arrested spread of vesicular stomatitis virus infections in vitro depends on interferon-mediated...

Arrested Spread of Vesicular StomatitisVirus Infections In Vitro Depends onInterferon-Mediated Antiviral Activity

Vy Lam, Karen A. Duca, John Yin

Department of Chemical and Biological Engineering, 1415 Engineering Drive,University of Wisconsin, Madison, Wisconsin 53706-1607;telephone: 608-265-3779; fax: 608-262-5434; e-mail: [email protected]

Received 27 October 2004; accepted 18 January 2005

Published online 15 April 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20467

Abstract: A quantitative understanding of the innateimmune response will enable its recruitment againstemerging, poorly characterized, or weaponized viralpathogens. To gain insights into how the innateresponses can limit viral spread, we used quantitativefocal infections to study how the spread of recombinantvesicular stomatitis viruses (VSV) onbabyhamster kidney(BHK) and delayed brain tumor (DBT) cell monolayers isaffected by innate cellular antiviral responses.We observ-ed that rates of infection spread correlated with one-stepgrowth rankings for four ectopic VSV strains: N1, N2, N3,and N4. However, this correlation was lost for M51R, arecombinant VSV mutant that lacks the ability to shut-offhost gene expression. In BHK cells, M51R spread at two-thirds the rate of the recombinant control virus, XK3.1,even though their one-step growth was comparable. InDBT cells, M51R infections failed to spread beyond thesite of inoculation. Addition of anti-interferon antibodyrestored M51R spread and one-step growth to wild-typelevels. Interestingly, the antibody enhanced the spread ofwild-type virus but not its growth. These results suggestthatwhile the rate of viral spread generally correlateswiththe rate of viral growth, the induction of cellular antiviralactivities can be in some cases, the overriding factor inboth spread and growth. In summary, focal infectionsenabled us to visualize and quantify how viral spreadwasinhibited by cellular antiviral activities. This study demon-strates a mechanism for quantifying how innate cellularresponses can mitigate infection spread in vitro.� 2005 Wiley Periodicals, Inc.

Keywords: vesicular stomatitis virus; virus–host interac-tions; interferon signaling; antiviral responses; infectionspread

INTRODUCTION

When viruses infect their host organisms they activate a

network of defensive reactions, within and between diverse

cells, that together define a host immune response. Activa-

tion of this response involves multiple cell types that

communicate through the secretion and sensing of diverse

soluble signaling molecules and modulators of cell growth

and differentiation. The overall response may be divided into

a nonspecific innate component, activated within minutes to

hours of the initial virus–host encounter, and a pathogen-

specific adaptive component that can require days or weeks to

develop. Specific activation of the adaptive response, largely

through the development and application of viral vaccines,

has contributed to the control of many diseases including

smallpox, polio, measles, hepatitis, and influenza. Despite

much effort, however, relatively little progress has been made

toward guiding the innate arm of the immune response. An

improved understanding of the innate responsewill enable its

recruitment towardminimizing the effects on human health of

emerging, poorly characterized, or weaponized viral patho-

gens. Further, because it influences the adaptive response,

advances in the controlled recruitment of the innate response

may also impact the development of more effective vaccines.

The innate response is mediated by cells of the immune

system, including monocytes, natural killer cells, and den-

dritic cells, as well as nonimmune cells. In their initial

encounter with a virus, both immune and nonimmune cells

respond by synthesizing a diversity of soluble signaling

molecules, or cytokines, including interferons (IFN) and

interleukins. These cytokines, upon binding to specific

receptors of other immune and nonimmune cells, trigger a

cascade of additional responses, including the activation of

further cytokines and the induction of antiviral mechanisms,

cell proliferation, cell differentiation, or cell death. The ex-

treme complexity of interacting cell types, cytokines, and

cellular function creates a significant barrier to elucidating

how the innate immunity develops as an integrated response

to a primary infection.

To facilitate advances and gain insights into how innate

responses in nonimmune cells work as a system to inhibit

viral spread, we are developing in vitro experimental ap-

proaches. Here we extend a focal infection method (Duca

et al., 2001) tomonitor the dynamics bywhich signaling from

�2005 Wiley Periodicals, Inc.

Karen A. Duca’s present address is Virginia Bioinformatics Institute(0477), Virginia Polytechnic Institute and State University, 1880 PrattDrive, Building XV, Blacksburg, VA 24061.

Correspondence to: John Yin

Contract grant sponsors: National Science Foundation; National Instit-

utes of Health; Merck Research Laboratories

Contract grant numbers: EIA-0331337; 5T32 GM08349

virus-infected cells bring about an antiviral state in nonin-

fected cells. The focal infection method, and a discussion on

how it compares to the traditional approach of quantifying

infection spread using plaque assay, was discussed in detail

by Duca et al. (2001). Briefly, in focal infections, cell mono-

layers are covered with an agar overlay prior to virus inocu-

lation (Fig. 1A). The overlay restricts the dispersion of the

virus particles and thus maintains a spatial segregation

between infected and noninfected cells. Over time, succes-

sive cycles of viral replication and spread to susceptible

noninfected cells drive spread of the infection across the

cell monolayer. Digital imaging and analysis of the areas of

infection at various times post inoculation yield a profile

of viral propagation in the cell monolayers. Induction of

antiviral activity in the cell monolayers can be inferred from

an inhibition of viral propagation, visualized as a decrease

in the rate of viral propagation. Quantitation of antiviral-

response mediated inhibition of infection spread reveals an

aspect of virus–host interactions that is not observable in

traditional in vitro studies, such as one-step growth experi-

ments (Fig. 1B), where homogeneously infected cell mono-

layers are used for the quantitation of viral replication and

cellular responses. Focal infections allow us to observe the

integration of cellular antiviral responses in the monolayer

over many cycles of viral replication while the responses of

homogeneously infected cell monolayers are limited by the

rate of viral replication and cytopathogenesis. Like in vivo

model systems, focal infections allow us to observe how

infections induce intercellular signaling and activate antiviral

activities that can restrict or arrest infection spread. Impor-

tantly, all antiviral signaling and effector activities in focal

infections are initiated from infected cells of the system

Figure 1. Experimental paradigms of focal infection and one-step growth experiments. (A) Focal infection experiment is used to quantify the extent of

infection spread as a function of time. The agar overlay localizes the virus particles to the site of infection and spatially segregates the infected cells from

noninfected cells. The duration of this experiment is not limited by the rate of viral pathogenesis but by the availability of host cells to support viral propagation.

(B)One-step growth experiment is used to quantify the expression of viral products or cellular antiviral proteins over the course of one infection cycle, i.e., from

the time of inoculation to the time of cell death. The duration of this experiment was determined by how quickly viral replication kills an infected cell.

794 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 90, NO. 7, JUNE 30, 2005

without the need for exogenous treatments. However, in

contrast with in vivo systems, focal infections lack the three-

dimensional structure of invivo tissues and can only visualize

the innate antiviral responses of one or a subset of selected

cell types.

In this study, we used focal infections to investigate how

the presence of IFN, induced by the localized addition of

vesicular stomatitis virus (VSV), affected viral propagation

on monolayers of murine delayed brain tumor (DBT) cells.

Delayed brain tumor cells respond to IFN and can be activat-

ed to resist the replication of encephalomyocarditis and

herpes simplex viruses (Zerial et al., 1982). As a control, we

also used baby hamster kidney (BHK) cells, which are

commonly used to study VSV replication processes and

are not known to produce or respond to IFN signaling

(Andzhaparidze et al., 1981; Kramer et al., 1983; Nagai et al.,

1981; Otsuki et al., 1979).

Vesicular stomatitis virus is a well-characterized virus that

is used extensively in medical research. Experimentally, it is

commonly used as a model virus for studying the evolution

(Domingo et al., 1996; Holland et al., 1982) and replication

strategies of important pathogens such as Ebola, rabies,

measles, and respiratory syncytial virus (Ball et al., 1999;

Lenard, 1999; van den Pol et al., 2002). Further, recombinant

VSV strains are being tested for use as oncolytic agents in

vivo (Balachandran andBarber, 2000; Fernandez et al., 2002)

and as vaccination vectors for many viruses including wild-

type VSV (Flanagan et al., 2001), HIV (McKenna et al.,

2003; Ramsburg et al., 2004), papillomavirus (Roberts et al.,

2004), and influenza (Roberts et al., 1998, 1999). Wild-type

VSVinfections are endemic in the US every 5 to 10 years and

cause foot-and-mouth disease-like lesions on infected live-

stock (Letchworth et al., 1999). Vesicular stomatitis virus

carries a single-stranded negative sensed RNA genome that

encodes for five viral proteins, ordered: 30—nucleocapsid

(N)—phosphoprotein (P)—matrix (M)—glycoprotein (G)—

large protein (L)—50. During viral replication the position ofthe genes, relative to the 30 terminus, dictates its level of

expression. Hence, during wild-type VSV replication the

expression levels of viral mRNAs and proteins rank:

N> P>M>G>L. High level of N expression in infected

cells is critical for the replication of viral genomic RNA and

production of viral progeny (Arnheiter et al., 1985; Patton

et al., 1984). Six VSV strains were used in our experiments:

N1, N2, N3, N4, XK3.1, andM51R. The N1, N2, N3, and N4

strains are recombinant viruses where the nucleocapsid (N)

gene is translocated from position one, next to the 30 terminus

of the genome, to downstream positions two, three, and four,

respectively. Translocation of the N gene further away from

the 30 terminus reduces expression of N protein and thereby

lowers the rate of viral replication in cells, as determined by

one-step growth experiments (Wertz et al., 1998). The

different replication rates of the N1 through N4 viruses

allowed us to examine the effect of viral replication on viral

spread. M51R is a recombinant strain of VSV that contains a

methionine-to-arginine mutation at position 51 of the matrix

protein (Black et al., 1993; Kopecky et al., 2001). While

expression of M protein normally shuts down expression of

host genes without affecting viral gene expression, theM51R

mutation inactivates M protein-mediated shut down of host

(Black and Lyles, 1992; Coulon et al., 1990). The growth and

propagation rates of M51R are compared to those of the

control recombinant virus, or the recombinant wild-type

virus, XK3.1. We observed that the absence of host shut-off

rendered M51R extremely sensitive to IFN-mediated anti-

viral activities in DBT cells. Not only was M51R replication

inhibited to a greater extent than XK3.1, M51R was not able

to propagate at all on the DBT monolayers.

MATERIALS AND METHODS

Cell and Virus Culture

Murine delayed brain tumor (DBT) cells were obtained from

Dr. J. Fleming (University of Wisconsin–Madison) and

grown as monolayers at 378C in a humidified atmosphere

containing 5% CO2. DBT growth medium was Dulbecco’s

Modified Eagle Medium (Celgro; Fisher Scientific, Pitts-

burgh, PA) containing 10% newborn calf serum (NCS;

Hyclone Laboratories, Logan, UT), 4 mMGlutamax I (Glu;Gibco, Invitrogen Corporation, Carlsbad, CA), and 15 mMHEPES (Sigma, St. Louis, MO). Baby hamster kidney

(BHK) cells were obtained from Dr. I. Novella (Medical

College of Ohio) and grown under the same environment as

the DBT cells. BHK growth medium was Minimal Essential

Medium with Earle’s salts (Celgro; Fisher) containing 10%

fetal bovine serum (FBS; Hyclone) and 2 mM Glutamax I(Glu; Gibco). Both BHK and DBT cells were subcultured

approximately every third day. For subculture, monolayers

were rinsed with Hanks Balanced Salt Solution (HBSS;

Fisher), incubated in 0.025% trypsin/26 mMEDTA (Fisher)for 5 min, dispersed through mixing, and then re-plated in

fresh growth medium at 1:15 (DBT) or 1:30 (BHK) dilution.

No antibiotics were used and cells were subcultured no more

than 4 months to minimize the effects of cell senescence.

Viability of cell populations, as determined by trypan blue

exclusion, at the time of experiments always approached

100%.

N1–N4 strains of vesicular stomatitis virus (VSV) were

obtained fromDr.G.Wertz (University ofAlabamaSchool of

Medicine). Their genomic structures are as follows: 30-N-P-M-G-L-50 (N1), 30-P-N-M-G-L-50 (N2), 30-P-M-N-G-L-50

(N3), 30-P-M-N-G-L-50 (N4). Vesicular stomatitis virus

strains XK3.1 and M51R were obtained from Dr. D. Lyles

(Wake Forest University School of Medicine). XK3.1 is

the control virus, or the recombinant wild-type, for M51R

which has amethionine-to-argininemutation at position51of

its matrix protein. Each strain of virus was grown on BHK

cells at multiplicity of infection (MOI) of 0.01 plaque-

forming units (PFU) per cell. Infected BHK cells were

incubated in infection medium (MEM/Glu/2% FBS) for 20–

24 h. At the end of the incubation period, the medium was

harvested and passed through a 0.2 mm filter. The filtered

solution was aliquoted and stored at �908C until use.

LAM ET AL.: QUANTITATION OF VSV FOCAL SPREAD IN VITRO 795

One-Step Growth Infection of Cell Monolayers

Cells were harvested, resuspended in growth medium, and

plated into six-well plates at a density of 5� 105 cells per

well (in 2 mL of culture medium). Plated cells were returned

to the incubator and allowed to grow overnight. Two repre-

sentative cell monolayers were harvested and counted to give

an approximate number of cells per well. Each monolayer

was then incubatedwith 200mLof virus inoculum (MOI of 3)

for 1 h to allow virus adsorption. The plates were rocked

gently every 20 min to encourage even virion distribution on

the monolayers. After the adsorption period, the monolayers

were rinsed twice with 1 mL of phosphate buffered saline

(PBS; Sigma) and then placed under 2 mL of infection

medium for incubation. Samples of the medium, at 200 mLeach, were taken from each well at 2, 3, 4, 6, 8, 10, and 20 h

post-inoculation. Samples were kept frozen at �908C until

quantification by plaque assay.

In experiments where interferon and anti-interferon anti-

body were used, mouse type 1 IFN (I1258; Sigma) or a

mixture of rat anti-mouse IFN alpha (I7662-07;United States

Biological, Swapscott, MA) and rat anti-mouse IFN beta

(I7662-10A, United States Biological) antibodies were

diluted and combined with the virus solution prior to ino-

culation. Interferons were diluted to 50 units of activity per

200 mL of inoculum while the antibodies were used at

12.5 units of anti-IFN alpha and 25 units of anti-IFN beta.

The same units of IFNs and antibody were added to the 2 mL

of post-inoculation incubation medium.

Plaque Assays

BHK cells were plated into six-well plates and cultured to

90% confluence. Culture medium was removed from each

well and replaced with 200 mL of serially diluted virus

samples. Inoculated monolayers were returned to the incu-

bator for 1 h of adsorption time. The plates were rocked

gently every 20 min. At the end of the adsorption time, the

inoculum was removed from each sample and then replaced

with 2 mL of agar overlay. The agar overlay consisted of

0.6% weight/volume (w/v) agar (Agar Nobel; Difco Labora-

tories, Livonia, MI). 5-Bromo-20-deoxyuridine or BrdU

(B5002; Sigma) was added, at 100 mg/mL, to the overlay of

N3 and N4 infected samples to reduce cell proliferation and

enhance plaque visibility (personal communication with

Dr. Gail Wertz). Without BrdU, N4 plaques on BHK mono-

layers were not visible by 24 h post infection. The presence of

BrdU did not increase the titer count of N3 infections (data

not shown). Following agar addition the plates were allowed

to cool at room temperature for 30 min, returned to the

incubator for 24 h, and then each samplewas fixed with 2 mL

offixative for 3 hr at room temperature. The fixative consisted

of 4% (w/v) paraformaldehyde (VWR International, West

Chester, PA) and 5% (w/v) sucrose (Sigma) in 10 mM

phosphate buffered saline (PBS; Sigma) at pH 7.4. The agar

overlay was then removed and each sample was rinsed twice

with 2mL of PBS. Gentian violet diluted inmethanol (0.01%

(w/v); Sigma) was used, at 1 mL each, to stain the samples

and the plaques were counted. Virus titers were the mean of

two or three independent replicates and the error bars

represent the standard error of the mean (SEM). Statistical

analysis of the N3 and N4 one-step growth profiles was done

using the paired t-test. At each time point, the difference

between each pair of N3 and N4 titers were normalized to the

corresponding mean of the N4 titer, i.e., (titerN4 � titerN3)/

meanN4, to account for the exponential increase in data

variance over the course of the experiment.

Focal Infection of Cell Monolayers

Cellswere harvested and plated into six-well plates at 5� 105

cells per well (in 2 mL of culture medium). Plated cells were

returned to the incubator for overnight growth. The next day,

the culture medium was suctioned from each well and

replaced with 4 mL of agar overlay. The agar overlay was

prepared byfirst combining agar powder (AgarNobel;Difco)

at 0.6% (w/v) with nanopure water (approximately 10% of

the final volume), autoclaving the mixture at 1218C for

20 min, and then combining it with infection medium (90%

of the final volume) that had been heated to 428C. After agaraddition, the plates were left at room temperature for 30 min

to allow the overlay to solidify. They were then returned to

the incubator for at least 60 min of recovery time before

inoculation.

To focally inoculate the cell monolayer under the agar

overlay, a virus deposition reservoir was made by punching a

hole in the center of the overlay using the tip of a Pasteur

pipette. The plug of agar inside the tip of the pipette was

subsequently removed by applying gentle suction through

the pipette. Five mL of virus inoculum at 1.6� 107 PFU/mL

was added to this reservoir to infect the exposed cells, giving

an estimatedMOI of 20. The inoculated plates were returned

to the incubator until the predetermined fixation times.

Where applicable, mouse type 1 IFN was diluted and

combined with the virus solution to give 50 units of IFN

activity per 5 ofmLof inoculum.Anti-IFN alpha and anti-IFN

beta antibody mixtures were diluted to yield 12.5 and 25

units, respectively.

Fixation and Immunocytochemistry

Representative focal infected monolayers were fixed at

selected time points with 3 mL of fixative per monolayer.

After 3 h of fixative exposure, the agar overlay was removed

and themonolayerswere rinsed twicewith 2mL/well of PBS.

They were stored in PBS at 48C until immunofluorescence

labeling.

Virus distributions were visualized by indirect immuno-

fluorescence labeling of VSV-glycoprotein. Fixed mono-

layers were washed once with 1 mL of PBS/0.1% (w/v)

saponin (PBS/SAP) and once with 1 mL PBS/SAP/5% natal

calf serum (NCS) for 10 min each. Monolayers were then

blocked for 20 min with 1 mL of PBS/SAP/NCS/0.2% (w/v)

bovine serum albumin (PBS/SAP/NCS/BSA). A primary

monoclonal murine antibody against VSV-G (V5507;


Sigma) was diluted 1:1000 in PBS/SAP/NCS, added at

0.5 mL to each monolayer, and incubated overnight at 48C.The following day the monolayers were washed twice with

PBS/SAP, once with PBS/SAP/NCS, and then blocked for

20 min with PBS/SAP/NCS/BSA. Cy3-conjugated donkey

anti-mouse F (ab0)2 secondary antibody (Jackson Immunor-

esearch Laboratories, West Grove, PA) was diluted 1:300 in

PBS/SAP/NCS and added to the monolayers at 0.5 mL each

and incubated for 60 min at room temperature (RT). Each

monolayer were then washed twicewith PBS/SAP to remove

unbound antibody, put under 2 mL of PBS, and stored at 48Cuntil imaging.

Image Collection, Processing, and Quantification

Images of the labeled monolayers were acquired using a

Nikon TE300 inverted epifluorescent microscope equipped

with a Nikon mercury light source, a Prior XYZ translation

stage driven byMetamorph 6.0 software (Universal Imaging,

Downington, PA), and a monochrome SensSys 4.0 cooled

CCD camera. The location and extent of infection was

manually identified in each well and the number of micro-

scope fields necessary to fully image the infected area at 4�magnification was estimated (4 to 120 fields). The system

was then programmed tomove the stage consecutively across

the entire infected area, acquire an image at each position,

and finally, combine the individual images into a single

montage.

Prior to quantification, all images from an experimentwere

pre-processed, using the same scaling factors, to enhance

contrast and re-sized to be accommodated easily on a com-

puter screen. Image preprocessing was done using Adobe

Photoshop 7.0 and quantification of the infection areas was

done using MATLAB 6.1 (MathWorks, Inc. Natick, MA).

The background illumination was estimated and subtracted

from each image before a threshold level was manually set to

optimize the inclusion of signal positive pixels across all

images of an experiment. In each image, neighboring signal

positive pixels were linked as one object and the total area

of this object, in pixels, was reported for the image. The

infection radius for each image was calculated from the area

of this object where the radius equals (area/p)1/2. The lengthof each radius was then converted from pixels to millimeters

using the scale bar on the image. At each time point, three to

four replicates were quantified and the mean was plotted as a

function of time. The standard error of themeanwas included

at each data point as error bars. Statistical analysis of the data

was done using the student’s t-test. In all cases, the null

hypothesis was that there was no difference between the

infection radii of control and treated samples.

RESULTS

Focal Propagation Rates of VSV Ectopic MutantsCorrelated With One-Step Growth Kinetics

The growth kinetics of the four ectopic VSV strains, N1, N2,

N3, and N4 on BHK cells, were investigated using one-step

growth experiments. Translocation of the viral N gene from

position one, thewild-type position, to downstream positions

two, three, and four on the viral genome reduced the pro-

duction of infectious progeny (Fig. 2A).Virus titers at all data

points beyond the initial 3-h lag time ranked N1>N2>N3>N4. In general, N1 yielded infectious titers that were

approximately 10-fold higher than N2, while N2 produced

about 5-fold higher titers than N3. The observed differences

between N3 andN4were relatively small, with N3 titers only

about 25% higher than N4 titers.

The spread of the N1 through N4 infections on BHK

monolayers were characterized using focal infections. Re-

presentative micrographs of focal infections on BHK mono-

layers at 72 h post infection are shown in Figure 3. Thevisible

rings of fluorescence intensity reveal the distribution of viral

glycoproteins and thus the extent of the infections. Estima-

tion of the infection radii from the micrographs showed that

N1, N2, N3, and N4 focal infections on BHK monolayers

increased linearly in time (Fig. 2B). Linear regression of the

mean infection radii yielded focal propagation rates that

ranked N1>N2>N3>N4. The ranking of the propagation

rates of N1, N2, N3, and N4 viruses correlated to the ranking

of their infectious progeny titers in one-step growth ex-

periments. The mean replication titers and fitted slopes of

propagation for N1–N4 on BHK are summarized in Table I.

On DBT cells, translocation of the VSV N gene also

reduced viral replication in one-step growth experiments

(Fig. 2C). However, the ranking on DBT cells was switched

for the N3 and N4 strains: N1 >N2>N4>N3. In general,

all viruses replicated more slowly and to lower titers on DBT

cells than on BHK cells (Fig. 2C, A). For example, at 8 h post

infection the number of infectious progeny produced per cell

by the virus strains on BHKs ranged from 44 to 2000 while

they ranged from 0.3 to 24 on DBTs (Table I).

Focal infections on DBT cells showed an overall different

pattern of spread as compared with infections on BHK cells.

While velocities of infection spread (slope of infection radius

versus time) on BHK cells were constant over the course of

the experiment (Fig. 2B), the velocities on DBT cells de-

creased two- to sixfold (Fig. 2D). Despite the different

expansion pattern and growth kinetics onDBT cells, the focal

propagation ranking of the VSV strains on DBT cells (N1>N2>N4>N3) correlated with their replication ranking

in one-step growth experiments. In short, the correlation

between focal propagation rates and one-step growth infec-

tious titers of the different VSV strains was preserved

independent of the host cell type.

Growth and Propagation Kinetics of XK3.1and M51R on BHK Were Different Fromthe N-gene Ectopic Strains

On BHK cells, XK3.1 (recombinant wild-type) and M51R

(pointmutant) strains replicated at similar rates and produced

titers that were between N1 and N4 strains (Fig. 4A, Table I).

However, the replication kinetics of XK3.1 and M51R were

more delayed than theN1 andN4 strains. At 8 h post infection


XK3.1 and M51R titers were 10-fold lower than their titers

at 20 h post infection, in contrast with the 3-fold difference

for the N-gene ectopic strains. Focal infections of XK3.1 on

BHK cells spread with a radial velocity that was lower than

N1 but higher than N4 (Fig. 4B, Table I), as one might expect

based on its growth kinetics. M51R, on the other hand,

propagated more slowly than N4.

M51R Failed to Propagate on DBT Cells

As was observed with the N-gene ectopic strains, XK3.1 and

M51R also replicated to lower titers on DBT cells than on

BHK cells (Fig. 4A, C; Table I). XK3.1 titers were 10-fold

lower while M51R titers were a 100-fold lower, resulting in

M51R titers that were comparable to those of N3. Surpris-

ingly, while XK3.1 focal infections spread to an extent

between N1 and N3, M51R failed to propagate outside of its

inoculated area (Fig. 4D). Photomicrographs of control

XK3.1 and M51R focal infection samples are shown in

Figure 5 (Fig. 5A, B). Detectable labeling of M51R glyco-

protein (M51R-G) within the site of inoculation, which was

one millimeter in radius, at 24 and 48 h post infection

(Fig. 5B) confirmed the successful inoculation of the cells by

M51R. However, by 72 h, there was no visible labeling of

M51R-G in the samples and phase contrast inspection of

the samples showed confluent monolayers of healthy cells

surrounding the inoculation sites.

Inhibition of Interferon Signaling EnhancedViral Spread on DBT Cells

To investigate whether interferon-signaling-induced anti-

viral activity contributes to the reduced propagation of the

six VSV strains on DBT cells, we added IFN neutralizing

antibody to the inoculum. Focal infections in the presence of

Figure 2. One-step growth and focal spread ofN1 throughN4VSVectopicmutants onBHKandDBTcells. In one-step experiments, bothBHK (A) andDBT

(C) cellswere inoculated atMOI of 3. The titer of viral progeny producedwas normalized by the number of inoculated cells. Each data point represents themean

of three replicates and error bars show the SEM.Differences betweenN3 andN4 titers onBHKandDBTcellswere significant beyond 99%as determined by the

paired t-test (analysis described inMaterials andMethods). In focal infections, BHK (B) and DBT (D) cells in the viral reservoir were inoculated at MOI of 20.

The same virus concentration (PFU/mL) was used for the inoculation of one-step and focal experiments; the difference in the per-cell MOI results from the

differences in inoculumvolume and number of exposed cells. Infection radii of theVSV strains were averaged over two (BHK,B) and four (DBT,D) replicates.

Differences in infection radii between N3 and N4 strains on DBT cells at 72 and 96 h were also significant beyond 99%.


anti-IFNenhanced the propagation of allVSVstrains (Fig. 6).

By 72 h, the increase in infection radii ranged from one (N4,

XK3.1) to slightly more than two (N3) millimeters. Most

notably, in the presence of the anti-IFN antibody M51R was

able to spread beyond 2 mm, comparable to the N3 and N4

control infections. A further intriguing result from this ex-

periment was the effect of the antibody onN3 propagation. In

the presence of anti-IFN antibody, N3 propagation was

increased to levels between N2 and N4 which resulted in the

propagation ranking of N1>N2>N3>N4 on DBT cells.

The phenotype of sequentially reduced viral propagation

in correlation with N gene translocation was apparently

Table I. One-step growth titers and infection spread of VSV strains on BHK and DBT cells.

BHK DBT

Titer PFU/cell

Spread rate mm/hrb

Titer PFU/cell Infect. radius mmc

8 HPIa 20 HPIa 8 HPIa 20 HPIa 96 HPIa

N1 2000� 200 4400� 600 73 24� 3 2200� 110 5.3� 0.1

N2 200� 29 750� 200 592 1.4� 1.5 450� 48 4.6� 0.06

N31 58� 7 460� 90 572 0.3� 0.03 35� 5 3.7� 0.06

N41 44� 2 300� 47 50 0.5� 0.06 59� 13 4� 0.08

XK3.13 280� 40 2400� 430 54 10� 0.5 190� 80 4� 0.1

M51R3 170� 43 2000� 400 41 0.7� 0.1 10� 4 0d

aHPI¼ hours post infection.bThe rate of infection spread on BHK monolayers were calculated from linear regression.cThe largest infection radii (96 h post infection) on DBT monolayers are shown since infection spread was arrested by 48 h.dThere were no visible fluorescence labeling of M51R glycoprotein on DBT monolayers beyond 48 h.1Differences between N3 and N4 one-step and AER profiles in both cell lines are significant to 99%.2Difference between the spread rate of N2 and N3 on BHK monolayers are not significant.3Differences between XK3.1 and M51R one-step and AER profiles in both cell lines are significant to 99%.

Figure 3. Representative fluorescencemicrographs of N1, N2, N3, and N4 focal infections on BHKmonolayers at 72 h. Viral glycoproteins (VSV-G) on cell

monolayers were visualized using indirect immunofluorescence labeling. Antibody pair used was mouse anti-VSV-G and Cy3TM conjugated donkey anti-

mouse.


recovered by the inhibition of IFN signaling, causing the

ectopic strains to rank onDBT cells as they did onBHK cells.

INTERFERON SIGNALING INHIBITED XK3.1 ANDM51R PROPAGATION IN DBT CELLS

The addition of IFN and antibody to the inoculum and

incubation medium did not affect the one-step growth of

XK3.1 on DBT cells (Fig. 7A). Replication titers of XK3.1

for all treatment conditions were comparable to control

infections. However, the propagation of XK3.1 in focal

infections was decreased by IFN and increased by anti-IFN

antibody (Fig. 7B). Representative photomicrographs of

XK3.1 infections with IFN and antibody are shown in

Figure 5C and 5E. In the presence of IFN, XK3.1 propagated

at a slower rate between 24 and 48 h and stopped propagating

earlier than control infections. While the infection radii of

control XK3.1 infections reached a plateau by 72 h post

infection (Fig. 7B), the plateau was established by 48 h in the

presence of IFN. When anti-IFN antibody was added, infec-

tion radii continuously expanded over the course of 96 h.

In contrast to XK3.1, the one-step growth of M51R on

DBT cells was susceptible to IFN and anti-IFN antibody

treatments (Fig. 7C). M51R titers were reduced by half with

the addition of IFN and increased fourfold with the addition

of anti-IFN antibody. Titers in samples treated with both IFN

and antibody (as a mixture) were equivalent to the samples

treated with antibody alone. Dramatic effects were observed

in focal infections for M51R where the addition of IFN

essentially eliminated cell infection (Fig. 7D). At 24 h post

infection, very few cells in monolayers inoculated with

M51R and IFN labeled positive for viral glycoprotein

(Fig. 5D). With the addition of anti-IFN antibody M51R

infections propagated to extents that were comparable to

those of control XK3.1 infections (Fig. 5F and 7D).

DISCUSSION

We investigated the in vitro one-step growth and spreading

dynamics of N1, N2, N3, N4, XK3.1, and M51R strains of

VSV on BHK and DBT cells. Viral growth was quantified

using single-generation, one-step growth experiments and

Figure 4. One-step growth and focal spread ofXK3.1 andM51RonBHKandDBT cells. BHK (A) andDBT (C) cells were inoculatedwithXK3.1 andM51R

viruses atMOI 3 for one-step growth studies. Themean of three replicates, and associated SEM, are shown at each data point. No datawere collected for XK3.1

andM51R one-step titers at 10 h post infection. BHK (B) andDBT (D)monolayerswere inoculatedwithXK3.1 andM51R viruses atMOI 20 for focal infection

experiments. (*) There was no visible labeling of M51R-G in M51R infections at 72 h. The mean and SEM of two replicates are shown.


compared with measurements of multigeneration virus

spatial spread in focal infections (Fig. 1). Results from the

studies of VSVectopic strains N1 through N4 indicated that

the rate of infectious progeny production in one-step growth

experimentswas a key determinant of the rate of focal spread.

Rankings of strains from one-step growth were preserved in

rankings based on focal spread rates, although rankings for

two of the strains, N3, andN4, depended on the host cell. This

correlation is consistent with previous observations where

faster replicating strains of VSV, including the N1 through

N4 viruses, formed larger plaques in plaque assays (Wagner

et al., 1963; Wertz et al., 1998).

Interestingly, the correlation between one-step and focal

growth did not apply for VSV strains XK3.1 and M51R,

indicating an opportunity for focal infections to reveal

aspects of virus-cell interactions that are not fully reflected

in one-step growth curves. On BHK cells, one-step growth

curves of XK3.1 and M51R were similar, supporting pre-

vious observations that the M51R mutation had negligible

effect on the intrinsic replicative ability of the virus (Black

et al., 1993;Kopecky et al., 2001).However, the rate ofM51R

focal spread was significantly slower than XK3.1. Moreover,

on DBT cells, M51R grew in one-step experiments but did

not spread in focal infections. The inhibition ofM51R spread

in focal infections on DBT and BHK hosts likely arose from

the failure of thevirus to shut off cellular gene expression. For

the DBT hosts, the inability of M51R mutants to inhibit the

host expression of INF-b reported by others (Ahmed et al.,

2003; Ferran and Lucas-Lenard, 1997) is consistent with our

observations that IFN addition reduces yields in one-step

infections (Fig. 7C), and that anti-IFN can enhance and

rescue M51R growth in one-step and focal infections,

respectively (Fig. 7C, D).

The potentiating effect of anti-IFN antibodies on XK3.1

focal spread but not one-step growth (Fig. 7A, B) suggests

that there is an integrating or priming aspect to IFN signaling

in the focal infections. While IFN signaling may not have

an immediate effect on the replication cycle of XK3.1 in

infected cells, it can apparently induce and reinforce the

antiviral activities in the neighboring noninfected cells,

hindering XK3.1 spread in focal infections. A similar con-

clusion was reached by Francoeur et al. in their study of a

Figure 5. Representative fluorescence micrographs of XK3.1 and M51R focal infections on DBT monolayers. VSV glycoprotein was labeled with mouse

primary antibody followed by Cy3 conjugated goat anti-mouse. Samples were fixed for labeling and imaging at 72 h post infection. The two exceptions are the

M51R control infection,which shows infection at 48 h, and theM51R infectionwith IFN,which shows infection at 24 h. Therewas novisible labeling ofM51R-

G in M51R control infections by 72 h and M51R infections with IFN by 48 h.


‘‘plaque interferon positive’’ phenotype of VSV mutants

(Francoeur et al., 1980). These mutants were defective in

their ability to inhibit host protein synthesis in infected cells,

allowing for the induction of IFN and a subsequent arrest in

plaque expansion.

Perhaps the most interesting result was the subtle yet

notable switch in the focal-spread ranking for the ectopic N1

through N4 strains on DBT cells in the presence or absence

of anti-IFN (Fig. 6). In the presence of anti-IFN, the strains

spread with the same ranking as they did on BHK hosts,

N1>N2>N3>N4, presumably reflecting the attenuation

one would expect from sequential translocation of the

nucleocapsid gene away from the 30 terminus. However, in

the absence of antibody, the switch in the propagation

rankings of N3 and N4 suggests that differences between the

strains give rise to differences in the level of IFN induction or

in the susceptibility of the strains to IFN-mediated inhibition.

The N3 andN4 strains, encoding gene orders P–M–N–G–L

and P–M–G–N–L, respectively, both have the gene for the

matrix (M) protein at the second position. Because of the

central role the matrix protein plays in the shut off of host

functions, including IFN responses, and the relatively early

and high level of M expression expected for strains N3 and

N4, relative to strains N1 or N2, one might have anticipated

that N3 and N4 would show minimal growth differences in

the presence and absence of anti-IFN. This was not the case.

We have initiated experimental and modeling studies to

better elucidate potential mechanisms for these and other

strain-dependent differences in IFN induction.

Although IFN exhibited prominent inhibitory effects on

viral propagation and replication in DBT cells, other host

antiviral activities likely also contributed to the one-step and

focal infection dynamics. We measured 100-fold lower titers

for all VSV strains grown onDBT cells, relative to their titers

on BHK cells. Possible causes for the reduced replication on

the DBT cells include IFN-independent antiviral pathways

such as calcium-mediated nitric oxide production and Toll-

like receptor activation. DBT cells may also have an intrin-

sically lower availability of accessible or active ribosomes,

nucleotides, or amino acids. Induction of IFN-independent

antiviral pathwayswould also explain the lower rate ofM51R

spread, relative to XK3.1, on BHK cells since BHKs are not

known to produce nor respond to IFNs during virus infections

(Andzhaparidze et al., 1981; Kramer et al., 1983; Nagai et al.,

1981; Otsuki et al., 1979). These reports were confirmed by

our own experiments where interferon treatment of BHK

cells did not reduce VSV growth (results not shown).

As one would expect, the rate of infection spread in focal

infection is mediated by the agar overlay which dictates

the diffusion rates of virus particles and cellular signaling

molecules. In general, as the concentration of agar increases

the size of the infections and the rate of infection spread

decreases. However, we do not expect that the reported

phenotypes, i.e., the relative ranking of the rate of infection

spread for the different VSV strains and the arrest of infection

spread by DBT cell monolayers, are dependent on the

properties of the agar overlay. These phenotypes have been

consistently observed in focal infection experiments that

utilize different types of agar (Duca et al., 2001) and at

different concentrations (results not shown).

Focal infections offer a potential systems biological view

of the innate antiviral responses in a cell population and their

effects on viral propagation in vitro. Preserved in each focal

infection sample are the diverse virus–host interactions that

are necessary for the cell monolayers to arrest viral pro-

pagation. These include the infection-mediated induction of

intercellular signaling events, inhibition of cellular responses

by viral factors, up-regulation and activation of cellular

antiviral effector proteins and ultimately, the arrest of viral

propagation. The approach contrasts with more established

reductionist approaches where cellular antiviral responses

are investigated as individual signaling or effector pathways,

and the focus tends to be on the isolation and characterization

of one or a few components of the system. The distinctive

nature of focal infection experiments is highlighted by

the propagation profiles of theVSV strains onDBT cells. The

gradual decrease in viral propagation suggests that the

establishment of the antiviral state in the DBT monolayers

was time dependent and required the propagation of inter-

cellular signaling events, particularly ones that activated the

IFN network.

In summary, we have shown how VSV spread in focal

infections is affected by the competition between viral repli-

cation and cellular antiviral responses. Our results suggest

that while the intrinsic rate of viral replication is an important

factor in determining the rate of viral spread, the degree of

induction of cellular antiviral activities can be a dominating

factor. Hence, an improved understanding of virus-mediated

Figure 6. Inhibition of interferon signaling enhanced viral propagation on

DBT cells. Bar plot shows the extent of propagation for the six VSV strains

on DBT cells with anti-IFN antibody (AIFN) and without (Ctrl) at 72 h post

infection. Antibody was combined with the virus inoculum, at 37 total

neutralizing units per 5 mL inoculum, prior to infection. Data points were

calculated from four replicate samples. Increase in infection radii in the

presence of the antibody was significant beyond 99% for all virus strains.

*Control infections with M51R showed no labeling of viral glycoprotein

protein by 72 H post infection.


antiviral activities may affect the application of viruses in

oncolytic therapies, gene therapies, and the development of

effective vaccines.

We thank Kwang-il Lim and Kristen Stauffer for their technical

assistance and Dr B. Edward Fulton Jr. for his helpful suggestions. We

are also grateful to Drs GailWertz and Douglas Lyles for providing the

recombinant vesicular stomatitis viruses.

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