INTRODUCTION:

Viperin enzyme is an anti-viral protein which is found in the endoplasmic reticulum of the cell. It is an interferon inducible enzyme by which it interferes with viral DNA and RNA replication process. It is also known as a RSAD2 (radical SAM domain-containing 2). It is also known as a multifunctional protein which is involved in antiviral mechanism and it can be involved in the mechanism by induction of interferon’s and variety of cell types by different cellular factors such as type I, II and III interferon, DNA and RNA viral proteins and polysaccharides¹. In innate signaling Viperin plays a main role by both direct inhibition and replication and by budding and it destroys the actin cyto-skeleton which further leads to increase HCMV. It is an best example to determine the antiviral property ².

Function:

Viperin is a cellular protein which is present in the endoplasmic reticulum of the mammalian cells and it is stimulated whenever the vitals infections attack to mammalian cells. It could inhibit many DNA and RNA viruses such as CHIKV, HCMV, HCV, DENV, WNV, SINV, Influenza, HIV LAI strain, and so on. It was first identified as an IFN-γ induced anti-viral protein in human cytomegalo virus (HCMV) infected macrophages, viperin is addressed to be induced by HCMV glycoprotein B in fibroblasts and it inhibits HCMV viral infection and which helps in down- regulating viral structural proteins, which are essential for viral assembling and budding. However, the viral induced reposition of viperin is found in HCMV infected cells, this reflects the mechanism of virus how it’s dodging the anti-viral activities of viperin. Viperin can be induced and then it can interacts with HCMV viral proteins and relocate to mitochondria in HCMV viral infected cells, and finally enhance viral infectivity by the disrupted cellular metabolism.¹

In the inhibition of influenza virus in budding and its release, the viperin could act by disrupting the lipid rafts which are present on the cell plasma membrane by decreasing the enzyme activities of farnesyl-di-phosphate synthase (FPPS), it is an essential enzyme which helpful in isoprenoid biosynthesis pathway. In addition to this viperin can also inhibit the viral replication of HCV by the interaction with host protein hVAP-33 and NS5A and disruption of the formation of the replication complex¹.

Structure:

Human viperin contains 361 amino acids which is a single polypeptide chain with a molecular mass of 42kDa. The first 42 residues of human viperin is N-terminalamphipathic S alpha-helix, which is less conserved in different species and shows effect on the antiviral ability of viperin against HCV, WNV and DENV. The N-terminal domain of viperin is also needed for the viperin localization in endoplasmic reticulum and the lipid droplets of the mammalian cells. The residues 77-209 contains the radical S-adenosyl methionine (SAM) domain, which consist of there are four conserved motifs. Motif 1 contains three conserved cysteines residues, CxxCxxC, and it is the Fe-S binding motif which is essential for the antiviral activities against HCV and HMCV infections. The 218-361 residues containing C-terminal domain of viperin, which is highly used in different species of viperin and it is essential for viperin dimerization. The last residue 361 of the C-terminal is a tryptophan, which is essential for the antiviral functions. Viperin has the ability in the formation of homodimers in endoplasmic reticulum, and this over expression of viperin could result in the formation of a crystalloid structure of endoplasmic reticulum^1,3.

Function and Mechanism of Viperin, A radical antiviral SAM protein:

The in vivo function of these new molecules is

1. Selective “poisoning” of viral RNA and DNA polymerases.

2. Modulation of cytidyl transferees using the CTP as a substrate, and these are also required for lipid biosynthesis e.g., phosphotidylethanolamine, phosphotidylcholine.

3. It also plays a major role as a novel signaling molecule.

All these probabilities are required for providing a unique mechanism of viperin for antiviral function, as each proposed mechanism depends on the radical-based enzymatic characteristics of viperin to regulate the fundamental processes of replication, membrane dynamics and signaling of all viruses. Viperin is an enzyme which helps in converting the CTP to ddhCTP its mechanism is showed in fig-1^.[4]

Fig 1: SAM dependent radical mechanism

Typical research studies from the literature are briefly described here focusing on novel targets for various viral infections by Viperin:

Human Macrophages induction by Viperin directly in HIV-1.

Viperin enhances the interferon stimulated genes by inhibiting the HIV-1 production through macrophages. It acts by the destruction of lipid rafts which are present on cell membrane through viperin induction. It redistributes the viperin into CD-8 1 compartments of HIV-1 by budding in MDMS. Exogenous farrnesol which is helpful in up regulating membrane protein phenylation. And it also act reversely by viperin mediated inhibition of HIV-1 production. It occurs by mutagenesis of transfected cell lines which shows that the internal s-adenosyl methionine domains of viperin which are essential for the activity^4,5.

Viperin promoter gene:

The type one interferon (IFNS) INF α and INF-β are key effectors molecules of the immune response to viruses. The interferon inducible genes have the anti viral action of the virus infected cells. It is a virus inhibitory protein which is present in endoplasmic reticulum and it can be induced by interferon’s. It is an interferon stimulated gene (ISG) which is induced by type I, II, III IFNS after the mammalian cells are infected with a broad range of DNA and RNA viruses. Viperin destroys a lipid rafts to block influenza virus by budding and release and it also interferes with the replication of hepatitis C virus by binding to lipid droplets⁶.

S–Adenosyl methionine (SAM) enzyme that impede viral replication. The Viperin is an interferon inducible radical:

Crystal structures of mouse viperin are prepared and its crystal gets complexed with S–adenosyl homocystine and L-Met. It contains a partial (βα) 6- barrel fold with a disordered N-terminal extension and partially ordered c-terminal extension that bind to form a bridge with a partial barrel to form an ultimate barrel fold structure which constitutes of CYS84, CYS88, CYS91 located after the first β strand and it bond to a [Fe-S4] cluster. This is the active site of viperin is bounded to a SAH or SAM cleavage product which acts by the mechanism of generation of radical S-adenosyl homocystine. Structural alignments prove that the viperin structure is similar to structure of radical SAM enzymes^3,4,5.

Viperin inhibits zika virus and tick borne encephalitis virus replication by targeting NS₃ which is majorly responsible for proteasomal degradation:

Flaviviruses are belongs to the arthropod borne viruses that are involved in causing the world health problems of millions of infectious diseases. Type I INFS are induced and they are stimulated by the interferon stimulated gene in response to viral infection. It also helps in encryption of viperin shows antiviral activity against a broad spectrum of viruses including flaviviruses. Here they described that a novel antiviral mechanism adopted by viperin against two prominent flaviviruses, tick borne encephalitis virus (TBEV) and zika virus (ZIKV) viperin. It interacts and distributes with structural proteins which are present in pre membrane and envelope of TBEV as well as with the non structural (NS) proteins NS2A, NS2B and NS3. Here viperin expressed by reducing the NS3 protein level and its stability of the other interacting with viral proteins occurs only in the presence of NS3. They also found that although viperin interacted with NS3 of mosquito-borne flaviviruses. Only ZIKV is sensitive to the antiviral effect of viperin. This sensitivity of ZIKV is correlated with viperin’s ability to induce proteasomal dependent degradation of NS3. ZIKV and TBEV replication was inhibited completely when NS3 was over expressed and it suggests that the viral NS3 is the specific target of viperin. Structural characterization says that viperin is a radical S-adenosyl–L-methionine (SAM) enzyme. Viperin contains a N-terminal trans-membrane helix a highly conserved C-terminus and a middle region containing a Cx3Cx2C motif, which is the characteristic feature of radical-S-adenosyl–L-methionine (SAM) enzymes up to now no structural characterization has been reported and reconstitution of the [4Fe-4S] cluster in viperin. All failed in dissecting the 361-residue human viperin into 12 fragments followed by extensive CD and NMR characterization of viperin [4S-361] was identified to be soluble and structured in buffers .Most importantly they have successfully reconstituted the [4Fe-4s] cluster in viperin (4S-361) thus provides the first experimental evidence for the confirmation of viperin structure is similar to a SAM enzyme. Further more studies shows that the C-terminus viperin (214-361) which is insoluble in buffer but it can be solubilized in salt free water and appears to be only partially folded their results says that the radial radical SAM enzyme activity may play a important role in the broad antiviral actions of viperin.^2,10.

The presence of iron sulfur motif is essential for the conformational structural stability of the antiviral protein, viperin:

Viperin is an antiviral protein that contains a cx (3) cx (2) motif which is used in the formation of radical s-adenosyl-methionine (SAM) enzyme family. A triple mutant which replaces these three cysteines with alanines has been shown to have defect in antiviral activity. Since the structural of Viperin is not available, they have used a combination of computational methods including multi template homology modeling & molecular dynamics stimulation to develop a low resolution predicted structure. The result proves that the Viperin is α-β protein which contains a iron sulfur cluster at the center pocket. For the verification of these predictions they have prepared, expressed & purified four mutant proteins. In three mutants individual cysteine residues were replaced by alanine residues while in the fourth all the cysteines were replaced by alanines. This structural conformation was done by using analysis of circular dichorism and steady state fluorescence spectroscopy to indicate that the mutant proteins are unfolded, confirmationally unstable and aggregation prone^.7.

mRNA Viperin is a novel target for the human RNAse MRP\RNase P endoribonuclease:

RNAse MRP is conserved endoribonuclease in humans consisting of a 267 nucleotide RNA associated with 7-10 proteins. Mutations in RNA component leads to several autosomal recessive skeletal dysplasia’s including cartilage hair, hypoplasia (CAH), Because the known substrates of mammalian RNAse , MRP, Pre- ribosomal RNA, RNA involved in mitochondrial DNA replication are not likely involved in CHH, they analyzed that the effects of RNAse MRP depletion on mRNase using DNA microarrays. They confirmed the up regulation of the interferon inducible viperin mRNA by RNA experiments and thus it appears to be independent of the interferon response. They detected two cleavage sites for RNAse MRP\ RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of an mRNA by RNAse MRP\RNAse P in human cells. Viral protein NS₃. They also found that although viperin interacted with NS₃of mosquito born flaviviruses. Only ZIKV was sensitive to antiviral effect of viperin. This sensitivity co-related with viperin’s ability to induce proteasome dependant degradation of NS₃. ZIKV & TBEV replication was rescued completely NS₃ was over expressed, suggested that the viral NS₃is the specific target of viperin¹⁰.

Viperin MTAP₄₄ and protein kinase R which leads to the interferon induced inhibition of bunyamwera or thobunya virus’s replication:

The first line of defense against viral replication interferon response involved in the expression of hundreds of proteins with expected antiviral activity and it must be overcome by a virus for successful replication. The non-structural NS5 protein is the primary IFN antagonist encoded by Bunyamwera virus (BUNV), it is the prototype of the ortho bunya virus genes which belongs to the family of Bunyaviridae. The NS5 protein interferes with RNA polymerase II which is mediated by transcription and it acts there by inhibiting cellular mRNA production and it also includes IFN mRNAs. A recombinant virus, gbundle NS5, that is unable to express the NS5 protein, does not inhibit cellular transcription and it is a strong IFN inducer. They says that cells stimulating antiviral activity by IFN β treatment were protected against wild type BUNV & gbundle NS5 infection but in addition of IFN β after infection had little effect on the replication cycle of virus. By screening a panel of cell lines that over expressed individual IFN stimulated genes they found that protein kinase R (PKR), MTAP44, & particularly viperin appreciably restricted BUNV replication. Thus the enzymatic activities of PKR & viperin were required for inhibitory activity of their data showed that the restriction of BUN replication is mediated by IFN & there is accumulated effect of atleast three IFN stimulated genes that probably act on different stages of the virus replication cycle^.8.

Identification of three interferon inducible cellular enzymes that inhibit the replication of hepatitis C virus:

Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis & is treated with IFN α based therapies. To identify the cellular proteins that mediate the anti-viral effects of IFN α they created a HEK 293 based cell culture system to induce & express individual interferon stimulated genes (ISGS) which are helpful in determining their effects against HCV. By screening 29 IGSS that are induced in HUH7 cells by IFN α or up regulated in HCV infected livers. They discovered that viperin ISG20, and double stranded RNA dependant protein kinase (PKR) none is cytolytically involves in inhibiting the replication of HCV. And its mechanism is inhibition of HCV replication by ISG20 & PKR depends on their 3¹-5¹ exo-nuclease and protein kinase activities. Their work shows that the strong evidence for suggesting that the viperin is a putative radical S- adenosyl -L-methionine (SAM) enzyme. In addition to demonstrating that the antiviral activity of viperin depends on its radical SAM domain, which are having the conserved motifs to co-ordinate [4FE-4S] cluster and its co-factor SAM is very much essential for its enzymatic activity, mutagenesis studies shows that viperin requires an aromatic amino-acid residue at its C-terminus for proper antiviral function. Although the N terminal 70 amino- acid residues of viperin are not absolutely required so detection of this region significantly shows the anti –viral activity against HCV. Their findings suggest that viperin represents a novel anti-viral pathway that works with other anti-viral protein, such as ISG20 & PKR, to mediate the INF response against HCV infection^{13, 14}.

CONCLUSION:

Viperin is an anti-viral protein that plays a main role in the prevention of viral infections. Viral infections are the major challenge now-a-days and virus changes its structure many times and that’s why its mechanism also varies simultaneously so it is very difficult to identify the particular mechanism of that virus causing infections the viral infections. So Pen state university states that Viperin converts the cytidine triphosphate (CTP) to 3¹-deoxy-3¹, 4¹-didehydro-CTP (ddhCTP) molecule which is a novel target for the viral infections, and it also spawn zika drug from our body^[14]. They said that the ddhCTP is a chain terminator for the RNA-dependent RNA polymerases from the multiple members of the flaviviruses genus, and suggest a partially unifying mechanism for the antiviral effects of viperin and it is based on the intrinsic enzymatic properties of the protein which involves in the generation of a naturally occurring replication chain terminator encoded by mammalian genomes^2,8,14.

REFERENCES:

1. https://en.wikipedia.org/wiki/Viperin

2. Fenwick. Compound made inside human body stops viruses from replication.PDB: 5VSM; PNAS 2017 114 6806-6811) Friday, February 1, 2019.

3. Katherine A. Fitzgerald. The Interferon Inducible Gene: Viperin. 2011 Jan; 31(1): 131–135.

4. Caitlyn Makins, Soumi Ghosh, Gabriel D. Roman Melendez, Paige A. Malecetal, Robert T. Kennedy, E. Neil G. Marsh. Does Viperin Function as a Radical S-Adenosyl-L-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity. J Biol Chem. 2016 Dec 23; 291(52): 26806–26815.

5. Karla J. Henbig and Michael R. Beard; The role of Viperin innate Antiviral Response; http;//dx.doi.org/10.1016/j.jmb.2013.10.019.

6. Michael K. Fenwick, Yue Lil, Peter Cresswell, Yorgo Modiset, and Steven E. Ealick , Structural studies of viperin, an antiviral radical SAM enzyme. Proc Natl Acad Sci U S A. 2017

7. Richard Lindqvist, Upadhyay, and, and Anna K. Överby. Tick-Borne Flaviviruses and the Type I Interferon Response Viruses. 2018 Jul; 10(7): 340.

8. Anthony S. Gizzi Tyler L. Grove Jamie J. Arnold, Rohit K Jangra; Scott J. Garforth, Quan Du,Sean M. Cahill, Natalya G. Dulyaninova, Kartik Chandran; Anne R. Bresnick, Cameron and Steven C. Almo. A naturally occurring antiviral ribonucleotide encoded by the human genome. Nature. 2018 Jun; 558(7711): 610–614.

9. Kaitlin S. Duschene and Joan B. Broderick. The antiviral protein viperin is a radical SAM enzyme. FEBS Lett. 2010 Mar 19; 584(6): 1263–1267.

10. Helbig KJ, Beard MRl The role of viperin in the innate antiviral response 2014 Mar 20; 426(6):1210-9. doi: 10.1016/j.jmb.2013. 10.019. Epub 2013 Oct 22.

11. Caitlyn Makins, Soumi Ghosh, Gabriel D. Roman Melendez, Paige A. Malecetal, Robert T. Kennedy, E. Neil G. Marsh. Does Viperin Function as a Radical S-Adenosyl-L-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity. J Biol Chem. 2016 Dec 23; 291(52): 26806–26815.

12. Kaitlin S. Duschene, Joan B. Broderick. Viperin: a radical response to viral infection. Biomol Concepts. 2012 Jun; 3(3): 255–266.

13. Richard Lindqvist, Upadhyay, and, and Anna K. Överby. Tick-Borne Flaviviruses and the Type I Interferon Response Viruses. 2018 Jul; 10 (7): 340.

14. Barbara Kennedy–Penn state; Enzyme from our bodies could spawn zika drug (June20th 2018).

15. Karla J. Henbig and Michael R. Beard; The role of Viperin innate Antiviral Response; http;//dx.doi.org/10.1016/j.jmb.2013.10.019.

Received on 04.02.2019 Modified on 18.03.2019

Research J. Pharm. and Tech. 2019; 12(6): 3063-3067.

DOI: 10.5958/0974-360X.2019.00519.5