I. Maxadilan insertion into GALV and KoRV-B env generally reduced infectivity, except when inserted after the signal peptide with Linker2 sequences, which allowed infection through native receptors. II. No Maxadilan constructs could infect via PAC1 receptors alone, suggesting a fusion block. III. In cells expressing both native and PAC1 receptors, Maxadilan env constructs showed lower titers, indicating interaction with PAC1 is deleterious to infectivity, likely by blocking fusion.

1. Retargeting gammaretroviruses for
gene delivery using Maxadilan
Specific delivery of curative genes to cell types of choice is an ongoing
challenge in gene therapy. We explore one strategy to re-target a gamma-retroviral
vector to neurons, by inserting a Maxadilan peptide into different regions of Gibbon
Ape Leukemia Virus (GALV) and a newly identified GALV-related koala retrovirus B
(KoRV-B) envelope protein env.
Various Maxadilan-GALV or KoRV-B env constructs were made and tested for
their ability to recognize, bind and mediate entry into PAC1 expressing cells via
binding and infectivity assays. We found that Maxadilan, flanked with ‘hinge’
sequences and inserted right after the signaling peptide of the GALV or KoRV-B env
did not disrupt the function of GALV or KoRVB env to infect susceptible cells that
express their receptors. Insertion of Maxadilan in KoRV-B allowed the env to bind
strongly to PAC1 receptors, but failed to mediate virus entry. Our data suggests that
the use of PAC1 to mediate entry, by KoRV-B env with Maxadilan inserted, is
blocked at the fusion step.
Background
Jan Clement Santiago, Wenqin Xu, Jill Russ & Maribeth V. Eiden
Section on Directed Gene Transfer,
National Institute of Mental Health, Bethesda, MD 20892
Abstract
Results
Conclusion
Methods
I. Maxadilan insertion generally reduces or neutralizes the infectivity of vectors
bearing GALV and KoRV-B env.
II. Maxadilan inserted after the signal peptide and flanked with Linker2 allows GALV
and KoRV-B to infect through their native receptors.
III. The infectivity of Maxadilan env constructs are reduced in susceptible cells
expressing PAC1 receptors.
IV. Since binding assays show that Maxadilan env constructs bind to PAC1, the block to
cell entry through PAC1 must be at the fusion step.
A. Virus particles binding to PAC1 and remaining “stuck” outside the cell could
account for the lower titers of Maxadilan env constructs in susceptible cells
expressing PAC1 receptors compared to cells that do not.
B. Although GALV-Maxadilan-Linker2 enveloped particles are infectious (Table 1),
GALV-MaxadilanV5-Linker2 was not binding to PAC1 (Fig 1). The V5 may be
affecting its functional conformation. We assumed previously that adding the
relatively small V5 tag would not drastically affect its properties. We may have to
compare infectious titers between GALV-Max2 and GALV-Max2-V5.
1
2
3
4
5
• GALV or KoRV-B env vectors with Maxadilan inserted all produced titers at least 1-
log lower than wild type (not shown), or none.
• Among the constructs though, B and E (Maxadilan+Linker2 inserted right after
signaling peptide) has the least detrimental effect on infectivity.
Ø They are still functional in mediating entry through their native receptors.
• No Maxadilan constructs could infect MDTF with PAC1 receptors only.
Ø Maxadilan inserts cannot mediate entry through PAC1.
• B and E are infectious, but their titers are significantly less in MDTF expressing also
PAC1. No titer reduction is seen in MDTF-PAC1 from envelopes without Maxadilan
(see A and D).
Ø Co-expression of PAC1 reduced susceptibility of MDTF cells to B and E,
suggesting the interaction of PAC1 with Maxadilan is deleterious to infectivity.
Gibbon Ape Leukemia Virus envelope Mean
&ters
on
MDTF
with
receptors:
Insert
Loca&on
PiT1
PiT1
&
PAC1
PAC1
A
V5+Linker2
5'
end
a/er
signaling
pep4de
2.60
x
105
5.89
x
105
<
10
B
Maxadilan+Linker2
5'
end
a/er
signaling
pep4de
2.53
x
105
2.10
x
104
<
10
C
Maxadilan+Linker2
replaces
Variable
Region
B
<
10
<
10
<
10
Koala Retrovirus B envelope Mean
&ters
on
MDTF
with
receptors:
Insert
Loca&on
THTR1
THTR1
&
PAC1
PAC1
D
V5+Linker2
5'
end
a/er
signaling
pep4de
3.12
x
104
3.33
x
104
<
10
E
Maxadilan+Linker2
5'
end
a/er
signaling
pep4de
1.78
x
105
7.04
x
103
<
10
F
Maxadilan
near
5'
end
a/er
signaling
pep4de
<
10
<
10
<
10
G
Maxadilan
replaces
Variable
Region
A
<
10
<
10
<
10
H
Maxadilan
replaces
Variable
Region
B1
1.00
x
101
3.12
x
102
<
10
I
Maxadilan
replaces
Variable
Region
B2
5.55
x
102
1.39
x
102
<
10
J
Maxalidan
+
Linker
2
replaces
H
of
PHQ
region
<
10
<
10
<
10
Binding of Env constructs to
MDTF-PAC1
K-MaxV5∆H
K-MaxV5-L2
KB-V5-L2
G-MaxV5-L2
Cellcounts
Intensity of bound probe (FL2-H)
To test if the Maxadilan insert
and PAC1 receptors are
indeed interacting, binding
assays were done. The
Maxadilan inserts were
tagged with V5 to allow for
anti-V5 antibody staining for
the envelopes that bound to
PAC1 receptors.
• KoRV-B V5-Linker2 is the negative control, because MDTF-PAC1 do not express the
corresponding KoRV-B receptor THTR1
• KoRV-B MaxadilanV5-Linker2 & KoRV-B MaxadilanV5∆H bind to MDTF-PAC1
• GALV-MaxadilanV5-Linker2 does not bind to MDTF-PAC1
Tables 2 & 3.
Infectious titers of GALV & KoRV-B envelopes with Maxadilan inserted at various sites
Table 3
GALV
env
inserts
Titers
in
MDTF-­‐PiT1
V5
8.03
x
105
V5
+
Linker1
3.62
x
105
V5
+
Linker2
1.18
x
106
V5
+
Linker3
7.05
x
105
Table 1. Infectious titers of GALV env with V5 peptide tag and linkers inserted
Adding Linker2 produced GALV env
insert construct with the highest titers.
Consequently, most env insertions later
will incorporate Linker2.
References6
Maxadilan1
– a potent 61 amino acid vasodilator peptide isolated from
sand fly salivary glands
– specifically activates the mammalian PAC1 receptor
– 500X more potent than the next most potent PAC1 agonist
GALV (Gibbon Ape Leukemia Virus) env
– widely used in gene therapy for its relative safety and efficacy
– receptor is PiT1 (phosphate transporter 1)
sand fly
Envelopes (env)
Env (envelope protein):
Enveloped Virus
Linker sequences2
– give space for an inserted epitope to “hinge” out and not inhibit the native
conformation of the envelope protein
– based on a published GALV construct
KoRV-B (Koala Retrovirus B) env
– closely related to GALV
– receptor is THTR1 (thiamine transporter 1)
1. Generate insert with overlapping
homologous regions to vector via PCR
2. Expose 5’ overhangs in insert & cut vector
by incubating each with T4 polymerase
• In absence of dNTPs, T4 polymerase have 3’
exonuclease activity that is stopped by adding dCTP
3. Incubate vector and insert together (with
Recombinase A)
• overlapping regions anneal to each other
4. Transform into E.coli
• cell repairs nicks and gaps in recombinant plasmid
Env constructs made via Sequence & Ligation Independent Cloning
1.) Transfect env to 293T cells
+ gag/pol
+ genome
(encodes β-galactosidase)
2.) Harvest viral
supernatant 48 hrs
post transfection
3.) Infect MDTF cells
4.) 48 - 72 hrs post infection,
stain then count β-gal -
expressing cells
Making virus particles for infectivity assays
40x magnification of β-gal transduced
MDTF (blue cells), using KoRVB-V5 env.
Binding assays
MDTF cell
PAC1 receptors
V5 tag
3.) Incubate MDTF-PAC1 with env supernatant for
30 mins at 37˚C
env
Maxadilan
ligand
4.) Probe with anti-V5 1˚ antibody
5.) Probe with 2˚ fluorescent antibody
6.) Detect fluorescing cells with FACS
1.) Transfect V5-tagged env only to 293T cells
2.) Collect supernatant 48 hrs after, concentrate 2X
Env surface unit
signal peptideN terminus variable
region A
variable
region B
1Lerner, E. A., Iuga, A. O., and Reddy, V. B. (2007). Maxadilan, a PAC1 receptor agonist from sand flies. Peptides 28, 1651-1654.
2Bahrami, S., Pagh K.,Ejegod, D., et al (2012). Construction of a Gammaretrovirus with a Novel Tropism and Wild-Type Replication Kinetics Capable of Using Human APJ as Entry Receptor. Journal of virology
86(19): 10621-106272012
variable regions are major determinants of receptor specificity