NS2-3 protease

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NS2-3 protease (of hepatitis C virus, HCV) is an enzyme responsible for proteolytic cleavage between NS2 and NS3, which are non-structural proteins that form part of the HCV virus particle. NS3 protease of hepatitis C virus, on the other hand, is responsible for the cleavage of non-structural protein downstream. Both of these proteases are directly involved in HCV genome replication, that is, during the viral life-cycle that leads to virus multiplication in the host that has been infected by the virus.

Contents

Background about Hepatitis C

Hepatitis C affects 170 million people around the world which includes 1.4 million people living in the U.S. Most of the people infected with this virus live in third-world countries which often tend to have poor sterilization of medical equipment, a common source of HCV infection. Education also plays a big part as a vast majority of people don't have access to the information about the virus, how it spreads and infects. Hepatitis C can enter human body through many ways which include sexual intercourse, blood transfusion and via HCV infected needles. HCV infection can lead to cirrhosis and liver cancer if interferon treatment fails. [1]

Viral genome

Hepatitis C virus is a single-stranded RNA virus in the family Flaviviridae . [2] The genome consists made up of about 10,000 nucleotides and encodes a single polyprotein. [3] Hepatitis C Virus (HCV) used host cell machinery to process its genome to synthesize 3 crucial viral proteases of which each has peptide cleaving role. These 3 proteases are also known as structural proteins. The HCV genome encodes 10 viral proteins: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. [4]

Discussion

NS2-3 protease is the enzyme responsible for proteolytic cleavage between the non-structural proteins NS2 and NS3. NS3 protease, on the other hand, is responsible for the cleavage of non-structural proteins downstream. Both of these proteases are directly involved in HCV genome replication. NS2-3 protease mechanism is essential for viral production, as shown by in-vitro chimpanzee studies where chimps which were inoculated with HCV with fully mutated NS2-3 protease activity didn't develop HCV infection. [5]

To this date, a promising cell-culture system has yet to be developed in a way that could support large scale need of future vaccine trials. The use of HCV non-structural proteins to initiate immune response in animal studies have shown promising results but the lack of robust tissue culture system and the ability of virus to mutate rapidly are still major hurdles. Interferon treatment has succeeded in only very small number of patients.[ citation needed ]

In the study discussed in this paper, Dentzer has succeeded in finding the actual viral protease domain and predicting possible ways to inhibit the protease mechanism. [6]

Inhibitors for protease such as Papain, Subtilisin and one from Sindbis virus capsid might share some similarities as they all are cysteine proteases. [7] If NS2-3 protease does share important similarities with other cysteine proteases, it might be possible to propose a model for inhibition of the NS2-3 cysteine protease activity, which might be able to offer a permissible approach to a robust vaccine for in-vitro animal model studies.[ citation needed ]

The team of researchers used native selenomethionine-containing protein that yielded crucial crystal forms. NS2 Pro (non-structural protease) monomer is made up of two sub-domains which is connected by a linker. Each monomer contains the anti-parallel alpha-helices and a loop of beta strands. NS2 dimer consists of two monomers each facing their N and C-termini toward each other. N-termini stay in close proximity while C-termini are farther apart from each other which resembles ‘butterfly’. NS2-3 protease is 42 kDa in length. Earlier studies also suggested that it was almost impossible to isolate NS2-3 protease due to hydrophobic nature of the native NS2.[ citation needed ]

Researchers have used a method called ‘Crystallization’ through which they have been able to isolate and further investigate role of NS2-3 protease. His143, Cys184 and Glu 163 are the three crucial resides responsible for proteolytic activity. These three residues together form an active site. Although NS2 protease has been proposed to have a unique fold, it is shown that superimposing three critical residues from other cysteine proteases revealed a major characteristic which would allow for more specific inhibitor studies. Researchers in this case used cysteine proteases such as papain and poliovirus 3C protease. NS2 dimer contains two active sites and requires dimerization for proteolytic activity.[ citation needed ]

There are also some critical residues including Pro 164 which help bend the peptide backbone of the Glu163 to offer the correct geometry so the catalytic process can occur. Cis-proline on the other hand offers dimer stability. The NS2-3 protease requires both NS2 and NS3 domain for proteolytic cleavage. Addition of Zinc is also required as NS2-3 protease is zinc dependent which releases N terminus of NS2. The tetrahedral geometry in active site hints at the zinc ion binding site. [8] Although NS3 domain's active role is not known yet, scientist propose that NS3 may interact with the active site of NS2 which would provided with catalytic environment necessary for polyprotein processing. The presence of zinc-binding site doesn't necessarily mean that a devoid of zinc in the process would inhibit the viral catalytic process.

An experimental model was devised to test if HCV full-length polyprotein sequence with a mutation (either H143A or C184A) would give NS2-3 cleaved polyprotein. Expression of two mutants in the same experimental model should yield NS2 and NS3 as it would provide with one active site. As the data shown in the paper, NS2 and NS3 were cleaved which meant that co-expression of two mutants did yield at least one functional active site. This experiment also provided a model for membrane association that would help with future studies regarding inhibitors for NS2-3 protease which is crucial for viral replication.[ citation needed ]

The article has proposed the structure for NS2-3 cysteine protease active site and provided with in-vitro co-expression mutation study results which would change the way researchers currently look at polyprotein processing.

Related Research Articles

<span class="mw-page-title-main">Protease</span> Enzyme that cleaves other proteins into smaller peptides

A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in many biological functions, including digestion of ingested proteins, protein catabolism, and cell signaling.

<span class="mw-page-title-main">Hepatitis C virus</span> Species of virus

The hepatitis C virus (HCV) is a small, enveloped, positive-sense single-stranded RNA virus of the family Flaviviridae. The hepatitis C virus is the cause of hepatitis C and some cancers such as liver cancer and lymphomas in humans.

<span class="mw-page-title-main">Cysteine protease</span> Class of enzymes

Cysteine proteases, also known as thiol proteases, are hydrolase enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad.

<i>Pestivirus</i> Genus of viruses

Pestivirus is a genus of viruses, in the family Flaviviridae. Viruses in the genus Pestivirus infect mammals, including members of the family Bovidae and the family Suidae. There are 11 species in this genus. Diseases associated with this genus include: hemorrhagic syndromes, abortion, and fatal mucosal disease.

<span class="mw-page-title-main">RNA-dependent RNA polymerase</span> Enzyme that synthesizes RNA from an RNA template

RNA-dependent RNA polymerase (RdRp) or RNA replicase is an enzyme that catalyzes the replication of RNA from an RNA template. Specifically, it catalyzes synthesis of the RNA strand complementary to a given RNA template. This is in contrast to typical DNA-dependent RNA polymerases, which all organisms use to catalyze the transcription of RNA from a DNA template.

<span class="mw-page-title-main">TEV protease</span>

TEV protease is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV). It is a member of the PA clan of chymotrypsin-like proteases. Due to its high sequence specificity it is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo.

<span class="mw-page-title-main">HIV-1 protease</span> Enzyme involved with peptide bond hydrolysis in retroviruses

HIV-1 protease (PR) is a retroviral aspartyl protease (retropepsin), an enzyme involved with peptide bond hydrolysis in retroviruses, that is essential for the life-cycle of HIV, the retrovirus that causes AIDS. HIV protease cleaves newly synthesized polyproteins at nine cleavage sites to create the mature protein components of an HIV virion, the infectious form of a virus outside of the host cell. Without effective HIV protease, HIV virions remain uninfectious.

<span class="mw-page-title-main">NS3 (HCV)</span>

Nonstructural protein 3 (NS3), also known as p-70, is a viral nonstructural protein that is 70 kDa cleavage product of the hepatitis C virus polyprotein. It acts as a serine protease. C-terminal two-thirds of the protein also acts as helicase and nucleoside triphosphatase. First (N-terminal) 180 aminoacids of NS3 has additional role as cofactor domains for NS2 protein.

<span class="mw-page-title-main">NS5A (hepacivirus)</span>

Nonstructural protein 5A (NS5A) is a zinc-binding and proline-rich hydrophilic phosphoprotein that plays a key role in Hepatitis C virus RNA replication. It appears to be a dimeric form without trans-membrane helices.

<span class="mw-page-title-main">NS2 (HCV)</span>

Nonstructural protein 2 (NS2) is a viral protein found in the hepatitis C virus. It is also produced by influenza viruses, and is alternatively known as the nuclear export protein (NEP).

Many major physiological processes depend on regulation of proteolytic enzyme activity and there can be dramatic consequences when equilibrium between an enzyme and its substrates is disturbed. In this prospective, the discovery of small-molecule ligands, like protease inhibitors, that can modulate catalytic activities has an enormous therapeutic effect. Hence, inhibition of the HIV protease is one of the most important approaches for the therapeutic intervention in HIV infection and their development is regarded as major success of structure-based drug design. They are highly effective against HIV and have, since the 1990s, been a key component of anti-retroviral therapies for HIV/AIDS.

<span class="mw-page-title-main">Simeprevir</span> Chemical compound

Simeprevir, sold under the brand name Olysio among others, is a medication used in combination with other medications for the treatment of hepatitis C. It is specifically used for hepatitis C genotype 1 and 4. Medications it is used with include sofosbuvir or ribavirin and peginterferon-alfa. Cure rates are in 80s to 90s percent. It may be used in those who also have HIV/AIDS. It is taken by mouth once daily for typically 12 weeks.

<span class="mw-page-title-main">Picornain 3C</span>

Picornain 3C is a protease found in picornaviruses, which cleaves peptide bonds of non-terminal sequences. Picornain 3C’s endopeptidase activity is primarily responsible for the catalytic process of selectively cleaving Gln-Gly bonds in the polyprotein of poliovirus and with substitution of Glu for Gln, and Ser or Thr for Gly in other picornaviruses. Picornain 3C are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Picornain 3C is encoded by enteroviruses, rhinoviruses, aphtoviruses and cardioviruses. These genera of picoviruses cause a wide range of infections in humans and mammals.

<span class="mw-page-title-main">PA clan of proteases</span>

The PA clan is the largest group of proteases with common ancestry as identified by structural homology. Members have a chymotrypsin-like fold and similar proteolysis mechanisms but can have identity of <10%. The clan contains both cysteine and serine proteases. PA clan proteases can be found in plants, animals, fungi, eubacteria, archaea and viruses.

<span class="mw-page-title-main">Discovery and development of NS5A inhibitors</span>

Nonstructural protein 5A (NS5A) inhibitors are direct acting antiviral agents (DAAs) that target viral proteins, and their development was a culmination of increased understanding of the viral life cycle combined with advances in drug discovery technology. However, their mechanism of action is complex and not fully understood. NS5A inhibitors were the focus of much attention when they emerged as a part of the first curative treatment for hepatitis C virus (HCV) infections in 2014. Favorable characteristics have been introduced through varied structural changes, and structural similarities between NS5A inhibitors that are clinically approved are readily apparent. Despite the recent introduction of numerous new antiviral drugs, resistance is still a concern and these inhibitors are therefore always used in combination with other drugs.

HPgV-2 is the second human pegivirus discovered. It was first identified in 2005 in blood of transfusion recipients and initially named hepegivirus 1 because it shared some genetic features with both pegiviruses and hepaciviruses. HPgV-2 was later independently discovered by another group in the blood of a HCV-infected patient who had undergone multiple blood transfusions and died from sepsis of unclear etiology. It was then named human pegivirus 2. HPgV-2 is now classified in the pegivirus genus as part of Pegivirus H species.

ORF1ab refers collectively to two open reading frames (ORFs), ORF1a and ORF1b, that are conserved in the genomes of nidoviruses, a group of viruses that includes coronaviruses. The genes express large polyproteins that undergo proteolysis to form several nonstructural proteins with various functions in the viral life cycle, including proteases and the components of the replicase-transcriptase complex (RTC). Together the two ORFs are sometimes referred to as the replicase gene. They are related by a programmed ribosomal frameshift that allows the ribosome to continue translating past the stop codon at the end of ORF1a, in a -1 reading frame. The resulting polyproteins are known as pp1a and pp1ab.

Planarian secretory cell nidovirus (PSCNV) is a virus of the species Planidovirus 1, a nidovirus notable for its extremely large genome. At 41.1 kilobases, it is the largest known genome of an RNA virus. It was discovered by inspecting the transcriptomes of the planarian flatworm Schmidtea mediterranea and is the first known RNA virus infecting planarians. It was first described in 2018.

<span class="mw-page-title-main">Nidoviral papain-like protease</span>

The nidoviral papain-like protease is a papain-like protease protein domain encoded in the genomes of nidoviruses. It is expressed as part of a large polyprotein from the ORF1a gene and has cysteine protease enzymatic activity responsible for proteolytic cleavage of some of the N-terminal viral nonstructural proteins within the polyprotein. A second protease also encoded by ORF1a, called the 3C-like protease or main protease, is responsible for the majority of further cleavages. Coronaviruses have one or two papain-like protease domains; in SARS-CoV and SARS-CoV-2, one PLPro domain is located in coronavirus nonstructural protein 3 (nsp3). Arteriviruses have two to three PLP domains. In addition to their protease activity, PLP domains function as deubiquitinating enzymes (DUBs) that can cleave the isopeptide bond found in ubiquitin chains. They are also "deISGylating" enzymes that remove the ubiquitin-like domain interferon-stimulated gene 15 (ISG15) from cellular proteins. These activities are likely responsible for antagonizing the activity of the host innate immune system. Because they are essential for viral replication, papain-like protease domains are considered drug targets for the development of antiviral drugs against human pathogens such as MERS-CoV, SARS-CoV, and SARS-CoV-2.

<span class="mw-page-title-main">Papain-like protease</span>

Papain-like proteases are a large protein family of cysteine protease enzymes that share structural and enzymatic properties with the group's namesake member, papain. They are found in all domains of life. In animals, the group is often known as cysteine cathepsins or, in older literature, lysosomal peptidases. In the MEROPS protease enzyme classification system, papain-like proteases form Clan CA. Papain-like proteases share a common catalytic dyad active site featuring a cysteine amino acid residue that acts as a nucleophile.

References

  1. ↑ Alter HJ, Seeff LB (2000). "Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome". Semin Liver Dis. 20 (1): 17–35. doi:10.1055/s-2000-9505. PMID   10895429.
  2. ↑ Bartenschlager R, Frese M, Pietschmann T (2004). "Novel insights into hepatitis C virus replication and persistence". Adv Virus Res. Advances in Virus Research. 63: 71–180. doi:10.1016/S0065-3527(04)63002-8. ISBN   9780120398652. PMID   15530561.
  3. ↑ Hoofnagle JH (2002). "Course and outcome of hepatitis C." Hepatology. 36 (5 Suppl 1): S21–9. doi: 10.1053/jhep.2002.36227 . PMID   12407573.
  4. ↑ Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H (1994). "Kinetic and structural analyses of hepatitis C virus polyprotein processing". J Virol. 68 (8): 5045–55. doi:10.1128/JVI.68.8.5045-5055.1994. PMC   236447 . PMID   8035505.
  5. ↑ Elmowalid GA, Qiao M, Jeong SH, Borg BB, Baumert TF, Sapp RK, Hu Z, Murthy K, Liang TJ (2007). "Immunization with hepatitis C virus-like particles results in control of hepatitis C virus infection in chimpanzees". PNAS. 104 (20): 8427–32. Bibcode:2007PNAS..104.8427E. doi: 10.1073/pnas.0702162104 . PMC   1895966 . PMID   17485666.
  6. ↑ Lorenz IC, Marcotrigiano J, Dentzer TG, Rice CM (2006). "Structure of the catalytic domain of the hepatitis C virus NS2-3 protease". Nature. 442 (7104): 831–5. Bibcode:2006Natur.442..831L. doi:10.1038/nature04975. PMID   16862121. S2CID   4319996.
  7. ↑ McPhalen CA, James MN (1988). "Structural comparison of two serine proteinase-protein inhibitor complexes: eglin-c-subtilisin Carlsberg and CI-2-subtilisin Novo". Biochemistry. 27 (17): 6582–98. doi:10.1021/bi00417a058. PMID   3064813.
  8. ↑ Love RA, Parge HE, Wickersham JA, Hostomsky Z, Habuka N, Moomaw EW, Adachi T, Hostomska Z (1996). "The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site". Cell. 87 (2): 331–42. doi: 10.1016/S0092-8674(00)81350-1 . PMID   8861916.
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