Peptide entry inhibitors of viruses
Emergence of viral diseases has posed a risk to the public health. It is necessary to develop antiviral strategies that would deal with the infections caused by the viruses. A current focus of the research agencies is to develop broad spectrum antivirals that would target the pathways of entry and replication of the viruses in the cellular environments. The scope of this article is to review the mechanism of peptide based entry inhibitors of viruses such as HIV, Dengue virus, Ebola, RVFV, Influenza that act by altering the physical chemistry of the membrane fusion proteins which in turn affect the fusion of the viral and cellular membranes.
PEPTIDE ENTRY INHIBITORS
The onset of worldwide AIDS epidemic in 1980s has led to the development of combinational antiviral therapy to combat the consequences of human immunodeficiency virus (HIV) and development of drugs against other viruses. There is a need for synthesis of antivirals which would be efficient not only against their targets but are active against multiple targets. The decreased availability of antiviral drug strategies combating multiple targets and the constant increase in drug resistant and multi drug resistant strains are the main reasons for which the development of a novel class of antivirals is required (Krepstakies et al. 2012)
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Human pathogenic viruses include many well known viruses like HIV, HPV, Influenza, herpes measles, SARS. These viruses follow a sequential multistep process of entering the host cell which includes I) binding to the host cell receptors II) fusing the viral membrane to the cellular membrane which involve fusion proteins of virus such as spike proteins or glycoproteins. Some viruses enter the host cell by the formation of endosome followed by endosomal acidification. (Porotto et al. 2010) One of the important strategy of development of broad spectrum antivirals against the enveloped viruses is to target the essential host cell components which would prevent the entry of the virus in to the host cell.
Viral fusion is mediated by fusion proteins that undergo various conformational rearrangement which involve exposure of a hydrophobic fusion peptide or loop and also presenting hydrophobic sequences for enhancing the fusion with the cellular membrane (H.Badani et al. 2010). The fusion process is mainly inhibited by structural changes in the fusion protein, by changes in the physical properties of the membrane, or by addition of antibodies or peptides which bind with the fusion protein thus inhibiting the binding of the protein to the cellular membrane. (Krepstakies et al. 2010) (H.Badani et al. 2010)
Various peptide entry inhibitors have been developed that block the entry of the virus in to the host. Peptides act as efficient entry inhibitors since they interact with the hydrophobic membrane-protein interfaces. (H.Badani et al. 2010) The present studies on peptide entry inhibitors have reported SALPs, a new class of synthetic anti lipopolysaccharide peptides showing broad spectrum antiviral activity. It was demonstrated that SALPs interfered with the de novo infection caused by various enveloped human pathogenic viruses involving HBV, HIV, HCV and HSV that pose a threat to major health concern. SALPs inhibit the viruses by targeting the cell surface HS for the initial attachment to the host cell thus may prove to be a broad spectrum inhibitor of enveloped viruses. It was demonstrated that antiviral activity is mainly influenced by the net positive charge on the peptide rather than the amphiphilicity of the peptide. (Krepstakies et al. 2010).
A) Entry inhibitor of HIV
HIV is the causative agent of AIDS which is a devastating disease affecting millions of people each year. The gp41 and gp120 are the envelope proteins of HIV which play a significant role in the entry process and thus intensely studied for the development of antiviral therapeutics. Peptide entry inhibitors which correspond to the viral fusion proteins act efficiently to prevent the entry of the viruses. (Egerer et al. 2010)
Recent advances in this approach has led to the development of the first entry inhibitor of Human immunodeficiency virus (HIV). Enfuvirtide (ENF/T-20/Fuzeon) is the first licensed entry inhibitor of Human immunodeficiency virus. ENF is a 36 amino-acid peptide which is based on the HR2 domain in the gp41 subunit of HIV-1 env protein. It has to be administered subcutaneously. ENF bind to HR1 and prevents its interactions with HR2 which is necessary for the membrane fusion. In order to determine the viral sensitivity to other inhibitors of the viral entry it is essential to quote the mechanisms for viral resistance and the consequences of ENF resistance. This is because virus resistant cases against ENF have been documented due to mutations in the HR1 domain. Hence new antivirals with increased specificity needs to be developed. It is shown that peptides derived from the membrane proximal region of gp41 are efficient inhibitors of HIV entry to the host. (Reeves et al. 2005).
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B) Dengue virus entry inhibitor
The fusion of viruses by formation of endosomes is seen in case of dengue virus and other flaviviruses. The entry takes place through uptake by clathrin mediated endocytosis. Class“II" fusion proteins exhibit membrane fusion by domain rearrangements of the envelope glycoprotein (E) followed by transitions of the dimers to trimers. The rearrangement occurs by external rotation of the ectodomain E and conformational changes include loss of dimer interface and formation of extended trimmers (Schmidt et al. 2010).
By a liposome fusion assay it was shown that the peptides which are obtained from the membrane proximal stem of dengue E protein does not bind to the prefusion dimer but binds to the trimeric form of the post fusion sE conformer in its conformational change which is triggered at low pH. This binding step of the peptide mimics the most significant step that occurs during the fusion process. The evidence provided suggest that the inhibition by the stem peptides takes place in two steps. Initially the stem peptides are shown to interact nonspecifically to the viral membrane followed by specific attachment to the E when the protein undergoes conformational changes since the binding surface on E is exposed only after a drastic drop in pH inside the endosome. The peptides derived from the stem also are shown to inhibit the viral infection. Their potency is determined by their affinity for E and this is achieved because of the specificity to block fusion by binding to postransfusion conformer sE. (Schmidt et al. 2010)
C) Avian influenza virus entry inhibitor
Avian influenza belonging to orthomyxoviridae family is the causative agent of bird flu and has resulted in high morbidity and mortality rates in poultry. The viral surface glycoproteins hemagglutinin and neuraminidase play an important role in its entry and pathogenicity. The prevention and treatment process is challenging due to the resistance offered by the influenza virus because of its mutative nature. (Rajik et al)
In the recent studies on PI antiviral peptide against avian influenza H9N2 using flow cytometry it was demonstrated to inhibit the viral attachment to the host cell irrespective of its effects on viral fusion. Thus this prevents the expression of early genes of the virus. It was determined that viral attachment takes place at even at 4ËšC but viral fusion proteins require 37ËšC for fusion with the cellular membrane. The efficiency of the viral inhibitor was confirmed by detecting the expression levels of nucleoprotein NP of influenza virus. The entry inhibitor PI peptide inhibits the viral replication by blocking its attachment and entry into the host cell. It’s been demonstrated that the PI peptide has the potential for studying the host- pathogen interactions and also has application in antiviral therapeutics. (Rajik et al. 2009)
D) Vaccinia virus entry inhibitor
Variola virus is the causative agent of smallpox which is now been eradicated. However concerns of Variola virus being used as a weapon for bioterrorism has led to studies involving development of antivirals against orthopoxviruses. Studies carried on anti Vaccinia virus have reported a novel peptide EB which blocks the VACV.
EB is the first non-antibody inhibitor of poxviruses. It has been obtained from the human signal sequence of FGF4 protein. The mechanism of action of EB was studied and it was reported that EB could block the entry of VACV in to the host cells in a sequence specific manner with EC50 of 15μM. EB was not virucidal and that it did not affect the attachment of the virus, instead blocked the entry of the cells was also reported. EB could block the viral fusion when been added after encountering low pH. Resistance against EB was confirmed when EB was added along with high EC50 concentrations. The inhibition of VACV by EB was found to be sequence specific and reversible. EB peptide has been identified with potentials clinical applications and therapeutics to prevent VACV infection. (Altmann et al. 2009).
E) Ebola virus entry inhibitor
Ebola virus belongs to the filoviridae family that produces filamentous enveloped particles and is the causative agent for hemorrhagic fever. There are currently no approved antivirals or vaccines to curb filovirus infections. The infection begins when the virus particles enter the host cell through the endosomes due to the fusion mediated by the viral glycoprotein GP. To demonstrate the inhibition of the virus studies were conducted using EboV-C peptide conjugated to the arginine rich Tat HIV-1 whose evidence has been found in the endosomes (Sequence Tat- Ebo: YGRKKRRQRRR-GSG-IEPHDWTKNITDKIDQIIHDFVDK). The antiviral activity was reported to be influenced by the Tat and native EboV CHR sequence. The antiviral activity of EboV could be enhanced by tagging the C-terminal with cholesterol moiety.
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The EboV peptide has been reported with broad entry inhibition antiviral activity with a potential to target few other filoviruses that cause infection mediated by filovirus GP protein. The endosome targeted C peptide acts as a fusion intermediate thus preventing the entry of the virus in to the host. (Miller et al. 2011)
F) Rift valley fever virus
Rift valley fever virus is an Arbovirus which is the causative agent of the rift valley disease amongst the humans and livestock in major parts of Africa. In order to study the inhibition of the virus, peptide (RVFV-6) analogous to the RVFV fusion protein stem region (Gc) was synthesized (Peptide sequence: WNFFDWFSGLMSWFGGPLK.) and tested for the entry inhibition process. It was reported that the peptide independent of the fusion protein binding could prevent the binding of the cells and the virions. It binds to the viral Gc protein following endosomal acidification and thus prevents the fusion of the virus.
From the studies carried out it was demonstrate that due to the hydrophobic and aromatic residues of amino acids in the viral and cellular membrane RVFV-6 binds to the viral and cellular membranes. This contributed to the internalization of the peptide into the virion. The low pH inside the endosome is known to trigger the Gc protein of the virion to undergo conformational changes and thus exposure of a hidden stem domain which is important for the fusion of the virus with the host. RVFV-6 which is analogous to the stem domain binds to the groove and thus inhibits the viral and host membrane interactions.
RVFV-6 has also been identified with potentials to target bunya virus such as ANDV and filovirus EBOV which fuses through class I fusion protein and rhabdovirus VSV whose fusion takes place thorough class III fusion proteins.RVFV-6 could also be used in competitive binding assays and therapeutics to study the infectivity of small drug molecules. (Koehler et al. 2013)
In this review the mechanism of entry inhibition of various viruses mediated by peptides has been considered. Preventing the viral entry in to the host mediated by using analogous peptides has been recognized as a novel strategy for developing antivirals against various viruses. This antiviral strategy would not only prevent the cell to cell spread but could also inhibit the replication and evolution of the virus. Since the fusion proteins of the virus are extracellular entry inhibition by means of using g peptides has been identified as a novel strategy in therapeutics since the drug molecules could easily target the proteins than the intracellular proteins of the virus. Development n of broad spectrum inhibitors that could target multiple viruses as well as with fewer side effects is anticipated in the near future.
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