Jpn. J. Infect. Dis., 63, 307-311, 2010

Invited Review A Virus-Host Interaction Involved in Viral Replication: toward the Identification of Antiviral Targets

Tetsuro Suzuki* Department of Infectious Diseases, Hamamatsu University School of Medicine, Hamamatsu 431-3192; and Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan (Received July 13, 2010) CONTENTS: 1. Introduction 4. Advances in HCV research concerning viral replica- 2. Treatment tion 3. Drugs in current clinical development 5. Future perspectives

SUMMARY: (HCV) infection is a leading cause of chronic liver disease. The current standard therapy for hepatitis C patients, which is based on a combination of pegylated and , results in viral clearance in about 50z of the treated individuals. Clinical trials of a variety of specific anti-HCV drugs, including several which target virus-encoded enzymes, are on-going, and some of these studies have reported impressive reductions of HCV levels in patients. However, the develop- ment of antivirals with diverse mechanisms of action is still required to eliminate this life-threatening vi- rus. Besides specific viral proteins, targeting host cellular factors that are key to efficient viral replica- tion could lead to the development of novel treatment strategies. Therapies against host factors are generally considered to present a low risk of generating drug-resistant viruses. The current under- standing of anti-HCV drugs in clinical development and of virus-host interactions implicated in the regulation of HCV replication is summarized.

fined by undetectable viral RNA in a sensitive quantita- 1. Introduction tive assay 6 months after the end of therapy. Treatment HepatitisCvirus(HCV)infectionisamajorcauseof of chronic HCV infection is currently based on inter- chronic hepatitis, liver cirrhosis, and hepatocellular car- feron (IFN)-alpha due to its potent antiviral and im- cinoma (HCC). Indeed, this virus is responsible for munomodulatory properties. The current standard pro- almost 75z of all cases of HCC in Japan (1,2). HCV is tocol for such treatment involves weekly injections of primarily transmitted by blood-borne routes, such as pegylated IFN-alpha together with twice-daily oral blood transfusion, shared needles, and blood products. ribavirin. Ribavirin, a member of the nucleoside an- A recent WHO estimate suggests that a minimum of timetabolite drugs, is a pleiotropic agent that may act 2–3z of the world's population is chronically infected against multiple targets, although the exact mechanism with HCV (3). Despite the fact that HCV is targeted by of its role in hepatitis C therapy remains elusive. innate, cellular, and humoral immune mechanisms, it IFN/ribavirin treatment often has serious side effects, can establish persistent infection in a majority of the in- including depression, flu-like symptoms, and hemolytic fected population. Unfortunately, a protective vaccine anemia (4). Furthermore, there are wide differences in is not yet available, and therapeutic options are still the response to the treatment depending on the charac- limited. teristics of the virus, such as viral loads and viral geno- types, as well as host genetic variations. Only around 50z of patients treated are estimated to achieve SVR 2. Treatment (5). Few treatment options are currently available for Successful treatment against hepatitis C is character- non-responders infected with HCV genotype 1, which is ized by a ``sustained virological response'' (SVR), as de- considered to be the most resistant to IFN therapy. It is noteworthy that IFN and ribavirin are not specific an- tiviral agents designed for HCV, which means that new * Corresponding author: Mailing address: Department of approaches to improve response rates are needed. The Infectious Diseases, Hamamatsu University School of development of novel and more effective antiviral drugs Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu to treat liver diseases with HCV infection is therefore a 431-3192, Japan. Tel: ＋81-53-435-2336, Fax: ＋81-53- major challenge. 435-2338, E-mail: tesuzuki＠hama-med.ac.jp

This article is an Invited Review based on a lecture 3. Drugs in current clinical development presented at the 19th Symposium of the National Institute HCV is a positive-strand RNA virus classified in the of Infectious Diseases, Tokyo, June 2, 2009. Hepacivirus genus within the Flaviviridae family. Its ap-

307 proximately 9.6-kb genome contains an open reading are effective and well tolerated, although some of them frame encoding a polyprotein precursor of approxi- have been discontinued after Phase II trials due to their mately 3,000 amino acids (aa) flanked by untranslated side-effects. regions (UTRs) at both ends. The 5? UTR, which is NS5A, which has no enzymatic activity but appears to ¿341 nucleotides (nts) long, contains an internal be a key player during regulation of the HCV lifecycle, ribosomal entry site (IRES), which is essential for cap- would appear to be another valid target for antivirals. independent translation of viral RNA and from which BMS-790052 (by Bristol-Myers Squibb) (21), which is in four highly structured domains (domains I–IV) are Phase I trials, was reported to cause an impressive produced (6–8). The 3? UTRvariesinlengthbetween reduction in HCV titers of all the genotypes tested at 200 and 235 nts, including a short variable region, a very low concentrations. Although its antiviral mechan- poly(U/UC) tract with an average length of 80 nts, and ism is still unclear, these promising results provide a a virtually invariant 98-nt X-tail region (9–11). This X proof of concept for targeting the non-enzymatic region forms three stable stem-loop structures and, as a properties of HCV proteins when developing new result, the HCV genome probably ends with a double- chemotherapies. strand stem structure. It appears that the 3? Xregion and the poly(U/C) tract are crucial for RNA replica- 4. Advances in HCV research concerning tion, whereas the remainder of the 3? UTR plays a role viral replication in enhancement of replication. The genome is translated into a single polypeptide of about 3,000 aa in which the An improved understanding of the viral lifecycle structural proteins Core, E1, and E2 reside in the N-ter- should contribute to the development of innovative minal region. A crucial function of Core involved in as- therapeutic and preventive strategies for liver diseases sembly of the viral nucleocapsid. The aa sequence of caused by HCV infection. In addition to specific viral this protein is well conserved among different HCV proteins, targeting host cellular factors which are vital strains in comparison with other HCV proteins. The for efficient viral replication may lead to the develop- non-structural (NS) proteins NS3–NS5B are considered ment of novel treatment strategies. In general, therapies to assemble into a membrane-associated HCV RNA against host factors are considered to present a low risk replicase complex, with NS3 possessing the enzymatic of generating drug-resistant viruses. This chapter sum- activities of serine protease and RNA helicase and marizes the interactions between HCV and host factors NS4A serving as a cofactor for NS3 protease. NS4B that may be possibly involved in viral replication. plays a role in the remodeling of host cell membranes, As mentioned above, IRES-dependent translation of probably to generate the site for replicase assembly. the viral open reading frame yields a polyprotein precur- NS5A has been considered to play an important but sorthatisprocessedbycellularandviralproteases(22). undefined role in viral RNA replication. Recently, we In addition to proteins involved in the translational and other research groups reported that NS5A is also a machinery, such as eukaryotic initiation factors, several key molecule for regulating the early phase of HCV par- host cell proteins, including polypyrimidine tract bind- ticle formation by interacting with Core protein (12–14). ing protein (PTB) (23), La autoantigen (24–26), RNA NS5B functions as the RNA-dependent RNA poly- helicase A, nuclear factors NF90, NF110, NF45 (27), merase (RdRp). heterogenous nuclear ribonucleoproteins (hnRNPs) As is the case with other viruses, such as HIV, inhibi- (28–30), and FUSE binding protein (31), have been tors targeting HCV-encoding enzymes that are essential reportedtointeractwiththe5?-or3? UTRs of the HCV to the viral life cycle have primarily been developed genome. Of these, hnRNP A1, which is implicated in as anti-HCV drugs by pharmaceutical companies several RNA metabolic process such as transport cellu- worldwide, as described in recent reviews (15–17). Many lar , was found to bind to both 5?-and3? UTR HCV-specific inhibitors are in preclinical development regions (30). This protein also interacts with the scaffold and several have reached clinical trials. Although protein septin 6 and HCV NS5B in cells, thus suggesting NS3-4A protease has received the most attention as a that hnRNP A1 and septin 6 play a key role in viral drug target, its unusual structural features, such as shal- replication by forming a complex with NS5B and the low active binding site, make it difficult to design small viral RNA genome. molecule inhibitors. The first HCV protease inhibitor to Cyclophilins (Cyps) and FKBPs are classified as im- enter clinical trials, namely BILN-2061 (by Boehringer munophilins that are capable of binding to the im- Ingelheim), showed impressive antiviral activity in munosuppressants cyclosporine and FK506, respec- chronic hepatitis C patients (18). However, it was later tively. Cyps are present with peptidyl-prolyl cis-trans withdrawn due to cardiotoxicity in animals. At present, isomerase (PPI) activity and are involved in de novo VX-950 (by Vertex) (19) and SCH503034 (by Schering- protein folding and isomerization of native proteins. Plough) (20), both of which have shown an ability to in- Furthermore, there is evidence to suggest that cyclophi- crease SVR rates in combination with IFN/ribavirin rel- lin B (CypB) is a positive modulator of HCV RdRp in ative to standard therapy, are the most advanced HCV the viral replication complex (32). CypB interacts specif- inhibitors. The inhibitors of another major target for ically with NS5B, thereby increasing the RNA binding anti-HCV drugs, namely NS5B, belong to two catego- property of NS5B. Recent studies demonstrated that cy- ries: nucleoside/nucleotide inhibitors target the catalytic clophilin A (CypA) functions as a cofactor for HCV in- site of the enzyme, whereas non-nucleoside inhibitors fection, with the PPI activity of CypA being shown to target the allosteric sites of the RdRp. Several com- play a key role in viral replication (33,34). Another im- pounds of both categories are currently in Phase I or II munophilin, FK506-binding protein 8 (FKBP8), inter- trials. Early clinical data indicate that several inhibitors acts specifically with HCV NS5A and recruits a molecu-

308 lar chaperone, heat shock protein 90 (Hsp90), to the through the formation of homo- and/or heterodimers viral RNA replication complex as a result of an interac- with VAP-A (42). VAP-A and VAP-B form hetero- and tion between the carboxylate clump structure of FKBP8 homodimers through their transmembrane regions and and the C-terminal MEEVD motif of Hsp90. Knock- interact with both NS5A and NS5B. VAP-C, the third down of FKBP8 reduced the replication efficiency of subtype of VAP, which is an alternative spliced isoform the HCV genome in replicon and HCV-infected cells, of VAP-B, has recently been shown to act as a negative thereby suggesting that FKBP8 is required for the repli- regulator for HCV propagation and to be partly in- cation of HCV via formation of the replication complex volved in the determination of the tissue specificity of (35,36). Several studies demonstrated that Hsp90 and HCV replication (46). Statins, which decrease the cochaperones may participate in formation of the HCV production of mevalonate by inhibiting 3-hydroxy-3- replication complex through interaction with NS5A or methylglutaryl CoA reductase, have been shown to in- other HCV proteins (37–40). The chaperone activity of hibit HCV RNA replication (44,47). This effect can be Hsp90 contributes to the refolding of an unfolded pro- reversed by adding geranylgeraniol, thus suggesting that tein in an ATP-dependent manner. Geldanamycin and viral replication requires geranylgeranylated proteins. its derivatives, which are known to be as specific Hsp90 The geranylgeranylated protein, FBL2, which contains inhibitors, are known to lead to degradation of the sub- an F-box motif and is therefore likely involved in pro- strate proteins. Human butyrate-induced transcript 1 tein degradation, plays a role in HCV RNA replication (hB-ind1), which possesses a significant homology with via its interaction with NS5A (45). the cochaperone p23, is also involved in the propagation Viral replication requires energy and macromolecule of HCV through interaction with NS5A and Hsp90. synthesis, and it is the host cells which provide the virus HCV NS5A has also been shown to interact with with the metabolic resources necessary for efficient other cellular proteins such as vesicle-associated mem- replication. It is therefore highly likely that the interac- brane protein (VAMP)-associated protein (VAP) sub- tion of viruses with host-cell metabolic pathways, in- types A (VAP-A) and B (VAP-B) and FBL2 (41–45). cluding energy-generating systems, affects the viral VAP-A is detected in a detergent-resistant membrane growth cycle. fraction containing the viral replication complex, We have recently demonstrated that creatine kinase B whereas the interaction of VAP-A with NS5A is re- (CKB), a key ATP-generating enzyme, interacts with quired for efficient replication of HCV genomic RNA HCV NS4A and is important for efficient replication of (41). VAP-B also participates in HCV replication the viral genome (48). Recruitment of CKB to the HCV

Table 1. HCV-interacting host factors involved in the regulation of the viral replication

Host factor Interacting HCV factor Reference

alpha-Actinin NS5B Lan et al. (53) ATM NS3/4A, NS5B Ariumi et al. (54), Lai et al. (55) hB-ind1 NS5A Taguwa et al. (38, 40) ChK2 NS5B Ariumi et al. (54) Creatine kinase B NS4A Hara et al. (48) Cyclophilin A/B NS5A, NS5B Liu et al. (33), Kaul et al. (34), Watashi et al. (32) DDX3 Core Ariumi at al. (56) FBL2 NS5A Wang et al. (45) FUSE binding protein NS5A Zhang et al. (31) FKBP8 NS5A Okamoto et al. (35, 36) Hsp27 NS5A Choi et al. (57) Hsp90 NS5A, NS3 Nakagawa et al. (37), Ujino et al. (39) La autoantigen UTR RNA Ali et al. (24, 25), Isoyama et al. (26) M2PK NS5B Wu et al. (58) Nuclear factors NF90, NF110, NF45 UTR RNA Isken et al. (27) Nucleolin NS5B Shimakami et al. (59) PI4K IIIalpha NS5A Berger et al. (60), Ahn et al. (61) PTB UTR RNA Anwar et al. (23) Rab5 NS4B Stone et al. (62) Raf-1 kinase NS5A Burckstummer et al. (63) RNA helicase p68 NS5B Goh et al. (64) hnRNP A1 UTR RNA Kim et al. (30) hnRNP C UTR RNA Gontarek et al. (28) hnRNP L UTR RNA Hahm et al. (29) Septin 6 NS5B Kim et al. (30) Sphingomyelin NS5B Sakamoto et al. (65) SYNCRIP UTR RNA Liu et al. (66), Kim et al. (67) TBC1D20 NS5A Sklan et al. (68) TRAF2 NS5A Park et al. (69) VAP-A/B/C NS5A, NS5B Gao et al. (41), Hamamoto et al. (42), Kukihara et al. (46)

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