Major roles of NS5A HCV protein, mutations, and its effects on treatment outcomes



Abstract:

Hepatitus C virus is the major agent of Hepatitus infection. It causes liver damage and different types of cancer like hepatocellular carcinoma. This review map out general view of HCV and main discussion is all about some roles of NS5A in cellular mitophagy (the process of transportation of damaged mitochondria to lysosomes for degradation), and n viral translation modulation. There is a brief discussion about role of NS5A on treatment outcomes like it mediates IFN resistance, mutations in its specific amino acids  and its  response to IFN based therapy. These mutations of NS5A protein also associated with the response of  antivirals agents like , daclatasvir (BMS-790052) and ledipasvir (GS-5885).

Introduction

HCV is a convincing cause of mortality and perniciousness around the world, give rise to a range of liver diseases, varying from acute to chronic, e.g. liver cirrhosis to hepatocellular carcinoma (HCC). (Ringehan et al. 2017). Contrary to Hepatitis B virus (HBV) and human immunodeficiency virus (HIV), infection caused by HCV is remediable.(Zhang,2016).

It was assessed that around 1.75 million new cases of HCV disease has been registered in 2015 (WHO, 2018).The most common genotypes are 1 and 3. In one examination, genotype 1 was found to be profoundly common throughout the world. The next most commonly dispersed genotype was genotype 3. The remainder of HCV cases in general was due to genotypes 2, 4, and 6. Minor cases of HCV were represented by genotype 5 worldwide. Genotype 3 has generally been seen overwhelmed in South Asia. (Messina et al. 2015).

It’s   Flaviviridae family to which the HCV belongs. The genome is deliver ed into the cytoplasm. The unique polyprotein is formed by interpreting the genome, which consists of 3000 amino acid units. Proteases cleaved this polyprotein to produce various proteins. Then, in the cytoplasm, the viral genome goes through replication process and then replicated (Delaney, 2013). Initial stages of translation of HCV carried out through internal ribosome entry site (IRES). Structural and non-structural proteins are the products after translation. After the whole process, it will be packed and assembled (Scheel & Rice, 2013) HCV with its ~9.6 kb long genome has a long open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). After the proteolytic cleavage, the translated protein product is processed into 10 proteins by viral proteases and host (Moradpour et al. 2007). Structural proteins encompases Core, envelope 1 (E1) and envelope 2 (E2)  while p7, NS2 protease, NS3/4 protease, NS4B, NS5A and the NS5B polymerase falls in the domain of  non-structural proteins. In the process of viral assembly and release, core protein plays its bit part. (Pawlotsky et al. 2007). E1 and E2 are the surface glycoproteins and complementary elements of HCV envelope. During the preliminary stages of infection, E2 plays its crucial role by acting as a target for neutralizing antibodies so making a way for virus to save from eradication (Khan et al. 2014). Within the cytoplasm, the virus-membrane fusion is arbitrated by E2 protein. In a nutshell, some extensive role of these envelope proteins is in viral entry and fusion, endoplasmic reticulum (ER) localization and heterodimer assembly. (Pawlotsky et al. 2007).

Non structural polyproteins segment also reflected as multifunctional part. Various proteins involved in it play its major part in alpha helical linkage of trans-membrane domains e.g. P7 (Sakai et al.2003), association of viral body with ER e.g. NS2 (Chevaliez et al. 2006), viral genome replication and post translational changes etc.

In the picture of this review, the discussion is all about NS5A non structural proteins, its critical roles, different mutations associated with them over the period of time and its susceptibility to antivirals.  

Role of NS5A in regulating cellular mitophagy

cellular homeostasis. In general, numerous steps compromised with mitophagy are overlapped by nonspecific autophagy, as both include the disposition of an autophagosome flooding the load and its development to an autophagolysosome for degradation. However, mitophagy does not resemble nonspecific autophagy that includes massive destruction of old or useless cytoplasmic substances, but requires additional regulators for the selective degradation of   mitochondria. Some of the key regulators that are chiefly localized in cytoplasm are Parkin, E3 ubiquitin ligase (Shimura et al. 2000), a serine-threonine kinase, and the PTEN-induced putative kinase 1 (PINK1) (Lazarou et al. 2012). In polarized mitochondria, the level of PINK1 that is exported to mitochondria is kept low by the presenilin-associated rhomboid-like protease (PARL) (Greene et al. 2012). PINK1 accumulates on the outer mitochondria membrane in depolarized mitochondria, and triggers the movement of Parkin to the mitochondria. At that point, parkin ubiquitinates itself and external mitochondrial proteins, consequently preparing mitochondria for autophagic degradation. (Jin et al. 2012). HCV disease has been recommended to modify mitochondrial dynamics, elevate Parkin movement to mitochondria, and along these lines activate mitophagy (Kim et al. 2013). But the exact fundamental tool and the significant effector protein responsible for HCV-induced mitoophagy is elusive. Due to the pleiotropic nature of ​​NS5A and its ability to regulate various cellular exercises, it may eventually instigate mitophagy.

Furthermore, induction of mitophagy via NS5A efflux caused an expansion in autophagic flux, as shown by an accumulation of LC3II in presence of the time-dependent decrease of the p62 protein and bafilomycin. . Interestingly, NS5A expression simultaneously increased the production of reactive oxygen species (ROS) and treatment with anti-oxidants limited the possibility of NS5A-induced mitophagy. These phenomena are also reaffirmed in the NS5A-expressing HCV subgenomic replicon cells. Expression of HCV core which has been documented to inhibit mitophagy by blocking the induction of mitophagy in both cells fostering HCV replicating subgenomes or expressing NS5A alone. Hence there is identification of another part of NS5A as a significant controller of HCV-initiated mitophagy (Jassey et al. 2019).

Role of NS5A in viral translation modulation

Non-structural (NS) protein 5A of hepatitis C (HCV) infection is a multifunctional phosphoprotein. NS5A exists as hypo and hyper-phosphorylated structures and the dynamic advances between these two states are associated with the functions of NS5A (kandangwa and Liu 2019). These two isoforms : hypo-phosphorylated and hyper-phosphorylated having the molecular weight of 56KDa (p56) and 58KDa (p58) respectively, in SDS-PAGE (Ross-thriepland and Harris 2015). Primarily, at six serine residues within the low complexity sequence I of NS5A, hyperphosphorylation occurs. Previous studies shows NS5A downregulates viral translation.  . By studying the impacts of phospho-ablative and phospho-mimetic mutants of the six serine residues on translation, it is cleared that NS5A hyperphospho-ablative mutation at all six serine residues can no longer downregulates viral translation process. After examining the effects of phospho-mutations at each of the six serine residues on translation, it is clear that  phosphorylation of S222, S225, S235 by NS5A is not involved in downregulation of translation. Conversely, S229D or S238D mutations have no effect but NS5A with alanine mutations at S229 or S238 can no longer downregulate translation. Interestingly, S232D NS5A, but not S232A, revokes the negative regulation of translation by NS5A. Since, NS5A dimerization do a significant work, so considering the effects of phospho-mutants S229, S232, and S238 on dimerization in a protein-protein interaction assay is important. The phospho-mimetic changes of S229D or S238D increase the dimerization of NS5A, although the phospho-ablative transformations of these two accumulations have no impact. There is no effect on dimerization by phospho-ablative or phopho-mimetic mutations of S232. This shows that NS5A phosphorylation in S229, S232, and S238 is associated with regulation of viral translation   and NS5A dimerization (kandangwa and Liu 2019).

Role of NS5A Protein on treatment outcomes

For a long time, the combination of IFN-α and ribavirin has been the approved therapeutic routine for persistent HCV disease. IFNs are a group of cytokines administered by host cells with regards to some sort of stimuli including some viral infections. IFNs induce the articulation of several antiviral effector proteins that neutralize viral infection by working against it (Stetson and Medzhitov, 2006). Treatment with IFN-α causes a rapid decrease in serum HCV RNA level (Carithers and Emerson, 1997). Ribavirin is a single nucleoside and has movement against some RNA and DNA viruses, however the specific system of activity of ribavirin against HCV is still unclear (Te et al, 2007). Although the combination of IFN-α and ribavirin treatment led to significant improvements in SVR rates (Scott and Perry, 2002) but further advancement of pegylated interferon alpha (Peg-IFN-α), in which a large atom of polyethylene glycol is covalently linked to the recombinant IFN-α, resulting in a functional particle with a longer half-life, better rate of virologic response with better  pharmacokinetic profile (Keating and Plosker, 2005).

  • NS5A Protein mediates IFN Resistance:

In vitro, HCV NS5A protein binds and inactivates PKR. This coupling is subject to the PKR binding domain (PKRBD, codons 2209-2274) (Gale et al, 1998). This association ventures to allow the synthesis of viral proteins during IFN treatment. Several reviews have shown inhibitory impacts of the HCV NS5A protein on the IFN-activated Jak / STAT signaling pathway (Kumthip et al, 2012). HCV NS5A has been shown to hinder the action of the ISRE promoter activity and IFN-induced phosphorylation of STAT1 and its nuclear movement caused an obstacle to the IFN-induced ISG expression (Lan et al, 2007). It has been recommended that interaction between the C terminal area of ​​NS5A and STAT1 is responsible for the action of the NS5A that encounters IFN  (Kumthip et al, 2012).

  • Amino acid variations in NS5A and response to IFN based therapy:

 A lot of interconnected mutations in an IFN-α sensitivity determinant region (ISDR; codons 2209-2248) encompassing 40 amino acids within the C-terminus of the NS5A protein and SVR to IFN- α in patients infected with HCV genotype 1b has been described in Japan (Enomoto et al, 1996). From patients who achieved SVR, the viruses are obtained and four mutations are seen within the ISDR in each case. Therefore, the relationship of changes in the ISDR with the reaction to IFN treatment has been studied by several batches in Japan, Europe and the United States. A significant number of reviews came from Japan and had the potential to affirm the strong connection between ISDR transformations and response to treatment (Shen et al, 2007). In any case, contrary information has been recorded from different parts of the world, especially from Europe and the United States. It was not possible to confirm the connection of the ISDR transformations with the IFN-α sensitivity (Munoz et al, 2008). The error could, in any case, be clarified to some extent by viral and host elements. Host factors, particularly race of subjects, have been reported to be important elements that add to treatment reactivity for HCV (Pascu et al. 2004). Despite the ISDR, the structure and number of changes found in the interferon and ribavirin resistance determining region (IRRDR, deposits 2334-2379, genotype 1) have been shown to be related to treatment response (El-Shamy et al, 2012). A dense collection of  modifications around such a large site (variable district 3; codons 2356-2379) within the C-terminal piece of NS5A was considered to correspond to the reaction to Peg-IFN / ribavirin treatment in HCV genotype 1 isolates (Kumthip et al, 2011). Furthermore, a hereditary analysis of HCV replicon cells immune to IFN treatment found that specific substitutions in the NS5A protein (M2174V, T2319A / N, T2242N, F2256L) are related to resistance against IFN activity. NS5A was imagined to block IFNs by collaborating with PKR, a two-stranded RNA-stranded protein kinase. The substitution of amino acids in the NS5A protein exerts the role of PKR. Similarly, it is considered that the possibility that some cellular factors, alone or in combination with viral variables, add to the acquisition of IFN resistance (Numba et al, 2004).

  • HCV NS5A mutations associated with the response to antiviral agents:

The NS5A inhibitors, daclatasvir (BMS-790052) and ledipasvir (GS-5885), target the official NS5A domain I gap (amino residues 1–213) and this coupling causes obstruction of RNA replication and virus assembly (Lim and Gallay, 2014). The strong antiviral movement of daclatasvir against HCV replicons of various genotypes has been displayed (Fridell et al, 2010). However, higher rates of virological reactions to daclatasvir were observed among HCV genotype 1b compared to genotype 1a (Sulkowski et al, 2014). The resistance profile of daclatasvir has been demonstrated to correlate with some amino acid changes in the NS5A I space at positions M28T, Q30R / H, L31V and Y93H for HCV genotype 1a and L31H and Y93H for genotype 1b. These substitutions have been distinguished within the replicon in vitro and show a strong connection with those observed in the clinical outcome (Lim and Gallay, 2014). Due to ledipasvir, the pretreatment pattern changes seen in HCV genotype 1a patients prior to the introduction of this drug are M28T, Q30R / H, L31M, Y93C / H. These changes are related to a decrease susceptibility to treatment. In genotype 1b, Y93H is the most relentlessly recognized mutation in 100% of all patients (Gao, 2013).

Conclusion

HCV is a main cause of liver damage and specific type of cancer all over the world. With the polyprotein strand that consists of 10 proteins including structural and non  structural proteins, it causes infectious diseases. NS5A Protein of HCV performs critical role. It plays some of its function in cellular mitophagy and most importantly in viral translation and modulation. Also different mutations associated with it results in different treatment outcomes. Amino acids mutations and its impact on IFN based therapy is important one. Meanwhile different antiviral drugs is now considered as a major source of treatment in HCV infection.