Characterisation of Viperin from the Saltwater Crocodile (Crocodylus porosus)

Natalie Milic1, Steven Davis2, Sally Isberg3 and Karla Helbig4.

1
School of Psychological and Clinical Science, Charles Darwin University, Darwin, Australia; 2Berrimah Veterinary Laboratories, Berrimah Farm,
Department of Primary Industry and Fisheries, Berrimah, Australia; 3 Centre for Crocodile Research, Noonamah, Australia; 4 School of Molecular and
Biomedical Science, The University of Adelaide, Adelaide, Australia.

Introduction
The Australian saltwater crocodile represents a prehistoric species of reptile
and to date little is known about its ability to control pathogens. The recent
acquisition of the full crocodile genome (International Crocodile Genomics
Working Group1) has afforded the opportunity to begin to unravel the
workings of the innate immune system in this ancient species, in particularly
its response to viral pathogens.
Viral infection results in the activation and expression of a cascade of
interferon-stimulated genes (ISGs), which aim to limit viral replication and
infection. Viperin is one of a few ISGs that has been shown to have a broad
antiviral effect against a range of viruses2. It is also a highly conserved
evolutionary host protein that has been identified in a variety of species
Figure 2: Viperin is a highly conserved protein.
Viperin is a highly conserved protein across multiple species2. (A) Clustawl
alignment of crocodile viperin re-iterated this, with the majority of the sequence
variation occurring within the N-terminal region. Crocodile viperin shares 84%,
80% and 75% similarity with chicken, human and lizard viperin proteins,
respectively. Interestingly, crocodile viperin was predominantly present as a splice
variant without its 6th exon (red boxed sequence). (B) Crocodile viperin contained
a conserved amphipathic helix (allows viperin to bind to the cytosolic side of
endoplasmic reticulum (ER) and to be present on lipid droplets), radial S-adenosyl
menthionine (SAM) domain, (binds iron sulphur clusters) and a highly conserved
C-terminal region (essential for some of the anti-viral properties displayed by
viperin1), which are seen in all viperin proteins.
Figure 4: Early pathogen recognition pathways of saltwater
crocodile.
LV-1 cells were incubated with 10μg/ml of poly I:C (synthetic dsRNA), dsDNA
or Sendai Virus (-ve sense ssRNA virus; SeV, 100HA units/ml) for 24 hours.
Real-time PCR was performed for the detection of known ISGs, in particularly
viperin, Mx1, ISG20L and OASL. Viperin was the only gene that was
responsive to all three stimuli, importantly the only gene that was responsive
to dsDNA. OASL was unresponsive to dsDNA, while ISG20L appeared
unresponsive to all stimuli (to date ISG20L has not been shown to be an
antiviral ISG).
OASL Viperin
1500 80

Relative fold change

Relative fold change

60
including mammals, avians and reptiles. This study aimed to investigate the A 1000
40
500
early innate immune response of crocodiles in the context of viral infection,

in mRNA

in mRNA
20
and ascertain the sequence homology and protein expression of viperin. 50 10

25 5

Figure 1: cDNA and amino acid sequence of Saltwater 0 0

Control dsDNA dsRNA SeV Control dsDNA dsRNA SeV
crocodile viperin.
Saltwater crocodile viperin was isolated from a crocodile liver-derived cell
line (LV-1) and subcloned into a pLenti6 D-Topo vector. The resultant clone
was 1017 nucleotides, with a corresponding 338 amino acid sequence (this
ISG20L Mx1
is shown by the single letter code below). They are numbered as indicated
20 20
at the left hand side. The ATG start codon and the TGA stop codon (*),

Relative fold change

Relative fold change
which are boxed and in bold, are at position 1 and 339, respectively. 15 15

in mRNA

in mRNA
1 ATGCTCCGCCTGCCCCTGGCGCTGGCCGCGCTGCTGCTGCGCGCCCTGCGGAGCCCGCTC 10 10

1 M L R L P L A L A A L L L R A L R S P L
5 5
61 AGCGTCCTGCGCTGGAGCCTGGCACACGTCTGGCCCCGGCTGCGGCTGGTGCTGGGGCGA
21 S V L R W S L A H V W P R L R L V L G R 0 0
Control dsDNA dsRNA SeV Control dsDNA dsRNA SeV

121 AGCGGCCCCGGCCCCGGCCCGACCCCCACCAGCGTCAACTACCACTTCACCCGGCGCTGC
41 S G P G P G P T P T S V N Y H F T R R C
Figure 5: Viral infection with Dengue virus-2.
181 AACTACAAGTGCGGCTTCTGCTTCCACACGGCCAAGACCTCCTTCGAGCTGCCCCTGGAG To assess the induction of ISGs in a spreading infection, we infected LV_1 cells
61 N Y K C G F C F H T A K T S F E L P L E with a Dengue-2 viral isolate (+strand ssRNA virus) and performed real-time
241 GAGGCGCGGCGGGGGCTCGCCATGCTGCGGGCGGCGGGTATGGAGAAAATTAATTTTTCA PCR at 24 and 48 hours following infection. Dengue virus infection induced a
81 E A R R G L A M L R A A G M E K I N F S marked upregulation of both viperin and OASL at 24 and 48 hrs, similar to that
301 GGAGGAGAACCATTTCTGACTGATAGAGGAGAATTTGTGGGCAAATTGGTCCAGTTTTGC
seen in Fig4. Interestingly, Dengue infection did not cause an upregulation of
101 G G E P F L T D R G E F V G K L V Q F C MX1, as seen with the extracellular dsRNA stimulation (Fig4).
361 AAGCAAGAATTAAAGTTACCAAGTGTCAGCATTGTGAGCAATGGCAGCTTGATTAGAGAA
121 K Q E L K L P S V S I V S N G S L I R E LV-1 infected with DEVN-2
(MOI=1)
421 AGATGGTTCAAGAATTACGGTGAATACCTAGACATCCTGGCAATTTCTTGTGATAGTTTT 500 Control

Reelative fold change

141 R W F K N Y G E Y L D I L A I S C D S F
400 24 hr
481 GATGAGGAAGTCAATGCTTTAATTGGCCGTGGGCAAGGGAAAAAAAGCCATGTTGAAAAT 300 48hr

in mRNA
161 D E E V N A L I G R G Q G K K S H V E N
200
541
181
CTGCAAAAATTGAGAAAATGGTGTCAAGAATATGGCGTGGCTTTTAAATTAAATTCAGTG
L Q K L R K W C Q E Y G V A F K L N S V
B 100

601 ATTAATAGATTTAATGTGGAAGAAGACATGAATGAACAGATTAAAGCACTGAACCCTGTT 2
201 I N R F N V E E D M N E Q I K A L N P V
0
661 CGTTGGAAGGTATTCCAGTGTCTGCTCATTGATGGGGAGAACGTAGGTGAAAATGCTCTG
ISG20L MX1 OASL Viperin
221 R W K V F Q C L L I D G E N V G E N A L Figure 3: Saltwater crocodile viperin localises to the endoplasmic
721 AGAGAAGCAGAGAAATTTGTTATCAGTGATGAGGATTTTGAACGATTCCTGGATCGCCAC reticulum via its N-terminal domain.
241 R E A E K F V I S D E D F E R F L D R H LV-1 cells were transiently transfected with a crocodile-viperin-pLenti6 (containing Conclusions
an MCherry tag on the N-terminus) and confocal microscopy was performed, -This is the first study to characterise saltwater crocodile viperin, which
781 AGAGATATCTCATGTATAGTGCCAGAGTCAAATCAGAAGATGAAAGATTCATATCTTATT
261 R D I S C I V P E S N Q K M K D S Y L I demonstrating localisation of viperin mainly to the endoplasmic reticulum (ER) contained the same evolutionary conserved regions (amphipathic helix, radical
and to lipid droplets as observed with other viperin proteins. (A) Bodipy stain SAM domain and antiviral C-terminus) as published viperin proteins. Further
841 CTGGATGAATATATGCGTTTTCTGAACTGTAGAAATGGACGAAAGGAGCCTTCCAAATCT
281 L D E Y M R F L N C R N G R K E P S K S
(binds to neutral lipids) (B) Viperin expression (C) Merge image of crocodile work will help characterise the ability of this protein to inhibit native crocodile
viperin pathogens.
901 ATTCTGGATGTTGGTGTTGAGCATGCTATAAAATTCAGTGGATTTGATGAGGAGATGTTT
301 I L D V G V E H A I K F S G F D E E M F A B C -We have shown that saltwater crocodile cells have activated early innate
961 CTGAAGAGAGGAGGAAAATACGTATGGAGTAAAGCAGATATGAAACTAGAATGGTAG immune signalling pathways in response to a viral infection, and that these
321 L K R G G K Y V W S K A D M K L E W * varying according to the stimuli; potentially indicating that the crocodile has
multiple PAMP recognition receptors (ie TLRs, RIG-I like receptors).
References
1. St John et al. Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes. -This research has the potential to improve outcomes within the crocodile
Genome Biol. 2012 Jan 31;13(1):415
2. Helbig and Beard. The role of viperin in the innate antiviral response. J Mol Biol. 2014 Mar 20;426(6):1210-9
industry, possibly decreasing both skin and systemic pathogen-related
problems.