The importance of Coronavirus RTC for antiviral signaling

Internalised viruses are recognised by pathogen recognition receptors (PRRs) which sense Pathogen-associated Molecular Patterns (PAMP). Generally, PRRs are located within the endosomes -as it the case for Toll-like receptor (TLR)-7 or MyD88 - or alternatively in the cytoplasm, as it the case for retinoic acid inducible gene I (RIG-I) like receptors, and melanoma differentiation associated gene 5 (MDA5). In the case of RNA viruses, viral RNA is recognised by proteins belonging to the cytoplasmic RNA-induced silencing complex (RISC), which consists of RIG-1, MDA-5, protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer. RIG-1, PACT, and MDA-5 recognize viral RNA and induce the expression of various cytokines, in particular Interferon(s) (IFN) and ultimately a broad array of IFN stimulated genes (ISG) as part of the antiviral response. In contrast, TRBP and Dicer process pre-microRNAs into mature microRNAs (miRNAs) by recruiting Argonaute 2 (Ago2) that target specific mRNA species -such as viral RNA- for regulation and targeting steroid-responsive promoters.

PACT, allowing the degradation of viral RNA as well as stimulating an immune response, can induce both TBRP and Dicer and thus targets viral microRNAs, viral small RNAs, and viral dsRNA.

ssRNA or dsRNA can induce an antiviral Interferon or NF-κB response via TLR or MAVS

In order to prevent host cell derived mRNAs from being recognised by cellular PRRs, cellular mRNA molecules not only possess a 5’ cap structure but are also methylated at their 5’ end; any RNA contained no 5’ cap, a non-methylated, or incompletely methylated 5’cap can therefore be recognised by cellular PAMP receptors and induce an antiviral response, in addition to viral dsRNA intermediates. In this system, RIG-1 preferentially recognizes short blunt end or 5’ triphosphate RNAs whereas MDA-5 recognizes dsRNA intermediates as well as non-methylated RNAs lacking 2’O methylated ribose. Antiviral signaling by these pathways can be inhibited by viruses using either the cellular machinery for capping viral RNAs, and thus prevent them from being recognised by the RISC, or alternatively encoding enzymes which cap the viral RNA in a manner similar to cellular enzymes.

In addition to stimulating the expression of antiviral genes, the recognition of single-stranded viral RNA by TLR-7 also induces the formation of autophagy vesicles via the interaction between the TLR adaptor MyD88 and the Beclin-1, the latter being required for the formation of the Phagosome/Autophagosomes.

Autophagy and the antiviral response

The formation of autophagy vesicles has been postulated to facilitate the presentation of viral antigens by the cellular MHC Class I (e.g. HSV-1 gB), MHC Class II complex (e.g. EBNA-1) and target viral RNA to endosomes (and thus being recognised by TLR-7) as well as facilitating the degradation of viral components. Indeed, infecting cells with a HSV-1 mutant deficient for the viral autophagy inhibitory ICP 34.5 protein not only fails to inhibit autophagy but also has decreased infectious titers, suggesting that autophagy degrades HSV-1 components in addition to facilitate the presentation of viral antigens.

Coronavirus RTC and  the antiviral response

In the case of Coronavirus infected cells, the major pathogen associated pattern recognised by the PARRs is the dsRNA intermediate and the viral ssRNA both which are located with the replication-transcription complexes (RTCs) derived from the ER and induced by the expression of the viral nsp-3,-, and -6 proteins as discussed earlier. In these RTCs, the viral RNAs are not only synthesized but also modified in order to prevent the induction of the antiviral response. In order to prevent the viral RNA from being recognised by RIG-1 or MDA-5, the Coronavirus RNA contain a 5’cap structure which is added to newly synthesized viral RNA within the RTC by the viral RNA-triphosphatase (nsp13), 2’O-methyltransferase (nsp16), as well as a N7-Methyltransferase (nsp14). Failure to methylate viral RNA in cells infected with a nsp16 deficient virus induces a IFN type 1, MDA-5 dependent, antiviral response concomitant with elevated levels of ISGs. In addition to bind MDA-5, uncapped viral RNA binds IFN induced protein with tetratricopeptide repeats (IFIT)-1, thus preventing the translation of viral RNA. So far the precise contribution of the viral N7-Methyltransferase remains unknown although the equivalent in West Nile Virus has been shown to be required for 2’O-methyltransferase mediated methylation of WNV viral RNA. The N-terminus of nsp14 encodes for the viral 3’ -to-5’ exoribo-nuclease (ExoN) which hydrolyses both ssRNA and dsRNA and excises nucleotide mismatches in dsRNA intermediates, thus providing proofreading of newly synthesized RNA. Accordingly, inhibiting or deleting ExoN increases the potency of antivirals such as 5-Fluorouracil.  Since both ExoN and 2’O-methyltransferase activity positively regulated by nsp10, inhibiting nsp10 might be an interesting target for antiviral therapy. In addition to ExoN, both the Arteriviridae and the Coronaviridae express another enzyme that can hydrolyze RNA, EndoU. In contrast to ExoN the function of EndoU (nsp15) is less well defined and it has been proposed that it might be involved in cleaving free -“mislocalised”-RNA in order to prevent the recognition by PRRs, although EndoU can be found both in RTCs as well as in the cytoplasm. One possibility, which the author of these lines suggests, is that EndoU might be associated with Endosomes that contain viral RNA (see below). These Endosomes might contain viral RNA as a result of the induction of the formation of autophagy vesicles by nsp-3,-4, and -6.

Although the Coronaviral nsps-3,-4, and -6 induce the formation of autophagy like (LC3-II negative) vesicles, so far the formation of autolysosomes and subsequent degradation of viral components in infected cells has not been demonstrated. Since the formation of LC3-II positive vesicles has been demonstrated in cells transfected with nsp6 derived from SARS-CoV, MHV, and avian IBV it might be possible that viral RNA derived from autophagy vesicles can be found in endosomes and induce TLR-7 mediated antiviral signaling and/or viral components transferred to multivesicular class II loading compartments. If this is the case, TLR-7/-8 mediated anitviral signaling might be inhibited by the orf4a protein (in the case of MERS-CoV and SARS-CoV) whereas the MHC- Class II mediated activation of cytotoxic T lymphocytes might not be inhibited and indeed contribute in particular to the disease outcome of SARS-CoV or MERS-CoV infected patients.  

Coronavirus nsp-3,-4, and -6 might contribute to the induction of autophagy vesicle induced antiviral response counteracted by orf4a

The infection of microglia cells with a neurotropic strain of MHV, MHV-JHM, indeed lead to a sustained up-regulation of both MHC Class I and Class II molecules not only during viral induced inflammation but also following viral clearance, similar to patients which have recovered from SARS. The formation of LC3-II positive vesicles therefore might induce an antiviral response that might be partially blocked by viral proteins. Some evidence suggests that the formation of LC3-II is more pronounced in cells transfected with SARS-CoV derived nsp6 compared to IBV derived nsp6 but if this has any implication for inducing an antiviral response has to be investigated.

Last but not least, a short note on STAT1 and 2 mediated signaling, STAT 1 and 2 signaling can be inhibited by SARS-CoV orf6 protein which in contrast to the above mentioned proteins is not residing within the RTC but at the ER and Golgi membrane where it sequesters STAT1 and 2 and thus prevents nuclear entry of these. In this case, orf6 therefore prevents the activation of IFN-β stimulated genes.

The emerging MERS-CoV has been shown to infect a wide variety of cells of the immune response, including dendritic cells, macrophages and T-Lymphocytes. It remains to be seen if the antiviral genes encoded by MERS-CoV are differ in their ability to block antiviral signaling compared to those found in SARS-CoV or HCoV-NL63. It also remains to be seen if the antiviral signaling in dromedary camels or in bats infected with MERS-CoV is less vulnerable to these proteins or not - not an easy task in dromedaries- and if the differences in the antiviral response account for the relatively benign outcome in these animals. In humans, it would be interesting to see if TLR-8 (restricted to myeloid dendrite cells, monocytes, and monocyte derived dendritic cells) is differently affected by MERS-CoV derived proteins than TLR-7 (plasmacytoid dendrite cells). One might argue that these are marginal questions, but I would argue that it might shed some light on the pathogenesis of MERS. 

The importance of antagonizing the antiviral response does not end at preventing the viral RNA from being recognised. A recent study indicated that the HCoV-OC43 Nucleocapsid protein binds microRNA9 and prevents a NF-κΒ dependent antiviral response and thus TLR-8 antiviral signaling; . In addition, the MERS-CoV derived orf4b and SARS-CoV N protein also block NF-κΒ. Moreover, the SARS-CoV nsp1 papain-like protease inhibits IRF3 induced expression of IFN-β. It remains of course to be seen if the mechanisms described for SARS-CoV or HCoV-OC43 also apply to MERS-CoV and more importantly if these mechanisms differ among various host species.

Coronavirus derived proteins block the antiviral signaling at various stages

As the reader of these lines can see, there is much to be learned from a virus family which until 2003 has been considered to be only a marginal virus by many.  

Further reading

Kindler E, & Thiel V (2014). To sense or not to sense viral RNA-essentials of coronavirus innate immune evasion. Current opinion in microbiology, 20C, 69-75 PMID: 24908561 

Zinzula L, & Tramontano E (2013). Strategies of highly pathogenic RNA viruses to block dsRNA detection by RIG-I-like receptors: hide, mask, hit. Antiviral research, 100 (3), 615-35 PMID: 24129118 

Redfern AD, Colley SM, Beveridge DJ, Ikeda N, Epis MR, Li X, Foulds CE, Stuart LM, Barker A, Russell VJ, Ramsay K, Kobelke SJ, Li X, Hatchell EC, Payne C, Giles KM, Messineo A, Gatignol A, Lanz RB, O'Malley BW, & Leedman PJ (2013). RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators. Proceedings of the National Academy of Sciences of the United States of America, 110 (16), 6536-41 PMID: 23550157 

English, L., Chemali, M., Duron, J., Rondeau, C., Laplante, A., Gingras, D., Alexander, D., Leib, D., Norbury, C., Lippé, R., & Desjardins, M. (2009). Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection Nature Immunology, 10 (5), 480-487 DOI: 10.1038/ni.1720 

English L, Chemali M, & Desjardins M (2009). Nuclear membrane-derived autophagy, a novel process that participates in the presentation of endogenous viral antigens during HSV-1 infection. Autophagy, 5 (7), 1026-9 PMID: 19556870 

Paludan C, Schmid D, Landthaler M, Vockerodt M, Kube D, Tuschl T, & Münz C (2005). Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science (New York, N.Y.), 307 (5709), 593-6 PMID: 15591165 

Cavignac Y, & Esclatine A (2010). Herpesviruses and autophagy: catch me if you can! Viruses, 2 (1), 314-33 PMID: 21994613

Taylor GS, & Rickinson AB (2007). Antigens and autophagy: the path less travelled? Autophagy, 3 (1), 60-2 PMID: 17102586 

Züst R, Cervantes-Barragan L, Habjan M, Maier R, Neuman BW, Ziebuhr J, Szretter KJ, Baker SC, Barchet W, Diamond MS, Siddell SG, Ludewig B, & Thiel V (2011). Ribose 2'-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nature immunology, 12 (2), 137-43 PMID: 21217758

Chen Y, Su C, Ke M, Jin X, Xu L, Zhang Z, Wu A, Sun Y, Yang Z, Tien P, Ahola T, Liang Y, Liu X, & Guo D (2011). Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex. PLoS pathogens, 7 (10) PMID: 22022266 

Cao J, & Zhang X (2012). Comparative in vivo analysis of the nsp15 endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses. Virus research, 167 (2), 247-58 PMID: 22617024 

Li T, Xie J, He Y, Fan H, Baril L, Qiu Z, Han Y, Xu W, Zhang W, You H, Zuo Y, Fang Q, Yu J, Chen Z, & Zhang L (2006). Long-term persistence of robust antibody and cytotoxic T cell responses in recovered patients infected with SARS coronavirus. PloS one, 1 PMID: 17183651

Hamo L, Stohlman SA, Otto-Duessel M, & Bergmann CC (2007). Distinct regulation of MHC molecule expression on astrocytes and microglia during viral encephalomyelitis. Glia, 55 (11), 1169-77 PMID: 17600339 

Frieman M, Yount B, Heise M, Kopecky-Bromberg SA, Palese P, & Baric RS (2007). Severe acute respiratory syndrome coronavirus ORF6 antagonizes STAT1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/Golgi membrane. Journal of virology, 81 (18), 9812-24 PMID: 17596301 

Lai FW, Stephenson KB, Mahony J, & Lichty BD (2014). Human coronavirus OC43 nucleocapsid protein binds microRNA 9 and potentiates NF-κB activation. Journal of virology, 88 (1), 54-65 PMID: 24109243

The nature of Viral Replication Centers: Coronavirus and Hepatitis C Virus RTCs

Positive strand RNA viruses are replicating in association with cytoplasmic membranes of infected cells which contains in general not viral RNA but also the machinery required for genome replication, namely the RNA dependent RNA Polymerase (RdRp) as well as accessory enzymes such as a viral RNA helicase. Whilst in most cases the RNA transcription complex (RTC) is localized and associated with the ER, in some cases the RTC is associated with the outer mitochondrial membrane (OMM) (Nodaviridae), the endosome/lysosome (Togaviridae), or even Peroxisomes and Chloroplasts (Tomboviridae); a special case are the lipid droplets formed upon Hepatitis C Virus infection, which are localized to the actin/microtubuli of the cytoskeleton. The replication enzymes -virus specific- become associated to the respective RTC early in infection process however not by the viral RNA but by the non-structural proteins (nsp’s) whose expression is sufficient to induce the formation of membrane structures which ultimately form the RTC - in other words, the replication proteins are targeted to the respective organelle by the nsp rather than the viral RNA. In order to form these structures, most of the nsp’s are integral proteins with multiple or single transmembrane domains (as in the case of Coronaviral nsp-3/-4/-6 proteins and Hepatitis C Virus NS4A/NS5A) or are attached to the membrane via a binding peptide (Semliki Forest virus nsp1). 

                         

Examples of Positive Strand RNA viruses inducing rearrangements of cellular membranes 

Coronavirus: Formation of DMV s containing the RTC adjacent to the ER

The RTC of the Coronaviridae consists of double-membrane vesicles (DMVs) in which the nsps required for the replication of the viral genome are anchored in addition to the viral genome and -once transcription of the genome starts- intermediate dsRNA can be detected by in situ hybridization or EU labeling of nascent newly synthesized RNA. In addition to viral proteins and viral RNA, the DMVs also contain host derived proteins such as the previously discussed EDAM1 or RNA binding proteins such as hnRNP, Polypyrimidine tract-binding protein (PTB), T-cell intracellular antigen-1 (TIA-1), and TIA-1-related protein (TIAR). RNA binding proteins are believed to be required to switch from replicating the viral genome to viral transcription by binding to the transcription regulating sequences; indeed, silencing of PTB has a negative effect on Coronaviral RNA accumulation but increases viral titer. The precise mechanism of targeting the viral RNA to sites of DMV formation/RTC assembly is not known but it has been postulated that the viral Nucleocapsid protein might be involved since it binds viral RNA and can be detected within DMVs.

The DMVs itself are derived from the cellular ER and regularly form a reticulovesicular network of modified ER membranes into which the viral nsp-3,-4, and -6 are inserted (also referred to as convoluted membranes) and induce a process known as “membrane zipping” or “membrane pairing”  which is succeeded  by the wrapping of membrane cisternae around cytoplasmic constituents in a process resembling the formation of autophagosomes or -to be more precise- the formation of  EDEMosomes in which the DMV forms a bud which eventually is released from the ER. Similar to the latter, the DMVs are believed to contain LC3-I although as I pointed out before it might be possible that the DMVs might be LC3-II positive. 
This notion is supported by evidence suggesting that the Coronaviral nsp6 protein can induce the formation of LC3-II positive  omegasomes that mature into autophagosomes; the induction of autophagy seems to be dispensable for the replication of SARS-CoV, MHV, and the avian Infectious Bronchitis Virus. If RTCs are also LC3-II positive than one of the question which needs to be answered is how the DMV/RTC escapes degradation - or maybe the answer in the end might be that LC3-II positive cannot be detected precisely because they are unstable and degraded. Since EDEMosomes are normally targeted for degradation in a LC3-II independent manner, the question remains why the EDEMosome-like Coronaviral DMVs are not degraded as well.

Further experiments might be worthwhile doing are to investigate if the DMVs undergo membrane fission in a RabGTPase dependent or independent manner. Here the use of the various RabGTPase mutants available might give us some answers - again is anybody up to the challenge?  Interestingly, two Coronavirus nsps, nsp-2 and -3, are located outside of the mature DMV are involved in inhibiting host cell pathways involving Prohibitin-1 and -, two proteins involved in modulating transcription as well as in maintaining mitochondrial function and morphology. It remains to be seen to which extent DMV bound nsp-2/-3 interferes with these processes.

Coronavirus nsp-3, -4, and -6 induce the formation of LC3-I positive EDEMosome like DMVsand hypothetically may form LC3-II positive DMVs

                    Hepatitis C Viruses: Role of the NS4B and NS5A proteins

The infection of cells with Hepatitis C Virus (HCV), a member of the Flaviviridae, triggers the formation of n membranous vesicles of heterogeneous size and morphology, which are associated with viral replication. Although originally it has been postulated that both structural and non-structural proteins are required for the formation of these vesicular structures, more recent results indicate that non-structural proteins NS4A and NS5B are inducing a membranous web and are able to recruit viral RNA independent of the structural Core-E1-E2-p7 complex as well as the NS3-4A complex. The entire HCV polyprotein however is required for membrane budding into the rER.

HCV NS4B is a 261 AA long protein, consisting of N- and C- terminal cytoplasmic domains and a central domain with four transmembrane helices and localizes to the ER in infected as well as transfected cells, although a small proportion of NS4B localizes either at the surface of Lipid droplets (LDs) or ER membranes associated with LDs.

Mutation analysis of full length NS4B showed that hydrophobic amino acids in amphipathic helix bind to and are critical for binding LDs and mutation of these residues caused a severe reduction in viral replication, suggesting that NS4B induced recruitment of LDs is critical for viral replication.  

In contrast to NS4B, the viral NS5A protein has multiple functions during the replication of HCV. As NS4B, NS5A localizes to the ER, where it interacts with viral proteases (NS2 and NS3/4A), the viral RdRp (NS5B) and the NS4B protein as well with the viral RNA (specifically 3’ untranslated region (UTR) ) and the viral Core protein. In addition, NS5A binds to at least one host cell protein that is found on the surface of LDs, Tail-Interacting Protein 47 (TIP47) via the N terminal PAT domain of TIP47, and thus relocalizes LDs to sites of HCV replication. siRNA mediated knockdown of TIP47 or mutations of NS5A prevent the recruitment of LDs and reduces viral replication whereas overexpression of TIP47 has the opposite effect, thus highlighting the importance of LDs for the replication of HCV. Although the function of NS5A and NS4B with regards to the recruitment of LDs seems to be similar on the first glance, NS5A binds to TIP47 localized on the surface of LDs followed by insertion of NS5A into the ER (although contradictory results suggest that only NS5A bound to viral RNA and/or Core recruits TIP47 positive LDs) by a mechanism that the author of these lines refers to as “capture”.

   

HCV NS4B and NS5A proteins "capture" LDs

So what is the role of TIP47? In non-infected cells, LDs are lipid storage organelles containing neutral lipids (triacylglycerides and sterol esters) which are surrounded by a phospholipid monolayer containing a number of associated proteins, including TIP47 that belongs to the LD associated Perilipin, ADRP and TIP47 (PAT) family. PAT domain containing proteins are coating the surface of LDs and involved in the turnover and stability of LDs. TIP47 is also found on late endosomes to which it is recruited by Rab9-GTPase which might facilitate the formation of transport vesicles. If these results stand the test of time, then the recruitment of TIP47 by NS5A to sites of viral replication might also recruit Rab9-GTPase, or the related Rab5-GTPase, which might be required for the formation of viral particles and trafficking to the endosome, the site of viral egress and released. Indeed, Rab9-GTPase expression is upregulated in HCV infected cells although NS5A does induce only a slight relocalisation of Rab9-GTPase suggesting that endosome-to-Golgi trafficking might be increased, whereas Rab5-GTPase co-localises with both NS5A and NS4B at the ER.  
TIP47 was originally described to function in the retrograde vesicular-membrane transport, whereby lysosomal enzymes are delivered to endosomes prior to recycling in the Golgi and thus be involved in the formation of transport vesicles. It seems feasible that this is precisely the role of TIP47 in HCV replication; to allow the transport of new viral particles. Personally I am curious to see if in TIP47 knockout cells transport vesicles are accumulating in the cytoplasm of the cell. It is well known that newly synthesized HCV particles are transported from early to late endosomes and subsequently to the Golgi, so is TIP47 maybe required for this step? Who knows….

One final word: the knowledge of the intricate details of the composition of the RTC not only of positive strand RNA viruses but also for DNA viruses, negative strand RNA viruses, or Retroviral agents might lead not only to better understanding of the interactions of viruses with the host cell but also lead to the discovery and development of new antiviral drugs as described for HCV. Until then, however, let us marvel in the wonderful world of viruses and their interactions with the host!

Further reading

Hagemeijer, M., Verheije, M., Ulasli, M., Shaltiel, I., de Vries, L., Reggiori, F., Rottier, P., & de Haan, C. (2009). Dynamics of Coronavirus Replication-Transcription Complexes Journal of Virology, 84 (4), 2134-2149 DOI: 10.1128/JVI.01716-09

Knoops, K., Kikkert, M., Worm, S., Zevenhoven-Dobbe, J., van der Meer, Y., Koster, A., Mommaas, A., & Snijder, E. (2008). SARS-Coronavirus Replication Is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum PLoS Biology, 6 (9) DOI: 10.1371/journal.pbio.0060226


Maier HJ, Hawes PC, Cottam EM, Mantell J, Verkade P, Monaghan P, Wileman T, & Britton P (2013). Infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes. mBio, 4 (5) PMID: 24149513


Sola I, Galán C, Mateos-Gómez PA, Palacio L, Zúñiga S, Cruz JL, Almazán F, & Enjuanes L (2011). The polypyrimidine tract-binding protein affects coronavirus RNA accumulation levels and relocalizes viral RNAs to novel cytoplasmic domains different from replication-transcription sites. Journal of virology, 85 (10), 5136-49 PMID: 21411518

Sola I, Mateos-Gomez PA, Almazan F, Zuñiga S, & Enjuanes L (2011). RNA-RNA and RNA-protein interactions in coronavirus replication and transcription. RNA biology, 8 (2), 237-48 PMID: 21378501

Cottam EM, Maier HJ, Manifava M, Vaux LC, Chandra-Schoenfelder P, Gerner W, Britton P, Ktistakis NT, & Wileman T (2011). Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate. Autophagy, 7 (11), 1335-47 PMID: 21799305

Maier HJ, & Britton P (2012). Involvement of autophagy in coronavirus replication. Viruses, 4 (12), 3440-51 PMID: 23202545

Verheije MH, Hagemeijer MC, Ulasli M, Reggiori F, Rottier PJ, Masters PS, & de Haan CA (2010). The coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes. Journal of virology, 84 (21), 11575-9 PMID: 20739524

Nagy PD, & Pogany J (2011). The dependence of viral RNA replication on co-opted host factors. Nature reviews. Microbiology, 10 (2), 137-49 PMID: 22183253

Egger D, Wölk B, Gosert R, Bianchi L, Blum HE, Moradpour D, & Bienz K (2002). Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. Journal of virology, 76 (12), 5974-84 PMID: 12021330

Tanaka T, Kuroda K, Ikeda M, Wakita T, Kato N, & Makishima M (2013). Hepatitis C virus NS4B targets lipid droplets through hydrophobic residues in the amphipathic helices. Journal of lipid research, 54 (4), 881-92 PMID: 23315449

Foster, T., Belyaeva, T., Stonehouse, N., Pearson, A., & Harris, M. (2010). All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding Journal of Virology, 84 (18), 9267-9277 DOI: 10.1128/JVI.00616-10

Vogt DA, Camus G, Herker E, Webster BR, Tsou CL, Greene WC, Yen TS, & Ott M (2013). Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein. PLoS pathogens, 9 (4) PMID: 23593007

Ploen D, Hafirassou ML, Himmelsbach K, Sauter D, Biniossek ML, Weiss TS, Baumert TF, Schuster C, & Hildt E (2013). TIP47 plays a crucial role in the life cycle of hepatitis C virus. Journal of hepatology, 58 (6), 1081-8 PMID: 23354285

 Lai CK, Jeng KS, Machida K, & Lai MM (2010). Hepatitis C virus egress and release depend on endosomal trafficking of core protein. Journal of virology, 84 (21), 11590-8 PMID: 20739534

Kohler JJ, Nettles JH, Amblard F, Hurwitz SJ, Bassit L, Stanton RA, Ehteshami M, & Schinazi RF (2014). Approaches to hepatitis C treatment and cure using NS5A inhibitors. Infection and drug resistance, 7, 41-56 PMID: 24623983