Non-insulin 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

Objective: The insulin/IGF superfamily is conserved across vertebrates and invertebrates. Our team has identified five viruses containing genes encoding viral insulin/IGF-1 like peptides (VILPs) closely resembling human insulin and IGF-1. This study aims to characterize the impact of Mandarin fish ranavirus (MFRV) and Lymphocystis disease virus-Sa (LCDV-Sa) VILPs on the insulin/IGF system for the first time.

Methods: We chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin like) forms of MFRV and LCDV-Sa VILPs. Using cell lines overexpressing either human insulin receptor isoform A (IR-A), isoform B (IR-B) or IGF-1 receptor (IGF1R), and AML12 murine hepatocytes, we characterized receptor binding, insulin/IGF signaling. We further characterized the VILPs' effects of proliferation and IGF1R and IR gene expression, and compared them to native ligands. Additionally, we performed insulin tolerance test in CB57BL/6 J mice to examine in vivo effects of VILPs on blood glucose levels. Finally, we employed cryo-electron microscopy (cryoEM) to analyze the structure of scMFRV-VILP in complex with the IGF1R ectodomain.

Results: VILPs can bind to human IR and IGF1R, stimulate receptor autophosphorylation and downstream signaling pathways. Notably, scMFRV-VILP exhibited a particularly strong affinity for IGF1R, with a mere 10-fold decrease compared to human IGF-1. At high concentrations, scMFRV-VILP selectively reduced IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation (Ras/MAPK pathway), while leaving Akt phosphorylation (PI3K/Akt pathway) unaffected, indicating a potential biased inhibitory function. Prolonged exposure to MFRV-VILP led to a significant decrease in IGF1R gene expression in IGF1R overexpressing cells and AML12 hepatocytes. Furthermore, insulin tolerance test revealed scMFRV-VILP's sustained glucose-lowering effect compared to insulin and IGF-1. Finally, cryo-EM analysis revealed that scMFRV-VILP engages with IGF1R in a manner closely resembling IGF-1 binding, resulting in a highly analogous structure.

Conclusions: This study introduces MFRV and LCDV-Sa VILPs as novel members of the insulin/IGF superfamily. Particularly, scMFRV-VILP exhibits a biased inhibitory effect on IGF1R signaling at high concentrations, selectively inhibiting IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation, without affecting Akt phosphorylation. In addition, MFRV-VILP specifically regulates IGF-1R gene expression and IGF1R protein levels without affecting IR. CryoEM analysis confirms that scMFRV-VILP' binding to IGF1R is mirroring the interaction pattern observed with IGF-1. These findings offer valuable insights into IGF1R action and inhibition, suggesting potential applications in development of IGF1R specific inhibitors and advancing long-lasting insulins.


Chrudinová M, Kirk NS, Chuard A, et al. A viral insulin-like peptide inhibits IGF-1 receptor phosphorylation and regulates IGF1R gene expression. Molecular Metabolism. 2024;80:101863.

Abstract: The human insulin receptor signalling system plays a critical role in glucose homeostasis. Insulin binding brings about extensive conformational change in the receptor extracellular region that in turn effects trans-activation of the intracellular tyrosine kinase domains and downstream signalling. Of particular therapeutic interest is whether insulin receptor signalling can be replicated by molecules other than insulin. Here, we present single-particle cryoEM structures that show how a 33-mer polypeptide unrelated to insulin can cross-link two sites on the receptor surface and direct the receptor into a signalling-active conformation. The 33-mer polypeptide engages the receptor by two helical binding motifs that are each potentially mimicable by small molecules. The resultant conformation of the receptor is distinct from—but related to—those in extant three-dimensional structures of the insulin-complexed receptor. Our findings thus illuminate unexplored pathways for controlling the signalling of the insulin receptor as well as opportunities for development of insulin mimetics.

Kirk NS, Chen Q, Wu YG, et al. Activation of the human insulin receptor by non-insulin-related peptides. Nat Commun. 2022;13(1):5695.

Objective: Members of the insulin/insulin-like growth factor (IGF) superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single-chain (sc), i.e., IGF-1-like, forms of the viral insulin/IGF-1-like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e., more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1) for the first time.
Methods: The dcVILPs were chemically synthesized. Using murine fibroblast cell lines overexpressing insulin receptor (IR-A or IR-B) or IGF1R, we first determined the binding affinity of dcVILPs to the receptors and characterized post-receptor signaling. Further, we used C57BL/6J mice to study the effect of dcVILPs on lowering blood glucose. We designed a 3-h dcVILP in vivo infusion experiment to determine the glucose uptake in different tissues.
Results: GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A and IR-B) and to the IGF1R, and for the latter, show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75 nmol/kg/min) stimulated a comparable glucose uptake in heart and skeletal muscle and brown adipose tissue, GIV dcVILP stimulated 2-fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin in WAT.
Conclusions: Our results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogs that specifically target the tissues and provide new insights into their potential role in disease.


Chrudinová M, Moreau F, Noh HL, et al. Characterization of viral insulins reveals white adipose tissue-specific effects in mice. Molecular Metabolism. 2021;44:101121.

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


  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.


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


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. 


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. 


  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 

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036-57 scMFRV-VILP 100 µg $497