of 32
*For correspondence:
walther.mothes@yale.edu (WM);
pradeep.uchil@yale.edu (PDU)
Competing interest:
See
page 28
Funding:
See page 28
Received:
20 October 2020
Accepted:
15 June 2021
Published:
05 July 2021
Reviewing editor:
Mark Marsh,
University College London,
United Kingdom
Copyright Haugh et al. This
article is distributed under the
terms of the
Creative Commons
Attribution License,
which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
In vivo imaging of retrovirus infection
reveals a role for Siglec-1/CD169 in
multiple routes of transmission
Kelsey A Haugh
1
, Mark S Ladinsky
2
, Irfan Ullah
3
, Helen M Stone
1
, Ruoxi Pi
1
,
Alexandre Gilardet
1
, Michael W Grunst
1
, Priti Kumar
3
, Pamela J Bjorkman
2
,
Walther Mothes
1
*, Pradeep D Uchil
1
*
1
Department of Microbial Pathogenesis, Yale University School of Medicine, New
Haven, United States;
2
Division of Biology and Biological Engineering, California
Institute of Technology, Pasadena, United States;
3
Department of Internal Medicine,
Section of Infectious Diseases, Yale University School of Medicine, New Haven,
United States
Abstract
Early events in retrovirus transmission are determined by interactions between
incoming viruses and frontline cells near entry sites. Despite their importance for retroviral
pathogenesis, very little is known about these events. We developed a bioluminescence imaging
(BLI)-guided multiscale imaging approach to study these events in vivo. Engineered murine
leukemia reporter viruses allowed us to monitor individual stages of retrovirus life cycle including
virus particle flow, virus entry into cells, infection and spread for retroorbital, subcutaneous, and
oral routes. BLI permitted temporal tracking of orally administered retroviruses along the
gastrointestinal tract as they traversed the lumen through Peyer’s patches to reach the draining
mesenteric sac. Importantly, capture and acquisition of lymph-, blood-, and milk-borne retroviruses
spanning three routes was promoted by a common host factor, the I-type lectin CD169, expressed
on sentinel macrophages. These results highlight how retroviruses co-opt the immune surveillance
function of tissue-resident sentinel macrophages for establishing infection.
Introduction
Retroviruses cause cancer and immunodeficiencies (
Blattner, 1999
). Once retroviruses establish viral
reservoirs, it is difficult to eliminate infection as retroviral genomes are permanently integrated into
host DNA. Despite the clinical relevance of early processes by which incoming retroviruses establish
infection by navigating complex host tissue architecture
en route
to their first targets, little is known
about these events (
Haase, 2010
;
Haase, 2011
;
Haase, 2014
). Retroviruses like the human immuno-
deficiency virus (HIV-1) can enter through the vaginal and rectal mucosa during sexual transmission,
orally via milk during mother-to-child transmission, and subcutaneously and intravenously through
needle stick injections during drug use and blood transfusions (
Haase, 2010
;
Friedland and Klein,
1987
). Most murine leukemia virus (MLV) transmission in mice occurs vertically from dam-to-pup via
ingestion of virus-containing milk through the gastrointestinal (GI) tract. MLV transmission can also
occur parenterally between male mice during infighting and via the venereal route between infected
male and female mice (
Portis et al., 1987
;
Buffett et al., 1969a
). Entry via different routes requires
retroviruses to navigate diverse host tissue architecture and overcome barriers for successful infec-
tion (
Uchil et al., 2019a
;
Pfeiffer, 2010
;
Fackler et al., 2014
). Whether retroviruses exploit common
host factors across these transmission routes remain to be clarified.
We have previously used MLV as a model retrovirus to understand how retroviruses establish
infection in mice through the lymph or blood following subcutaneous and intravenous delivery,
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RESEARCH ARTICLE
respectively (
Sewald et al., 2015
;
Uchil et al., 2019b
). We found that sentinel macrophages lining
blood/lymph-tissue interfaces such as the subcapsular sinus in lymph nodes or the marginal zones in
the spleen function to filter out incoming retroviruses from
Sewald et al., 2015
;
Uchil et al., 2019b
.
The ‘fly-paper’-like activity of sentinel macrophages has been observed for various incoming viruses
and pathogens (
Kastenmu
̈
ller et al., 2012
;
Iannacone et al., 2010
;
Junt et al., 2007
). The frontline
position of sentinel macrophages allows them to orchestrate downstream innate, cell-mediated, and
humoral immune responses to incoming pathogens in the lymph and blood (
Uchil et al., 2019a
;
Uchil et al., 2019b
;
Honke et al., 2012
;
Martinez-Pomares and Gordon, 2012
;
Perez et al.,
2017
). These macrophages naturally express the I-type lectin Siglec-1/CD169, which specifically
interacts with sialosides present on retroviral membranes (
Sewald et al., 2015
;
Izquierdo-
Useros et al., 2012
;
Puryear et al., 2013
). CD169 expression allows sentinel macrophages to cap-
ture retroviruses and limits their dissemination (
Uchil et al., 2019b
). However, retroviruses like MLV
and HIV-1 exploit their CD169 to promote infection of target lymphocytes that sample antigens cap-
tured by sentinel macrophages (
Sewald et al., 2015
;
Uchil et al., 2019b
;
Pi et al., 2019
). Whether
the observed roles for CD169
+
macrophages following subcutaneous and intravenous delivery are of
any relevance for natural mother-to-offspring transmission when viruses enter via the GI tract has
remained unknown.
Identifying portals of entry such as Peyer’s patches (PP) in underdeveloped intestines can be very
challenging, given the complexity, functional diversity, and length of the GI tract, which can measure
>7 cm even in neonatal mice (
Torow et al., 2017
). Here, we developed a whole-body biolumines-
cence imaging (BLI)-based approach to illuminate areas where MLV concentrates to traverse into the
gut tissue from the lumen. We implemented BLI by developing a series of MLV-based reporter
viruses to enable observation of specific stages of the retrovirus life cycle in vivo, including viral parti-
cle flow, entry into the cytoplasm, first infection events, and spread. We first validated this system by
testing its ability to uncover new insights into previously studied subcutaneous and intravenous
transmission routes (
Sewald et al., 2015
;
Uchil et al., 2019b
;
Pi et al., 2019
). Second, BLI permitted
temporal tracking of various steps of virus infection for orally administered retroviruses along the GI
tract as they traversed the lumen through the PP to reach the draining mesenteric sac (mSac). Finally,
we show that capture and acquisition of lymph-, blood-, and milk-borne retroviruses spanning three
routes was promoted by a common host factor, CD169, expressed on sentinel macrophages. Our
results highlight how retroviruses co-opt the immune surveillance function of tissue-resident sentinel
macrophages for establishing infection. Understanding these events will inform design of improved
prophylactic strategies that target prevention of virus acquisition and establishment of infection.
Results
Generation of reporter viruses to enable visualization of individual
stages of retrovirus infection in vivo
We established a BLI-directed approach for studying individual stages of retroviral infection in vivo
by strategically inserting reporters into unique sites in the MLV genome (
Figure 1A
). To track virus
particle flow, we generated bioluminescent virus particles by introducing nanoluciferase (Nluc) into
the proline-rich region (PRR) of the MLV envelope (Env). Nluc-Env-tagged virus particles produced
using a tripartite plasmid system (encoding Nluc-tagged MLV Env, MLV Gag-Pol, and MLV-LTR)
exhibited 200 times more luciferase activity per virus particle (0.2 RLU/virion) compared to viruses
generated using the full-length MLV genome (0.001 RLU/virion) (
Figure 1B
). To monitor virus entry
into the cytoplasm of cells, we fused firefly luciferase (Fluc) to the C-terminus of MLV Gag (MLV
Gag-Fluc Env
WT
) and exploited the ATP-dependence of Fluc activity, which is restricted to the host
cell cytoplasm in vivo. This strategy ensured that Fluc activity was exhibited when both detergent
(Triton X-100) and ATP were present (
Figure 1C
). A Gag-Fluc-labeled virus in which MLV envelope
was replaced by the fusion-defective SFFV gp55 envelope (MLV Gag-Fluc Env
FD
) served as a nega-
tive control. To monitor single-round virus transduction, we utilized a replication-defective virus gen-
erated by co-transfecting a dual BLI and GFP reporter (pMIG-Nluc-IRES-GFP) in conjunction with
MLV Gag-Pol and Env (
Ventura et al., 2019
). In addition, we generated red-shifted reporter viruses
encoding Antares, which is a bioluminescence resonance energy transfer (BRET) reporter that ena-
bles superior deep-tissue sensitivity over Nluc in vivo (
Chu et al., 2016
). Finally, to permit
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longitudinal monitoring of progressing infection, we generated replication-competent MLV reporter
viruses by introducing a shortened internal ribosome entry site (IRES), 6ATRi, to drive Nluc expres-
sion downstream of viral Env (MLV 6ATRi-Nluc) (
Ventura et al., 2019
;
Alberti et al., 2015
;
Logg et al., 2001
). This strategy enabled bi-cistronic expression of Env and Nluc in infected cells.
U3 R U5
U3 R U5
gag pol env
5’-LTR
3’-LTR
Env-Nluc
Gag-Fluc
Cytoplasmic
Nluc
luminescent
virus
particle
1. Flow
2. Entry
3. Infection
4. Spread
replication
defective
(
D
gag-pol-env)
replication
competent
Proline
rich region
(PRR)
6ATRi
Fluc activity
(O
2
+ATP)
A
- ATP
+ ATP
1
10
0
1
10
1
1
10
2
1
10
3
RLU (Firefly)
0.1% BSA/PBS
Triton X-100
p=0.0013
ns
Fluc activity of MLV
Gag-Fluc viruses
HEK293
Virions
p65 Gag
p30 Gag
B
C
D
F
ul
l
-
l
eng
t
h
1×10
-3
1×10
-2
1×10
-1
1×10
0
RLU
(
Nl
uc
)
RLU/infectious virus
FrMLV Env-Nluc
p<0.0001
0.2
0.001
Single-
round
luminescent
infected
cells
cytoplasmic
access
Monitor:
WT 6ATRi-
Nluc
0.0
0.5
1.0
1.5
Relative Infectivity
I
nf
e
ct
iv
ity
relative
t
o WT
p=0.100
1
0.75
Figure 1.
Construction and characterization of reporter viruses for visualizing individual stages of the retrovirus life cycle in vivo. (
A
) A scheme denoting
the location of inserted reporters into unique sites in the Friend murine leukemia virus (FrMLV) genome. (1) To monitor particle flow, Nluc was inserted
in-frame into the proline-rich region (PRR) of envelope. (2) To monitor virus entry into the cytoplasm of cells, Gag-Firefly luciferase (MLV Gag-Fluc;
D
Pro-
Pol) was employed as Fluc requires both oxygen and ATP in the presence of substrate D-luciferin for its activity. (3) To monitor infection, Nluc was
expressed in the cytoplasm using a viral LTR-driven construct (single round;
D
gag-pol-env). (4) To monitor spreading infection, Nluc was expressed from
a short internal ribosome entry site (IRES) element (6ATRi) downstream of the envelope gene that resulted in replication-competent virus. (
B
) A graph
comparing brightness (relative light units [RLU]) per infectious unit of full-length FrMLV Env-Nluc viruses with single-round MLV generated using gag-
pol, env-Nluc, and LTR-GFP constructs. Infectious units for both viruses were estimated using DFJ8 cells followed by flow cytometry. RLU per infectious
virion was determined by measuring Nluc activity in sedimented virus. The error bars denote standard deviations between triplicate samples. p values
derived from Student’s t-test. (
C
) A graph showing detectable Fluc activity in intact (0.1% BSA/PBS) or lysed (0.1% BSA/PBS, Triton X-100) MLV Gag-Fluc
virions in the presence or absence of ATP and substrate D-luciferin (15 mg/mL in PBS). The error bars denote standard deviations between triplicate
samples. p values derived from Student’s t-test; ns: not significant. (
D
) A graph comparing released infectivity of replication-competent FrMLV Nluc
reporter virus (6ATRi-Nluc) and WT FrMLV (WT). Viruses were produced by transfecting equal amounts of virus-encoding plasmids into HEK293 cells in
triplicate. 48 hr post-transfection released infectivity in culture supernatants was determined using DFJ8 target cells followed by flow cytometry with
antibodies to MLV GlycoGag to enumerate infected cells. Released infectivity of WT FrMLV was set to 1. The error bars denote standard deviations
between triplicate samples. Western blot analyses of sedimented virus from culture supernatants and HEK293 cell lysates for a similar experiment as
shown in the graph above using antibodies to MLV Gag.
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Infectivity measurements revealed that MLV 6ATRi-Nluc was
~
60% as infectious as wild-type MLV
(
Figure 1D
).
BLI-driven characterization of blood-borne retrovirus infection
MLV infection via the intravenous route (retroorbital [r.o.]) is a well-studied infection route. We previ-
ously showed that r.o.-delivered MLV is first captured by CD169
+
metallophilic marginal zone macro-
phages before infecting follicular, marginal zone, and transitional B cells (
Uchil et al., 2019b
). We
revisited this route at the whole-body level by applying these new reporter viruses. We challenged
mice r.o. with MLV Env-Nluc reporter viruses. BLI-driven virus tracking immediately after challenge
showed that MLV rapidly reached both liver and spleen within 30 s after administration and accumu-
lated predominantly at the spleen after passing through the liver (2–3 min) (
Figure 2A, B
,
Figure 2—
video 1
). This was consistent with the spleen being the main blood-filtering organ in mice. Interest-
ingly, the decaying luminescence over time (>30 min) in the spleen was revived when Nluc substrate
(furimazine) was readministered (
Figure 2—figure supplement 1
). This indicated that viruses remain
captured at the spleen, and the exhaustion of substrate contributed to the decay in the signal. We
next investigated virus entry into the host cell cytoplasm by utilizing MLV Gag-Fluc-tagged viruses
with wild-type (Env
WT
) or fusion-defective Env (Env
FD
). Inocula were equalized by measuring the lucif-
erase activity (relative light units [RLU]) in detergent-lysed viral preparations (
Figure 2C
). Mice chal-
lenged r.o. with MLV were monitored at 5 min, 30 min, and 1 hr post infection (hpi) using BLI. In
contrast to animals infected with fusion-defective MLV, we observed Fluc signal emerging at the
spleen (1 hpi) in animals infected with reporter viruses carrying wild-type MLV envelope
(
Figure 2D, E
; p=0.0061). Taken together, our data indicated that blood-borne MLV was filtered
rapidly at the spleen within 2–3 min and entered the cytoplasm of cells by 60 min after capture.
We next infected mice with WT FrMLV or MLV 6ATRi-Nluc reporter virus and compared infection
levels at 7 days post infection (dpi). In vivo infectivity of MLV 6ATRi-Nluc virus was reduced in com-
parison to WT FrMLV (
Figure 2F
). This was not unexpected based on the reduced released infectiv-
ity of MLV 6ATRi-Nluc in vitro (
Figure 1D
), and the known effect of genomic reporter insertions on
retrovirus fitness (
Logg et al., 2001
). To visualize the first round of infected cells and virus spread at
the whole animal level, we challenged mice with single-round MLV reporter virus (pMIG-Antares) as
well as replication-competent reporter MLV and monitored replication dynamics using BLI every 2–3
days over the course of 2 weeks (
Figure 2G
). In contrast to the decline of luminescent signal
observed with single-round Antares-encoding MLV, luminescent signal in organs infected by MLV
6ATRi-Nluc increased over time, indicative of fresh rounds of infection (
Figure 2G, H
). We observed
infection in auricular and inguinal LN in addition to the spleen and liver. As time progressed, there
was gradual decline of luminescent signal in mice infected with MLV 6ATRi-Nluc (
Figure 2G, H
). This
decline was not due to loss of the Nluc reporter as RT
2
-qPCR analyses of viral RNA isolated from
blood and spleen indicated that the ratio of Gag to Nluc did not significantly change during the
observed course (3–14 dpi) of infection in mice (
Figure 2I
). The Gag:Nluc ratio was similar to that
seen from RNA isolated from DFJ8-infected cells (30 hpi). These data suggested that Nluc reporter
was retained within the viral genome throughout the observed course of infection. Therefore, the
decline in the signal is consistent with the immune control of MLV infection in C57BL/6J (B6) mice,
observed in previous studies (
Sewald et al., 2015
;
Uchil et al., 2019b
;
Pi et al., 2019
;
Sewald et al., 2012
;
Nowinski, 1976
). Taken together, these results demonstrate the utility of our
bioluminescent reporter viruses in monitoring particle flow, capture, cytoplasmic entry, transduction,
and subsequent virus spread following intravenous infection of mice. The validation of this reporter
system also set the stage for applications to other infection routes.
BLI-driven characterization of lymph-borne retrovirus infection
Intrafootpad (i.f.p.) infection is widely used to study subcutaneous (s.c.) infection and to model anti-
gen trafficking to draining lymph nodes via lymphatics (
Sewald et al., 2016
;
Chatziandreou et al.,
2017
). The draining lymph node for i.f.p. infection is the popliteal lymph node (pLN)
(
Iannacone et al., 2010
;
Junt et al., 2007
;
Gonzalez et al., 2010
). Previous studies using multi-pho-
ton intravital microscopy indicated that incoming lymph-borne viruses were captured by CD169
+
sentinel macrophages, resulting in accumulation at the subcapsular sinus of the pLN within a few
minutes following viral challenge (
Sewald et al., 2015
;
Iannacone et al., 2010
;
Junt et al., 2007
;
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0:30
0:30
1:30
2:30
3:30
3.0
2.5
2.0
1.5
x10
5
4:30
5:30
6:30
7:30
C
MLV Gag-Fluc Env
wt
MLV Gag-Fluc Env
FD
Virus particle flow: FrMLV Env-Nluc (r.o.) (min:s)
In vitro
Fluc activity of
input virus (lysed
)
0 1 2 3 4 5 6 7 8
1
10
6
5x10
6
Time (min)
Flux (p/s)
Liver
Spleen
n = 3
Virus particle flow
FrMLV Env-Nluc (r.o.)
B
5 min 30 min 60 min
0.0
5.0
10
4
1.0
10
5
1.5
10
5
2.0
10
5
Spleen Flux (p/s)
Cytoplasmic entry (spleen)
MLV Gag-Fluc (r.o.)
p=0.0061
ns
ns
E
WT 6ATRi-
Nluc
10
3
10
4
10
5
10
6
10
7
# infected cells/spleen
In vivo
infectivity
7 dpi (r.o.)
F
A
1
3
5
7
9
1
10
-3
1
10
-2
1
10
-1
1
10
0
1
10
1
1
10
2
1
10
3
1
10
4
Flux (p/s)
relative to day 1 infection
MLV pMIG-Antares
Spleen
Auricular LN
Inguinal LN
1
3
5
7
9
11
13
15
dpi
MLV 6ATRi-Nluc
H
n=3
n=8
4
7
8
11
12
13
14
Replication
competent
MLV 6ATRi-Nluc
0.5
1.0
1.5
dpi: 4
7
8
11
12
13
14
3 5
6
7
8
11
Longitudinal Infection Dissemination (r.o.)
Single round
MLVpMIG-Antares
2
3
5
7
9
11
5
dpi: 1
2
6
7
9
10
6
4
0.6
x10
5
G
spleen
inguinal
LN
auricular
LN
Liver
x 10
4
DFJ8
D3
D7
D10
D14
D14
spleen
0.90
0.95
1.00
1.05
1.10
1.15
1.20
In vivo
Nluc stability
MLV 6ATRi-Nluc (r.o.)
C
t
M
L
V gag :
C
t
Nluc
(cDNA from viral RNA)
ns
ns
blood
I
min:
5
30
60
MLV Gag-Fluc
Env
FD
MLV Gag-Fluc
Env
wt
3000
4000
5000
6000
D
MLV Gag-
Fluc Env
wt
MLV Gag-
Fluc Env
FD
0
50
100
150
RLU (Firefly
)
Figure 2.
Real-time visualization of individual steps of retrovirus infection in vivo during retroorbital challenge. (
A
) A scheme showing the path of
murine leukemia virus (MLV) Env-Nluc particles after intravenous retroorbital (r.o.) challenge. Furimazine (Nluc substrate)-administered mice were
challenged retroorbitally with 1

10
5
IU of MLV Env-Nluc and monitored using IVIS at 30 s (s) intervals. Images from one representative experiment
from three biological replicates (n = 3) are shown. (
B
) Quantification of MLV Env-Nluc bioluminescent signal in the spleen and liver, displayed as photon
Figure 2 continued on next page
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Murooka et al., 2012
). CD169
+
macrophages did not get infected but rather promoted
trans
-infec-
tion of susceptible lymphocytes such as innate-like B1-a cells and CD4
+
T cells during the first round
of infection. B-1a cells then spread the virus to naı
̈
ve B-2 cells via virological synapse during the
expansion phase of the infection (
Pi et al., 2019
;
Sewald et al., 2015
). To gain further insight into
behaviors of incoming virus particles in this well-studied route, we performed BLI imaging of incom-
ing virus particle flow from the administration site to the target organ by infecting mice i.f.p. with
MLV Env-Nluc. Incoming viruses accumulated rapidly at the pLN, with detectable signal occurring
within 1 min 30 s pi (
Figure 3A, B
,
Figure 3—video 1
). This was indicative of lymph flow-mediated
dissemination of MLV to pLN and was consistent with previous multiphoton microscopy studies
(
Sewald et al., 2015
). However, we observed that most of the incoming virus particles localized to
the injection site at the footpad (
Figure 3A, B
). Quantification of virus particle accumulation in the
footpad and pLN, displayed as photon flux (photons/s), revealed that virus accumulation in the foot-
pad was over 200-fold higher than that in the pLN. This observation remained constant for the entire
imaging window. Even when virus eventually accumulated in the pLN, plateauing at
~
9 min pi, the
level of virus in the footpad remained
~
150-fold above that of the pLN (
Figure 3A, B
). Thus, our
imaging analyses showed that only a small fraction of incoming viruses surpass tissue barriers includ-
ing collagen fibrils, muscle tissue, and antigen-capturing cells at the footpad to reach the draining
site.
We next explored the location where viruses fused and gained access to the host cell cytoplasm.
We challenged mice with MLV Gag-Fluc Env
WT
or MLV Gag-Fluc Env
FD
via the i.f.p. route and moni-
tored these events by BLI. Bioluminescent signal in the mouse footpads infected with MLV Gag-Fluc
Env
FD
minimally increased over time (
Figure 3C, D
). In contrast, luciferase activity in footpads chal-
lenged with MLV Gag-Fluc Env
WT
significantly increased over time, indicating progressive virus
access to cell cytoplasm in vivo (
Figure 3C, D
). We observed that fusion-competent viruses gained
access to the host cell cytoplasm in the footpad as early as 3 min post-challenge (
Figure 3C, D
,
Fig-
ure 3—figure supplement 1A, B
). Despite accumulation of virions in the pLN within minutes follow-
ing challenge (
Figure 3A, B
), we were unable to detect Fluc activity in the first 40 min of the
Figure 2 continued
flux (photons/s) in each organ following r.o. challenge from the experiment described in (
A
). Curves represent mean flux over time, and error bars
denote standard deviation. (
C
) A plot showing Fluc activity (relative light units [RLU]) associated with MLV Gag-Fluc Env
WT
or MLV Gag-FLuc Env
FD
(fusion defective) inocula after lysis. Mice were subsequently inoculated with 2

10
4
RLU of indicated unlysed virus preparations. Error bars denote
standard deviations from mean. (
D
) Representative images of MLV Gag-Fluc Env
WT
entry into the target cell cytoplasm at the spleen observed via BLI at
the indicated time points following r.o. infection. Mice challenged similarly with MLV Gag-Fluc Env
FD
served as negative controls for determination of
background signals. Images from one representative experiment from 4 to 7 biological replicates are shown. (
E
) Quantification of MLV cytoplasmic
entry in the spleens (photon flux/s) of mice (n = 4–7) following r.o. infection with MLV Gag-Fluc Env
WT
or MLV Gag-Fluc Env
FD
at indicated time points
from the experiment as in (
D
). p values derived from non-parametric Mann–Whitney test; ns: not significant; bars represent mean values and error bars
denote standard deviations from mean. (
F
) A plot showing total number of infected cells per spleen of B6 mice (n = 3–4) challenged with 5

10
5
IU of
FrMLV (WT) and replication-competent reporter FrMLV (MLV 6ATRi-Nluc) at 7 days post infection (dpi) (r.o). Infected cells in single-cell suspensions of
spleen were determined using flow cytometry with antibodies to MLV GlycoGag. Horizontal lines represent mean values. (
G
) Mice were infected r.o.
with 5

10
5
IU of replication-competent reporter FrMLV (MLV 6ATRi-Nluc or single-round MLV pMIG-Antares) as a control for tracking dissemination of
transduced infected cells. Dissemination of viral infection was monitored via bioluminescence imaging (BLI) at the indicated time points. Infected
organs are indicated in the schematic. Images from one representative experiment are shown. (
H
) Quantification of virus dissemination in indicated
organs is displayed as photon flux in mice infected with MLV 6ATRi-Nluc or MLV pMIG-Antares for the experiment described in (
G
). Antares, n = 3;
6ATRi-Nluc, n = 8. Images in (
G
) are from one representative experiment. Symbols represent mean values, and error bars denote standard deviations
from mean. Scale bars that accompany the images showing luminescence denote radiance in photons per second per square centimeter per steradian
(p/s/cm
2
/sr). (
I
) In vivo longitudinal stability of Nluc within the MLV 6ATRi-Nluc genome was determined via RT
2
-qPCR in B6 mice. RNA extracted from
blood or spleen of mice (n = 5) r.o. infected with MLV 6ATRi-Nluc (2

10
6
IU) at the indicated time points. Infected spleens were harvested at 14 dpi.
RNA from DFJ8-infected cells (MLV 6ATRi-Nluc; 2

10
5
IU) and uninfected cells served as positive and negative controls for PCR. RT
2
-qPCR was
performed on viral RNA extracted from infected samples using primers for Nluc and MLV gag. Stability of Nluc within the viral genome was determined
by ratios of gag:Nluc C
t
values over time.
The online version of this article includes the following video and figure supplement(s) for figure 2:
Figure supplement 1.
Stable retention of murine leukemia virus (MLV) Env-Nluc in the spleen demonstrated by readministration of furimazine substrate
after retroorbital (r.o.) challenge.
Figure 2—video 1.
Murine leukemia virus (MLV) particle flow through the liver and spleen in the retroorbital infection route.
https://elifesciences.org/articles/64179#fig2video1
Haugh
etal
. eLife 2021;10:e64179.
DOI: https://doi.org/10.7554/eLife.64179
6 of 32
Research article
Microbiology and Infectious Disease