulated, cytosolic, calcium-dependent, group IVA phospholipase A2 (PLA2G4A) as a novel HCV dependency factor. Inhibition of PLA2G4A activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. Moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. Exogenous addition of arachidonic acid, the cleavage product of PLA2G4A-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. Strikingly, production of infectious Dengue virus, a relative of HCV, was also dependent on PLA2G4A. These results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define PLA2G4A-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny.

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The hepatitis C virus (HCV) viroporin p7 is crucial for production of infectious viral progeny. However, its role in the viral replication cycle remains incompletely understood, in part due to the poor availability of p7-specific antibodies. To circumvent this obstacle, we inserted two consecutive hemagglutinin (HA) epitope tags at its N terminus. HA-tagged p7 reduced peak virus titers ca. 10-fold and decreased kinetics of virus production compared to the wild-type virus. However, HA-tagged p7 rescued virus production of a mutant virus lacking p7, thus providing formal proof that the tag does not disrupt p7 function. In HCV-producing cells, p7 displayed a reticular staining pattern which colocalized with the HCV envelope glycoprotein 2 (E2) but also partially with viral nonstructural proteins 2, 3, and 5A. Using coimmunoprecipitation, we confirmed a specific interaction between p7 and NS2, whereas we did not detect a stable interaction with core, E2, or NS5A. Moreover, we did not observe p7 incorporation into affinity-purified virus particles. Consistently, there was no evidence supporting a role of p7 in viral entry, as an anti-HA antibody was not able to neutralize Jc1 virus produced from an HA-p7-tagged genome. Collectively, these findings highlight a stable interaction between p7 and NS2 which is likely crucial for production of infectious HCV particles. Use of this functional epitope-tagged p7 variant should facilitate the analysis of the final steps of the HCV replication cycle.

Hepatitis C virus (HCV) particles closely mimic human very-low-density lipoproteins (VLDL) to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/β hydrolase domain containing protein 5, also known as CGI-58) as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis.

IntroductionThe human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, is the etiologic agent of a severe and fatal lymphoproliferative disorder of CD4 ϩ T cells, the adult T-cell leukemia (ATL), and the neurodegenerative, inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). [1][2][3][4] These diseases develop as a consequence of prolonged viral persistence in T cells.As emerged from many observations, HTLV-1 has developed a unique strategy for lifelong persistence in the presence of an active immune system. It is characterized by (a) replication of the virus mainly in its provirus form, (b) stimulation of cell division by the virus, and, as a consequence, (c) clonal amplification of infected cells. Evidence that HTLV-1 rarely replicates via reverse transcription but mostly by division of infected cells using cellular DNA polymerase is provided by the high HTLV-1 reverse transcriptase error rate, which is comparable to other retroviruses, 5 and the low mutation rate of the HTLV-1 genome. 6 The stimulation of T-cell proliferation in patients by viral gene expression was substantiated recently by cell dynamic studies. 7 They revealed the correlation of the in vivo proliferation rate of CD4 ϩ CD45RO ϩ cells with viral expression (ex vivo). Another indication that HTLV-1 stimulates growth and survival of T lymphocytes is provided by their expansion to detectable clones, which can persist over many years even in nonleukemic individuals. 8,9 Finally, the virus' ability to stimulate permanent T-lymphocyte growth in vitro, resulting in T-cell immortalization, 10 completes the arguments in favor of viral gene functions as cause of host cell proliferation and clonal expansion of patients' lymphocytes.Besides structural proteins, HTLV-1 encodes the regulatory proteins Tax and Rex, which are essential for viral replication, 11 and the accessory proteins p12, p30, p13, 12,13 and HBZ. 14 While Tax strongly enhances viral mRNA synthesis by transactivating the HTLV-1 long terminal repeat promoter, Rex controls the synthesis of the structural proteins on a posttranscriptional level. 15,16 Although the accessory proteins are important for viral infectivity and replication, 12,17 p12, p13, p30, and HBZ are not required for lymphocyte immortalization. [18][19][20] Tax is able to stimulate transcription by interacting with various signaling pathways. It activates nuclear factor kappa B (NF-B) by 2 pathways. While the canonical pathway is induced by binding and stimulating IKK␥, a component of the inhibitor of kappa B kinase, 10,17 the activation of the noncanonical pathway is less well understood. Transactivation of various cellular promoters is mediated by Tax via direct contact with transcriptional activators CREB and SRF and with the coactivators p300/CBP. 11,21 Tax confers the transforming properties on HTLV-1. 10 The protein can immortalize T lymphocytes 22,23 and induce leukemia in transgenic mice. 24 Biochemically, several Tax functions may contribute to its transforming capacity....

bHepatitis C virus (HCV) is highly variable and associated with chronic liver disease. Viral isolates are grouped into seven genotypes (GTs). Accumulating evidence indicates that viral determinants in the core to NS2 proteins modulate the efficiency of virus production. However, the role of the glycoproteins E1 and E2 in this process is currently poorly defined. Therefore, we constructed chimeric viral genomes to explore the role of E1 and E2 in HCV assembly. Comparison of the kinetics and efficiency of particle production by intragenotypic chimeras highlighted core and p7 as crucial determinants for efficient virion release. Glycoprotein sequences, however, had only a minimal impact on this process. In contrast, in the context of intergenotypic HCV chimeras, HCV assembly was profoundly influenced by glycoprotein genes. On the one hand, insertion of GT1a-derived (H77) E1-E2 sequences into a chimeric GT2a virus (Jc1) strongly suppressed virus production. On the other hand, replacement of H77 glycoproteins within the GT1a-GT2a chimeric genome H77/C3 by GT2a-derived (Jc1) E1-E2 increased infectious particle production. Thus, within intergenotypic chimeras, glycoprotein features strongly modulate virus production. Replacement of Jc1 glycoprotein genes by H77-derived E1-E2 did not grossly affect subcellular localization of core, E2, and NS2. However, it caused an accumulation of nonenveloped core protein and increased abundance of nonenveloped core protein structures with slow sedimentation. These findings reveal an important role for the HCV glycoproteins E1 and E2 in membrane envelopment, which likely depends on a genotype-specific interplay with additional viral factors.