Toll-like receptors (TLRs) bind pathogen-specific ligands early in infection, initiating signaling pathways that lead to expression of multiple protective cellular genes. Many viruses have evolved strategies that block the effector mechanisms induced through these signaling pathways, but viral interference with critical proximal receptor interactions has not been described. We show here that the NS3/4A serine protease of hepatitis C virus (HCV), a virus notorious for its ability to establish persistent intrahepatic infection, causes specific proteolysis of Toll-IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF or TICAM-1), an adaptor protein linking TLR3 to kinases responsible for activating IFN regulatory factor 3 (IRF-3) and NF-kappaB, transcription factors controlling a multiplicity of antiviral defenses. NS3/4A-mediated cleavage of TRIF reduces its abundance and inhibits polyI:C-activated signaling through the TLR3 pathway before its bifurcation to IRF-3 and NF-kappaB. This uniquely broad mechanism of immune evasion potentially limits expression of multiple host defense genes, thereby promoting persistent infections with this medically important virus.

https://www.pnas.org/content/pnas/102/8/2992.full.pdf

Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF

Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease–helicase

Infection with hepatitis C virus (HCV) is a major medical problem with over 170 million people infected worldwide. Substantial morbidity and mortality are associated with hepatic manifestations (cirrhosis and hepatocellular carcinoma), which develop with increasing frequency in people infected with HCV for more than 20 years. Less well known is the burden of HCV disease associated with extrahepatic manifestations (diabetes, B-cell proliferative disorders, depression, cognitive disorders, arthritis and Sjogren's syndrome). For patients infected with genotype 1 HCV, treatment with polyethylene glycol decorated interferon (peginterferon) α and ribavirin (PR) is associated with a low (40-50%) success rate, substantial treatment-limiting side effects and a long (48-week) duration of treatment. In the past 15 years, major scientific advances have enabled the development of new classes of HCV therapy, the direct-acting antiviral agents, also known as specifically targeted antiviral therapy for hepatitis C (STAT-C). In combination with PR, the HCV NS3-4A protease inhibitor telaprevir has recently been approved for treatment of genotype 1 chronic HCV in the United States, Canada, European Union and Japan. Compared with PR, telaprevir combination therapy offers significantly improved viral cure rates and the possibility of shortened treatment duration for diverse patient populations. Developers of innovative drugs have to blaze a new path with few validated sign posts to guide the way. Indeed, telaprevir's development was once put on hold because of its performance in a standard IC(50) assay. Data from new hypotheses and novel experiments were required to justify further investment and reduce risk that the drug might fail in the clinic. In addition, the poor drug-like properties of telaprevir were a formidable hurdle, which the manufacturing and formulation teams had to overcome to make the drug. Finally, novel clinical trial designs were developed to improve efficacy and shorten treatment in parallel instead of sequentially. Lessons learned from the development of telaprevir suggest that makers of innovative medicines cannot rely solely on traditional drug discovery metrics, but must develop innovative, scientifically guided pathways for success.

Discovery and development of telaprevir: an NS3-4A protease inhibitor for treating genotype 1 chronic hepatitis C virus

The family Flaviviridae contains three genera: Hepacivirus, Flavivirus, and Pestivirus. Worldwide, more than 170 million people are chronically infected with Hepatitis C virus and are at risk of developing cirrhosis and/or liver cancer. In addition, infections with arthropod-borne flaviviruses (such as dengue fever, Japanese encephalitis, tick-borne encephalitis, St. Louis encephalitis, Murray Valley encephalitis, West Nile, and yellow fever viruses) are emerging throughout the world. The pestiviruses have a serious impact on livestock. Unfortunately, no specific antiviral therapy is available for the treatment or the prevention of infections with members of the Flaviviridae. Ongoing research has identified possible targets for inhibition, including binding of the virus to the cell, uptake of the virus into the cell, the internal ribosome entry site of hepaciviruses and pestiviruses, the capping mechanism of flaviviruses, the viral proteases, the viral RNA-dependent RNA polymerase, and the viral helicase. In light of recent developments, the prevalence of infections caused by these viruses, the disease spectrum, and the impact of infections, different strategies that could be pursued to specifically inhibit viral targets and animal models that are available to study the pathogenesis and antiviral strategies are reviewed.

https://cmr.asm.org/content/cmr/13/1/67.full.pdf

Perspectives for the Treatment of Infections with Flaviviridae

The flaviviruses dengue, West Nile, and Japanese encephalitis represent three major mosquito-borne viruses worldwide. These pathogens impact the lives of millions of individuals and potentially could affect non-endemic areas already colonized by mosquito vectors. Unintentional transport of infected vectors (Aedes and Culex spp.), traveling within endemic areas, rapid adaptation of the insects into new geographic locations, climate change, and lack of medical surveillance have greatly contributed to the increase in flaviviral infections worldwide. The mechanisms by which flaviviruses alter the immune and the central nervous system have only recently been examined despite the alarming number of infections, related deaths, and increasing global distribution. In this review, we will discuss the expansion of the geographic areas affected by flaviviruses, the potential threats to previously unaffected countries, the mechanisms of pathogenesis, and the potential therapeutic interventions to limit the devastating consequences of these viruses.

/pdf/flaviviruses-an-expanding-threat-in-public-health-focus-on-1v4ixb2iuc.pdf

Flaviviruses, an expanding threat in public health: focus on dengue, West Nile, and Japanese encephalitis virus

The major etiological agent of non-A, non-B hepatitis was identified in 1989 and named hepatitis C virus (HCV) (8, 23). Presently, it is estimated that approximately 1% of the human population is infected by HCV (42). Exposure to HCV results in an overt acute disease in only a small percentage of cases, while in most instances the virus establishes a chronic infection which causes liver inflammation and slowly progresses to liver failure and cirrhosis (24). In addition, seroepidemiological surveys have indicated an important role of HCV in the pathogenesis of hepatocellular carcinoma (27). The absence of a protective vaccine and the limited efficacy of alpha interferon treatment (55) have raised considerable interest in developing alternative anti-HCV therapies.

The genetic organization of HCV is similar to that of flaviviruses and pestiviruses (9, 37), and therefore HCV was assigned to a separate genus of the family Flaviviridae (43). The HCV genome consists of a single-stranded RNA of about 9.5 kb in length encoding a precursor polyprotein of 3,010 to 3,033 amino acids (8, 9, 26, 50). Individual viral proteins are produced by proteolysis of the precursor: the putative structural proteins (C, E1, E2, and p7) span the amino-terminal third of the precursor and are generated by cleavages probably mediated by the endoplasmic reticulum signal peptidase (21, 44), and the remaining part of the precursor contains the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which presumably form the virus replication machinery and are released from the nascent precursor by two virus-encoded proteases. A zinc-dependent protease associated with NS2 and the N terminus of NS3 is responsible for the cleavage between NS2 and NS3 (16, 19, 39). A distinct serine protease located in the N-terminal domain of NS3 is responsible for proteolytic cleavages at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B junctions (3, 17, 52).

Substantial efforts have been devoted to the characterization of the HCV serine protease, which is contained within the amino-terminal 180 amino acids of NS3 (3, 13, 17, 20, 52). Although the NS3 protease domain possesses enzymatic activity, the 54-amino-acid NS4A protein is required for cleavage at the NS3/NS4A and NS4B/NS5A sites and increases cleavage efficiency at the NS4A/NS4B and NS5A/NS5B junctions (2, 14, 33, 51). The central domain of NS4A, encompassing amino acids 21 to 32, was shown to be sufficient for activation of the protease (30, 34, 46, 53). In transfected cells, NS3 and NS4A assemble into a stable heterodimeric complex whose formation requires both the amino-terminal and the central domains of NS4A, as well as about 30 amino acids at the amino terminus of NS3 (4, 14, 30, 34, 45, 51). The determination of the crystal structure of the NS3 protease domain uncomplexed and complexed with central domain of NS4A (29, 36, 56) has confirmed the characteristics of this enzyme predicted by molecular modelling and biochemical studies (12, 40). The enzyme adopts a chymotrypsin-like fold and features a tetrahedrally coordinated metal distal to the active site. The central domain of NS4A forms a β strand which contributes to the formation of an eight-stranded β barrel with the amino-terminal domain of NS3 and plays a significant role in stabilizing NS3. Thus, NS4A is considered an integral structural component of the enzyme. For this reason, we here refer to the HCV serine protease as the NS3-NS4A protease. This protease cleaves the viral polyprotein in a precise temporal order which is probably critical for virus replication: the NS3/NS4A cleavage is the first event and occurs only in cis, and this is followed by cleavage at the NS5A/NS5B, NS4A/NS4B, and NS4B/NS5A sites, which can also occur in trans (2, 14, 33). An additional peculiar feature of the protease domain of NS3 is that it is covalently attached to an RNA helicase possessing ATPase activity (18, 25, 28).

This overwhelming amount of data makes the NS3 protease an attractive candidate for developing effective HCV therapies. Indeed, several in vitro assay systems have been developed and are being used for the identification of specific inhibitors.

Protease inhibitors have proved to be good therapeutic agents in the case of the human immunodeficiency virus protease. However, the long-term clinical efficacy of these drugs is potentially limited by the existence of inhibitor-resistant protease variants which are found in untreated subjects and emerge both in vivo during treatment and during selection in culture (10, 22, 32). Apparently, the ability of the virus to produce inhibitor-resistant protease variants depends largely on the ability of the protease to tolerate substitutions in critical subsites. Thus, attempts to subvert viral resistance should take this feature in account and concentrate on the search for inhibitors active against a broad range of variants. These attempts greatly benefit from the possibility of using in vitro cell culture systems for the selection and characterization of virus variants with decreased sensitivity to inhibitors.

Sequence analysis of several HCV isolates indicates that there are multiple HCV genotypes and subtypes and that even in the same individual the virus exists as quasispecies (6, 47). Accordingly, a number of sequence differences are found in the portion of the genome encoding the NS3-NS4A protease. Only a few variants have been characterized biochemically, and these show similar kinetic parameters. On the other hand, the lack of an efficient in vitro infection system prevents a large-scale comparison of the different protease variants present in various HCV isolates and also precludes testing the sensitivities to inhibitors of the different variants in cell culture.

We have recently described the generation of stable Sindbis virus (SBV)-HCV chimeric viruses whose propagation depends on the activity of the serine protease of HCV (15). Here we report the use of these viruses as a genetic system for the identification of functional variants of the NS3-NS4A protease which could be used to identify and characterize protease variants with decreased sensitivity to inhibitors. Since there are no selective inhibitors of the NS3-NS4A protease presently available, we investigated whether variants of the NS3-NS4A protease could be selected in the absence of a specific selective pressure, taking advantage of the high rate of spontaneous mutations of the SBV replication machinery. We selected more-infectious virus mutants by serial passaging on BHK cells, and almost all of them produced an NS3-NS4A protease different from those encoded by the original chimeras. All of these mutant enzymes displayed a measurable activity when assayed in vitro and efficiently processed the chimeric polyprotein in infected cells. These results imply that HCV-SBV chimeric viruses can be used under the appropriate conditions of selective pressure as a surrogate system for the identification and characterization of inhibitor-resistant variants of the NS3-NS4A protease.

Paola Delmastro

Papers

Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric Sindbis viruses

Rational design and functional expression of a constitutively active single-chain NS4A–NS3 proteinase