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Secreted viral Insulin-like peptides activate human insulin and IGF-1 receptor signaling

Disulfide Bridges and Domain Structure in Human Insulin, IGF1 and VILPs

Disulfide bridges and the domain structure in human insulin, igf1,and four VILPs

 

sequence comparison LCDV, IGF-1


Sequence Comparison of LCDV1, Insulin, IGF1, and IGF2

sequence comparison LCDV

 

 

Insulin (Human) and GIV Sequence Alignment

sequence comparison giv insulin

 

 

Insulin (Human), Des (1-3), IGF-1 and LCDV-Sa Sequence Alignment

sequence comparison LCDV-Sa


Viral insulin-like peptides

Viruses are the most abundant biological entities and carry a wide variety of genetic material, including the ability to encode host-like proteins. Here we show that viruses carry sequences with significant homology to several human peptide hormones including insulin, insulin-like growth factors (IGF)-1 and -2, FGF-19 and -21, endothelin-1, inhibin, adiponectin, and resistin. Among the strongest homologies were those for four viral insulin/IGF-1-like peptides (VILPs), each encoded by a different member of the family Iridoviridae VILPs show up to 50% homology to human insulin/IGF-1, contain all critical cysteine residues, and are predicted to form similar 3D structures. Chemically synthesized VILPs can bind to human and murine IGF-1/insulin receptors and stimulate receptor autophosphorylation and downstream signaling. VILPs can also increase glucose uptake in adipocytes and stimulate the proliferation of fibroblasts, and injection of VILPs into mice significantly lowers blood glucose. Transfection of mouse hepatocytes with DNA encoding a VILP also stimulates insulin/IGF-1 signaling and DNA synthesis. Human microbiome studies reveal the presence of these Iridoviridae in blood and fecal samples. Thus, VILPs are members of the insulin/IGF superfamily with the ability to be active on human and rodent cells, raising the possibility for a potential role of VILPs in human disease. Furthermore, since only 2% of viruses have been sequenced, this study raises the potential for discovery of other viral hormones which, along with known virally encoded growth factors, may modify human health and disease.

Emrah Altindis, Weikang Cai, Masaji Sakaguchi, et al, Proceedings of the National Academy of Sciences. 2018;:201721117.

Insulin-like growth factors (IGFs) play crucial roles in regulating cell differentiation, proliferation and apoptosis. In this study, a novel IGF homologue gene (IGF-like) encoded by Singapore grouper iridovirus (SGIV) ORF062R (termed SGIV-IGF), was cloned and characterized. The coding region of SGIV-IGF is 771 bp in length, with a variable number of tandem repeats (VNTR) locus at the 3'-end. We cloned one isoform of this novel gene, 582 bp in length, containing the predicted IGF domain and 3.6 copy numbers of the 27 bp repeat unit. SGIV-IGF was an early transcribed gene during viral infection, and SGIV-IGF was distributed predominantly in the cytoplasm with a diffused granular appearance. Intriguingly, overexpression of SGIV-IGF was able to promote the growth of grouper embryonic cells (GP cells) by promoting G1/S phase transition, which was at least partially dependent on its 3'-end VNTR locus. Furthermore, viral titre assay and real-time quantitative PCR (RT-qPCR) analysis proved that SGIV-IGF could promote SGIV replication in grouper cells. In addition, overexpression of SGIV-IGF mildly facilitated apoptosis in SGIV-infected non-host fathead minnow (FHM) cells. Together, our study demonstrated a novel functional gene of SGIV which may regulate viral replication and cellular processes through multiple mechanisms that appear to be cell type-dependent.

Yan Y, Cui H, Guo C, et al, J Gen Virol. 2013 Dec;94(Pt 12):2759-70. doi: 10.1099/vir.0.056135-0. Epub 2013 Sep 23.

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease, which has been reported to occur in over 100 different fish species worldwide. LCDV is a member of the family Iridoviridae and the type species of the genus Lymphocystivirus. The virions contain a single linear double-stranded DNA molecule, which is circularly permuted, terminally redundant, and heavily methylated at cytosines in CpG sequences. The complete nucleotide sequence of LCDV-1 (flounder isolate) was determined by automated cycle sequencing and primer walking. The genome of LCDV-1 is 102.653 bp in length and contains 195 open reading frames with coding capacities ranging from 40 to 1199 amino acids. Computer-assisted analyses of the deduced amino acid sequences led to the identification of several putative gene products with significant homologies to entries in protein data banks, such as the two major subunits of the viral DNA-dependent RNA polymerase, DNA polymerase, several protein kinases, two subunits of the ribonucleoside diphosphate reductase, DNA methyltransferase, the viral major capsid protein, insulin-like growth factor, and tumor necrosis factor receptor homolog.

Tidona CA, Darai G. Virology. 1997 Apr 14;230(2):207-16.

Chemical Syntheses of Viral Insulin-like Peptides by Selective Formation of Disulfide Bridges.

Presented at the Relaxin Conference in May 2018

Introduction:

  1. Insulin and Insulin-like growth factors play a central role in regulating cell metabolism, growth and development. Recently, several homologues of human growth factors encoded in viral genomes, called Virus Insulin-like peptides (VILPs), has been showed bind to human insulin and IGF1 receptors . However, the reported affinity of VILPs to human receptors were 10 ~100 fold lower than the human insulin and IGF-1 [1]. 
  2. Human microbiome studies also reveal the presence of these virus in blood and fecal samples. Thus, a possibility of secreted VILPs may participate into human disease or modulate the human physiology [1, 2].
  3. The disulfide bridges in these peptides has been predicted to have a conformation of human insulin or IGF-1. However, it is not know whether the truncated-variants or more precised disulfide bond pairing synthetic peptide can make a higher affinity to Insulin or IGF-1 receptors than the C-peptide containing VIPLs. It will be better to use precise chemical synthesis to investigate its bioactivity. In the current work, bioinformatics and chemical synthesis are used to enable synthesis of large quantity of peptide and afforded the opportunity to do more structure and functional studies.
  4. The sequence alignment of A-Chain and B-Chain region of VILPs and insect peptide, Hsal-ILp2 region showed 38-60% homology to human insulin and IGF1.
sequence comparison of 6 Insulin like proteins
The insulin superfamily of peptides is characterized by the presence of a set of three disulfide bonds in a unique disposition. With the exception of insulin-like growth factors I and II, which are single chain peptides, the remaining members of the human insulin superfamily are two-chain peptides containing one intramolecular and two intermolecular disulfide bridges. Technically, such structural features have long made the chemical synthesis of the peptides a considerable challenge, in particular, including their correct disulfide bond pairing and formation. Also, it is not known whether the secreted VILPs in vivo has been a structure like insulin which eliminate the C-peptide or similar to IGF-1 that contains C-peptide.

Methods:

Method 1: As one of the examples in VILPs, the synthesis of LCDV-1 (lymphocyctis disease virus) is demonstrated here. The linear LCDV-1, {ITAEILCSAHLVAALQRVCGNRGVYRPPPT-RRRSTRNGTT-GIATKCCTTTGCTTDDLEKYCN, with Cys-Cys(1-4, 3-5 and 2-6)}, was synthesized by Fmoc-solid-phase method using 3 different Cysteine side chain protected groups, such as Trityl, tert-Butyl and Acm. These protected groups can be cleaved by TFA, CF3SO3H and cyclized to form the Cys-Cys by iodine in HOAc respectively. In order to achieve an exactly folding of disulfide-bridges in LCDV1, the procedures are shown on the diagram below. 
Full length LCDV synthesis


Method 2: The A- and B-chain of VILPs without the C-peptide linkage was synthesized separately as described in the synthesis of DILP-8 [4]. This method was used to synthesize Des-C-peptide LCDV1, Des-C-peptide LCDV-Sa, Des-GRR-GIV, and Des-GRR-SGIV. 
As an example of such synthesis, a procedure for exact folding of disulfide-bridge is shown in the diagram below.

Des C peptide LCDV-1 sequence synthesis



Results from Method 1: LC-MS profile of LCDV-1


LC-MS profile of LCDV-1


LC-MS profile for Des-C-peptide LCDV-Sa
LC-MS profile for Des-C-peptide LCDV-Sa
LC-MS profile for Des-GRR SGIV
LC-MS profile for Des-GRR GIV

Conclusions: 

A full length of LCDV-1 and four of the Des-C-peptide VILPs (LCDV-1, LCDV-Sa, GIV, and SGIV) has been synthesized. It has been confirmed that all synthetic peptides showed 95% of purity and correct molecular weight. 
Current methodology proved to have a high synthetic yield. By using this approach, the preparations of pure VILPs in large quantity (10-20mg) were produced to support the exploratory bioassays. Meanwhile, these peptides can be used to raise antisera for peptide levels quantification. 

Summary

Instead of folding the linear peptide with 6 free Cys-SH in high pH buffer for days, pure VIPLs can be synthesized by using the selective Cys-Cys bond formation. In this example, the chemical synthesis of LCDV-1 with predicted disulfide bonds and with/without connecting peptide had been accomplished. 
Human microbiome studies reveals the presence of these iridoviridae in blood and fecal samples. Since these VILPs has been showed with different extent of activity like Insulin or IGFs, the bioactivity of VILP mutant is worthy to further explore. Further investigation by using both bioinformatics and chemical synthesis for insulin/IGFs structure similar peptides may have a great impact on the treatments of disease. 

References:

  1. Altindis E. et al. Viral insulin-like peptides activate human insulin and IGF-1 receptor signaling: A paradigm shift for host-microbe interactions. Proc Natl Acad Sci U S A, 2018 Mar 6;115(10):2461-2466.
  2. Yan Y. et al. An insulin-like growth factor homologue of Singapore grouper iridovirus modulates cell proliferation, apoptosis and enhances viral replication. J Gen Virol. 2013 Dec;94(Pt 12):2759-70.
  3. Tidona C.A., and Darai G. The complete DNA sequence of lymphocystis disease virus. Virology, 1997 Apr 14;230(2):207-16.
  4. Sabirov A, et al.. A total solid-phase synthesis of DILP8. Ital J Anat Embryol. 2013;118(1 Suppl):13-4 


Related Products

Catalog# Product Standard Size Price
036-55 Des-GRR-GIV 100 µg $350
036-56 Des-GRR-SGIV 100 µg $350
036-58 Des-C-peptide-LCDV-1 100 µg $350
036-61 LCDV-1 100 µg $400
B-036-58 Des-C-peptide-LCDV-1 - Biotin Labeled 20 µg $259
T-036-58 Des-C-peptide-LCDV-1 - I-125 Labeled 10 µCi $734
036-59 LCDV-Sa 100 µg $350