1. ACKNOWLEDGEMENTS
We would like to thank J. Sodroski and M.L. Linial for reagents,
the NaConal Science FoundaCon, the M.J. Murdock Charitable
Trust, and the SeaGle University College of Science and
Engineering Dean’s Office for the funding of this work.
REFERENCES
1. C. Meiering and M.L. Linial. 2001. Clin. Microbiol. Rev. 14:165–
176.
2. A. Gessain, et. al. 2013. J. Virol. 435:187–199.
3. C. Stenbak, et. al, J Virol. 2014. 88(2): 982–991.
4. G.A. Engel and L. Jones-­‐Engel. Am. J. Primatol. 2012. 74(6):
543-­‐50.
5. D.G. Gibson, et. al. 2009. Nat. Methods. 6:343-­‐345.
FUTURE DIRECTIONS
• Complete construcCon of infecCous clone of SFVsqu
• Transfect SFVsqu clone into mammalian cells to study
the viral life cycle
FIGURE 3. A) Plasmid map of SFVsqu. The reconstructed
genome is composed of a 2.6 kb pUC19 vector and
fragments ranging from 300 bp to 6 kb. B) Mechanism of
Gibson reacCon used to clone the SFVsqu genome.
Fragments with complementary ends are sealed in a three
step process mediated by an exonuclease, polymerase, and
ligase. C) SchemaCc of intended use of SFVsqu plasmid. The
SFVsqu genome can be transfected into mammalian 293T
cells where cellular machinery will express the viral
proteins. Virus parCcles will then self-­‐assemble and be
released from the cells. These virus parCcles can be used to
study SFVsqu in subsequent experiments.
MONKEYING
AROUND
WITH
VIRUSES:
CONSTRUCTION
OF
A
SIMIAN
FOAMY
VIRUS
INFECTIOUS
CLONE
Cooper K. Hayes, Israel Lopez, and Carolyn R. Stenbak
Department of Biology, SeaGle University, SeaGle, Washington 98122
WHY STUDY NEW WORLD MONKEY FOAMY VIRUSES?
FIGURE 1. Geographic distribuCon of representaCve old world and new world
monkeys.
FIGURE 2. SchemaCc of outcomes of foamy virus infecCons in humans. OWM SFV can establish
persistent infecCons in humans, but NWM SFV do not.
METHODS
2.6 kb
300 bp
1 kb
4 kb
6 kb
pUC19 vector
14,105 bp
RESULTS/DISCUSSION
T5 exonuclease chews back 5’ ends
Strands anneal, polymerase fills in gaps
Ligase seals nicks
B
A
New World
Primates
Old World
Primates
VS
Figure 4. Gel electrophoresis of
SFVsqu genome fragments.
Fragments were PCR amplified
and verified before incubaCon
with the Gibson assembly mix.
New World Monkeys
Old World Monkeys
Lifelong Persistent InfecCon
InfecCon is Cleared
HOW?
INTRODUCTION
Ladder
Vector
Fragment1
Fragment2
Fragment3
Fragment4
SFVsqu Genome Fragments
kb
10
3.0
1.0
0.5
C
Foamy viruses (FV) are among the most ancient vertebrate RNA
viruses (1). FVs infect a wide range of hosts, including primates, horses,
and cats, but do not cause any disease in their natural hosts (1). Simian
foamy viruses (SFV) have been found to be zoonoCcally transmissible to
humans, sekng up lifelong, persistent infecCons (2). To date, the vast
majority of research on human FV infecCon has focused on Old World
Monkey (OWM) strains of SFV, which infect a group of primates including
chimpanzees, macaques, and baboons. By comparison, liGle is known
about human infecCon by New World Monkey (NWM) strains of SFV (3).
Recent work in the field has taken an interest in the zoonoCc
transmission of SFV from NWMs to humans. PopulaCons that cohabitate
with or spend large amounts of Cme with primates are at the highest risk
for transmission (4). A recent study surveyed primatologists for SFV
infecCon and found that while some humans have detectable levels of
anCbody to NWM SFV, indicaCng exposure to the virus, there is no
evidence of persistent infecCon (5). This finding is in contrast to human
infecCon by OWM SFV, which does persistently infect humans.
Understanding how the human immune system combats infecCon with
NWM SFV will shed light on immune mechanisms that could be used to
combat other retroviral infecCons.
In order to study NWM SFV infecCon of human cells, we have
begun construcCon of an infecCous clone of the squirrel monkey strain of
SFV (SFVsqu). By fusing together pieces of the SFVsqu genome previously
isolated, we will be able to transfect mammalian cells that will in turn
produce virus parCcles. ConstrucCon of the whole genome is carried out
using molecular cloning techniques, including a Gibson assembly
reacCon. The individual fragments of the genome are PCR amplified with
ends complementary to the adjacent fragments (Figure 3A). These
products are then incubated with a Gibson reacCon enzyme mix
containing a polymerase, T5 exonuclease, and ligase. This isothermal,
one-­‐tube reacCon seals together the fragments into a circular piece of
DNA (Figure 3B) (5).
GeneraCng Virus ParCcles
Expression à Packaging à Release
• All four SFVsqu genome fragments have been PCR-­‐
amplified.
• The vector backbone fragment has been successfully
PCR-­‐amplified.
• Fragment sizes have been verified using gel
electrophoresis