Hepatitis C Virus: Overview, Morphogenesis, Infection, Therapies

Abstract

Chronic hepatitis C (CHC) is a big cause of liver related fibrosis and cirrhosis. The level of fibrosis is usually in the past (established by histology). The prognosis is estimated using fibrosis progression rates (FPRs; annual probability of progressing across histological stages). However, new noninvasive options are quickly replacing biopsy. As the rates of HCV cirrhosis goes on increasing and hence remains high the morbidity and mortality of HCV- related HCC. Reduced complication form cirrhosis, including HCC is one of long term goal of antiviral. The new directly acting antiviral development with high rates of virological clearance has restructured the cure of HCV infection. Fewer patients remain at risk for heptocelluar carcinoma, especially those with the severe fibrosis and cirrhosis in spite of development of HCC in HCV patients who achieve disease sustained virologic response is reduced. This review put lights upon the overview, morphogenesis, infection, therapies.

Introduction

Hepatitis C virus (HCV) infection is a major cause of liver-related deaths, cirrhosis, and hepatocellular carcinoma in the United states. In the year 1989 HCV was discovered, but till 1992 the blood supply was not screened for HCV; hence, before 1992, primary cause of infection was the contaminated blood products. Nowadays, Primary cause of HCV infection is percutaneous blood exposure, usually through injection drug use. As safe, tolerable and curative therapies are available since 2011, Clinical and public health frameworks related to the prevention, control and clinical management of HCV have extensively modified. According to World Health Organization (WHO), annually there are about 3-4 million new cases of infection. Till date there is no vaccine reported for HCV as the genotype of virus observed in different areas is different. HCV virus exists at least in 6 distinct genetic forms (genotypes) with multiple subtypes. Almost 50 subtypes have been identified. A global vaccine be developed so as to protect against all these variants of the virus. Even if several anti-viral drugs, including HuIFN-alpha-Le and nucleoside analogue reverse transcriptase inhibitors, have been used for the hepatitis B treatment. Still huge issues remain as including moderate efficacy, dose-related adverse effects, and drug effective due to resistance. Along these lines remarkable medical requirement for safe and effective anti- HCV drugs exists and finding new anti- HCV agents remains a challenge. HCV is a tiny engulfed virus has a single-stranded RNA genome, positive-sense that encodes a large polyprotein of 3010 amino acids. The polyprotein is co- and post translationally processed by cellular and virally encoded proteases to form the mature structural and non-structural (NS) proteins. Among the NS proteins, the NS3 serine-like protease enzyme and the RNA-dependent RNA polymerase (RdRp) are important for viral maturation and replication, and therefore represent ideal targets for the development of small molecule anti-HCV compounds.

Genome (HCV)

The HCV genome is approximately 9600 nucleotides long. It consist of two highly conserved untranslated regions (UTR) 5’-UTR and 3’-UTR that flank a single open reading frame (ORF). The ORF might contain from 9030 to 9099 nucleotides and it is coding for a single polyprotein precursor of 3010 to 3033 amino acids (aa), respectively depending on the genotype. Their occurs translation in the endoplasmic reticulum and it is initiated by IRES at the 5’ UTR. 3’-untranslated region. It is said HCV replication involves 400 nucleotides long 3’-UTR region. The region is highly conserved. It is divided into three functionality parts: A variable sequence of 40 nucleotides, a variable internal poly (U/UC) rich tract of 30 to 80 nucleotides (depending on the HCV strains), which is followed by a highly conserved 98-nucleotide X-tail containing three stable stem loop (SL) structures called: 3’SL1, 3’SL2, 3’SL3. 5’-untranslated region. It is the special site to control the HCV genome replication and the viral polyprotein translation and is highly conserved. It contains four distinct domains (I-IV) and the region is 341 nucleotides long. The first 125 nucleotides of 5’UTR spanning the domains I and II have been shown to be essential for the viral RNA replication. The domains III-IV composes an internal ribosomal entry site (IRES) involved in ribosome binding which can initiate viral polyprotein translation in a cap-independent manner. The ordered assembly of ribosomal pre-initiation complexes, beginning with the association of the small (40S) ribosomal subunit with a messenger RNA (mRNA) is required for the initiation of HCV protein synthesis. The cap-independent translation starts with the 40S ribosome binding and the scanning to the initiation codon which is followed by association with the 60S ribosomal subunit to form an active 80S ribosome. The HCV IRES is folded into highly structured domains which are called II to IV. The mutational analysis of the IRES domains suggested that a structural integrity was necessary for an efficient protein synthesis both in vitro and in vivo.

Core protein (p22)

Translation of the core protein of HCV as an immature protein of 22 kDa, that it is composed of 191 amino acids (aa) take place. It is removed from the polyprotein in the endoplasmic reticulum (ER) by a cellular signal peptidase (SP). An additional cleavage of the immature protein forms mature 21 kDa core protein. There are three domains in the core protein. The first domain spans the N-terminal region of 117 aa (aa 1-117). Which consists of mostly basic residues and there are two short hydrophobic regions. It results in binding to the viral RNA. The second domain between the aa 118 and 174 is more hydrophobic and less basic which is engaged in the creation of links with the lipid droplet (LD). LDs are intracellular structures that are used for the lipid storage. The third domain is localized between the aa 175 and aa 191 and it contains the signal sequence for the ER membrane translocation of E1 ectodomain. In vivo, the mature core proteins are believed to form homo-multimers that are accumulated mainly at the ER membrane and can be self-assembly into the HCV-like particles. The protein also interacts with the HCV RNA during the viral capsid assembly. Besides that, the core protein poses regulatory functions during the RNA translation. It is stated after the analysis of HCV core protein expression indicate that additionally it may be involved in several processes in cells such as apoptosis, lipid metabolism fatty liver and the development of HCC. It has been shown, that if the core protein is mutated, the association with LDs might be disrupted. A decrease of this association has a negative effect on the HCV cc production. The HCV core protein was also shown to be involved in the Ca2+ regulation. Thus, it is not only a basic structural element of HCV, but it is an active player in a significant amount of additional processes, including signaling pathways regulation.

Viral proteins

The translation in the endoplasmic reticulum is initiated by IRES at the 5’UTR. 17 A single polyprotein precursor is processed by cellular and viral proteases into ten proteins. Core, E1 and E2 are the three structural proteins located at the amino-terminal part of the polyprotein and are essential components of the virions while in the remaining part of polyprotein locates seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). These proteins are involved in particle morphogenesis, RNA replication and in regulate on of cell functions. E1 (gp35) and E2 (gp70) Envelope proteins Essential component of HCV virion envelope are necessary for viral entry and fusion are E1 & E2 envelope glycoproteins. Molecular weight of E1 and E2 are 33–35 and 70–72 kDa, respectively, and noncovalent heterodimers assembly. The E1 and E2 transmembrane domains consist of two stretches of hydrophobic aa which is separated by a short polar region containing fully conserved charged residues. Membrane anchoring, ER localization and heterodimer assembly are numerous functions they possess. Even if intramolecular disulfide bonds have not been observed still E1 and E2 ectodomains contain numerous cysteine proline and residues. E1 and E2 are highly glycosylated. It contains up to 5 and 11 glycosylation sites, respectively. Also, E2 contains hypervariable regions with aa sequences differing up to 80% between HCV genotypes and between subtypes of the same genotype. Hypervariable region 1 (HVR1) contains 27 aa and is a major (but not the only) HCV neutralizing epitope. Despite the HVR1 sequence variability, the physicochemical properties of the residues at each position and the overall conformation of HVR1 are highly conserved among all known HCV genotypes, suggesting an important role in the virus lifecycle. Crucial role is played by E2 in early steps of infection. It is observed that their take place the viral attachment to be initiated via E2 interaction with one or several components of the receptor complex. As HVR1 is a basic region with positively charged residues located at specific sequence positions, it can theoretically interact with negatively charged molecules at the cell surface. This interaction could play a role in host cell recognition and attachment, as well as in cell or tissue compartmentalization. It was shown recently that human serum facilitated infection of Huh7 cells by HCV pseudoparticles, apparently mediated through an interplay between serum high-density lipoproteins (HDL), HVR1 and the scavenger receptor B type I (SR-BI).

Nonstructural Proteins

P7 is a 63 aa polypeptide, small and is shown to be integral membrane protein and is composed of two transmembrane domains that are organized in α-helices and connected by a cytoplasmic loop. p7 appears to be essential, because mutations or deletions in its cytoplasmic loop suppressed infectivity of intra-liver transfection of HCV cDNA in chimpanzees. p7 belongs to the viroporin family and could act as a calcium ion channel was suggested by invitro studies. However, results are yet to confirm in vivo. NS2 is a non-glycosylated transmembrane protein of 21–23 kDa and consist of two internal signal sequences at aa positions 839–883 and 928–960. ER membrane association is prime role of NS2. 30 Together with the amino-terminal domain of the NS3 protein, the NS2-3 protease, NS2 constitutes a zinc-dependent metalloprotease that cleaves the site between NS2 and NS3. It is a short-lived protein that loses its protease activity after self-cleavage from NS3 and is degraded by the proteasome in a phosphorylation-dependent manner by means of protein kinase casein kinase. In addition to its protease activity, NS2 could interact with host cell proteins, such as the liver specific pro-apoptotic cell death-inducing DFF45-like effector (CIDE-B), and affect reporter genes controlled by liver and non-liver-specific promoters and enhancers. However, the consequences of such interactions within the context of the HCV lifecyle are not clear. NS3 is a multi-functional viral protein and is a cofactor of NS3 protease activity. It contains serine protease domain in its N-terminal third and a helicase/NTPase domain in its C-terminal two-thirds. NS3-4A also bears additional properties through its interaction with host cell pathways and proteins that may be important in the lifecycle and pathogenesis of infection. Not surprisingly, the NS3-NS4A protease is one of the most popular viral targets for anti-HCV therapeutics. The NS3-NS4A protease is essential for the HCV lifecycle. NS3-NS4A catalyzes HCV polyprotein cleavage at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions. The 3D structure of the NS3 serine protease domain complexed with NS4A has been determined. The catalytic triad is formed by residues His 57, Asp 81 and Ser 139. The central region of NS4A (aa 21–30) acts as a cofactor of NS3 serine protease activity, allowing its stabilization, localization at the ER membrane as well as cleavage-dependent activation, particularly at the NS4B/NS5A. Recently, HCV NS3-NS4A was shown in vitro to antagonize the dsRNA-dependent interferon regulatory factor 3 (IRF-3) pathway, an important mediator of interferon induction in response to a viral infection. NS3-NS4A also appears to prevent dsRNA signaling via the toll-like receptor 3 upstream of IRF-3. One potential mechanism includes a blockade of the intracellular double-stranded RNA sensor protein (RIG-I) pathway by NS3-NS4A. Thus, HCV could utilize NS3-4A protease to circumvent the innate immune response at the early stages of infection. In addition, NS3 was also reported to induce malignant transformation of NIH3T3 cells, suppress actinomycin D-induced apoptosis in murine cell lines, and to be involved in hepato carcinogenesis events, although the exact mechanisms are not clear. The NS3 helicase-NTPase domain consisting of the 442 C-terminal aa of the NS3 protein is a member of the helicase superfamily-2. Its three-dimensional structure has also been determined. 45,46, 47 The NS3 helicase-NTPase has several functions, including RNA-stimulated NTPase activity, RNA binding, and unwinding of RNA regions of extensive secondary structure by coupling unwinding and NTP hydrolysis. During RNA replication, the NS3 helicase has been suggested to translocate along the nucleic acid substrate by changing protein conformation, utilizing the energy of NTP hydrolysis. A recent study proposed that the helicase directional movement step is fueled by single-stranded RNA binding energy, while NTP binding allows for a brief period of random movement that prepares the helicase for the next cycle. In addition, NS3 helicase activity appears to be modulated by the NS3 protease domain and the NS5B RdRp51 An integral membrane protein of 261 aa with an ER or ER-derived localization membrane is NS4B. NS4B is predicted to harbor at least four transmembrane domains and an N-terminal amphipathic helix that are responsible for membrane association. NS5A is a 56–58 kDa phosphorylated zinc-metalloprotein that probably plays an important role in virus replication and regulation of cellular pathways. The N-terminal region of NS5A (aa 1–30) contains an amphipathic α-helix that is necessary and sufficient for membrane localization in perinuclear membranes as well as for assembly of the replication complex. Downstream of this motif, the NS5A protein was predicted to contain three domains, numbered I to III. Domain I, located at the N-terminus, contains an unconventional zinc-binding motif formed by four cysteine residues conserved among the hepacivirus and pestivirus genera. HCV replicon RNA replication was inhibited by mutations in the NS5A sequence and abolished by alterations of the zinc-binding site. The mechanisms by which NS5A regulate HCV replication are not entirely clear. NS5A associates with lipid rafts derived from intracellular membranes through its binding to the C-terminal region of a vesicle-associated membrane-associated protein of 33 kDa (hVAP-33). This interaction appears to be crucial for the formation of the HCV replication complex in connection with lipid rafts.

Another report suggested that the level of NS5A phosphorylation plays an important role in the viral lifecycle by regulating a switch from replication to assembly, whereby hyper phosphorylated forms function to maintain the replication complex in an assembly-incompetent state. Furthermore, NS5A can interact directly with NS5B, but the mechanism by which NS5A modulates the RdRp activity has not been elucidated. In addition, NS5A was reported to interact with a geranyl geranylated cellular protein. This is potentially significant considering that assembly of the viral replication complex has been shown to require geranyl geranylation of one or more host cell proteins. Multiple have been assigned to NS5A based on its interactions with cellular proteins. For instance, NS5A appears to play a role in interferon resistance by binding to and inhibiting PKR, an antiviral effector of interferon-α. NS5A also bears transcriptional activation functions and appears to be involved in the regulation of cell growth and cellular signaling pathways. However, these observations remain to be confirmed in vivo. In addition to ten proteins described above, it has been reported that there are frame shift (F) or alternate reading frame protein (ARFP), or “core+1” protein. The ARFP is the result of a -2/+1 ribosomal frame shift between codons 8 and 14 of the adenosine-rich region encoding the core protein. ARFP ends have different stop codons depending on the genotype. Thus, its length may vary from 126 to 161 amino acids. In the case of genotype 1a, the protein contains 161 amino acids. However, the situation with ARFP is more complicated, since alternative forms of this protein such as ARFP/DF (double-frame shift) in genotype 1b, and ARFP/S (short form) were recently described Short living protein is located in the cytoplasm70 in associated with the endoplasmic reticulum. Detection of anti-ARFP antibodies in sera of HCV-positive subjects indicates that the protein is expressed during infection but is likely not involved in the virus replication. Some findings suggested engagement of ARFP in the modulation of dendritic cells function and stimulation of the T cell responses the implication of ARFP in the viral life cycle remain to be elucidated.

Mode of transmission

The essential mode of HCV transmission is blood borne transmission. Hazardous infusion hones in healthcare settings and recreational infusion medicate utilize are especially vital for HCV transmission around the world. In joined together states in 1992 earlier to the screening of blood items for HCV starting, healthcare-associated transmission of HCV happened more habitually; in any case, 33 healthcare episodes including more than 239 outbreak-associated cases were detailed to the Centers for Illness Control and Avoidance [CDC] from 2008–2015. Vertical transmission can happen in ~6% of newborn children born to HCV-infected moms and transmission may be twice as likely to happen in newborn children born to HCV/HIV co-infected moms or HCV mono-infected moms with tall viral loads. Sexual transmission is for the most part wasteful; in any case, an expanding number of cases of sexually transmitted disease have been detailed among HIV-infected men who have sex with men [MSM]. At last, HCV transmission has too been detailed within the setting of non-injection sedate use as well as within the setting of unregulated tattoos.

Testing and diagnosis

Research facility determination of persistent HCV contamination within the Joined together States as of now requires the utilize of two sorts of tests: immunoglobulin (Ig) G antibody chemical immunoassays (anti-HCV) and nucleic acid tests (NAT). HCV testing ought to be started with an anti-HCV counter acting agent test. People without hazard variables for HCV and a non-reactive anti-HCV counter acting agent require no advance assessment for HCV contamination. Additional testing may be suitable for certain populaces with seriously compromised resistant frameworks or current dangers for HCV introduction such as infusion sedate utilize or hemo dialysis. A responsive anti-HCV counter acting agent requires affirmation with a HCV NAT to decide the nearness of HCV RNA and current HCV contamination. People with a reactive anti-HCV antibody and a positive HCV NAT are infected with HCV and ought to be connected to suitable HCV therapeutic care and treatment.

Diagnostic

For HCV diagnosis both serologic and nucleic acid-based tests were developed. Serologic tests are adequate when constant hepatitis C is anticipated, with a sensitivity of more than 99% in case utilizing the 3rdgeneration assays. Positive serologic comes about require extra HCV RNA or (with somewhat decreased sensitivity) HCV center antigen estimations in arrange to distinguish between chronic hepatitis C and settled HCV disease from the past. When an acute hepatitis C is considered, a serologic screening alone is inadequate, since mature anti-HCV antibodies are developed late after transmission of the infection. Because of low sensitivity, poor specificity and low efficacy compared to serologic and nucleic acid-based approaches morphological methods like immune histo-chemistry, in situ hybridization or PCR from liver specimens does not play any relevant role.

HCV core antigen assay

There are 5 different antibodies targeted the HCV core in the assay. The test is equally effective for different HCV genotypes and is highly specific (99. 8%), and shows a relatively high sensitivity for the determination of chronic hepatitis C (corresponding to 600-1000 IU/ml HCV RNA). Even though, HCV core antigen correlated well, still not fully linearly, with HCV RNA serum levels, and false-negative results may be obtained in patients with an impaired immunity. Another study has shown that the HCV core antigen quantification could be an alternative to the HCV RNA quantification for on-treatment antiviral response monitoring. Here, a HCV center antigen below the limit of quantification at treatment 1 wk was strongly predictive of RVR, while patients with a less than 1 log10 decay in HCV core antigen at treatment 12 wk had a high probability of achieving non response. The new HCV core antigen assay may be a cheaper, in spite of the fact that to some degree less sensitive, elective for nucleic acid testing. Nucleic acid testing for HCV. The early diagnostic of acute hepatitis C should be done and considered as mandatory as it is detectable in few days of infection. The nucleic acid based tests are available and are efficient. The HCV RNA measurement is besides vital in determination of the HCV genotype, selection of treatment procedure, therapy duration and assessment of the treatment success HCV RNA estimation. For a number of antiviral combination treatments, the HCV RNA follow-up thinks about are fundamental to characterize the result of the treatment and encourage helpful techniques, if vital. Traditionally, in order to assess whether a sustained virologic response (SVR) has been achieved the tests should be repeated 24 wk after treatment completion. However, the new time point for assessment of final virological treatment outcome is 12 wk after the end-of-treatment as the probability of a virologic relapse is similar after 12 and 24 wk. Both qualitative and quantitative PCR-based detection assays are available. The initial diagnostic of hepatitis C is done using qualitative PCR tests being sensitive, for screening of blood and organ donations and for confirming SVR after treatment completion. Quantitative reverse transcriptase (RT) real-time PCR-based assays can detect and quantify the HCV RNA over a very wide range, from approximately 10 IU/ml to 10 million IU/ml. In the treatment monitoring when the virus load is gradually reducing the measurements are essential.

HCV genotyping

For every patient who considers antiviral therapy HCV genotyping is mandatory. The determination of HCV genotypes and even subtypes is important because of significantly distinct barriers to resistance on the HCV subtype level for DAA-based therapies. However, the importance for the HCV genotyping may decline with the availability of highly and broadly effective all oral combination therapies in the future. Both direct sequence analysis and reverse hybridization technology allow the HCV genotyping. To analyze exclusively the 5’UTR, which was burdened with a high rate of misclassification especially on the subtype level initial assays were designed. Analyzing the coding regions, in particular the genes encoding core protein and the NS5B, both of which provide non-overlapping sequence differences between the genotypes and subtypes current assays were improved.

Therapies

The current treatment of Hepatitis C virus is combination of medicines. Based on the genotype of infection is the choice of medication and duration of treatment. Newest available agents to treat HCV’s are direct acting antivirals. These medications works by targeting specific steps in HCV’s life cycle to disrupt the reproduction of viral cells. The treatment for chronic HCV was lengthy and uncomfortable before the availability of DAA’s. 8-12 weeks is the average duration of treatment. The interferon and ribavirin based regimens where the mainstay of HCV therapy historically. Of which where ued sparingly because of poorly tolerated side effects and modest efficacy with sustained virologic response (SVR) rates of approx 50%. The two NS3/4A protease inhibitors were used used in combination with interferon based regimens for chronic HCV treatment Additional drugs comprising interferon-free DAA regimens with cure rates >90% have become available. Other viral components such as the NS5A protein inhibitors and NS5B polymerase inhibitors are targeted by these drugs. Additionally, in June 2016, a new treatment regimen effective against all HCV genotypes became available, which has the potential to further simplify HCV treatment protocols.

Conclusion

HCV is infectious. The most common reason is unsafe injections, transmitted by percutaneous blood exposure. It seems to be endemic in most part of the world. Long term complications of HCV infection include cirrhosis and hepatocellular carcinoma. Fundamental studies on virus-cell interactions and studies directing towards development of the prophylactic vaccine should be intensified. There are several advances in HCV treatment have created new opportunities for reducing HCV-associated morbidity and mortality. These treatments are safe, well-tolerated, and highly effective; however, the benefits cannot be realized without a significant increase in the number of persons tested for HCV so that all chronically infected individuals can be aware of their diagnosis and linked to appropriate clinical care.