New mRNAs Are Preferentially Translated
during Vesicular Stomatitis Virus Infection
Zackary W. Whitlow, John H. Connor and Douglas S. Lyles
J. Virol. 2008, 82(5):2286. DOI: 10.1128/JVI.01761-07.
Published Ahead of Print 19 December 2007.
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JOURNAL OF VIROLOGY, Mar. 2008, p. 2286–2294
0022-538X/08/$08.00⫹0 doi:10.1128/JVI.01761-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 5
New mRNAs Are Preferentially Translated during Vesicular
Stomatitis Virus Infection䌤
Zackary W. Whitlow,† John H. Connor,‡ and Douglas S. Lyles*
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
Received 11 August 2007/Accepted 4 December 2007
erentially translated. Understanding the mechanisms behind
preferential translation of viral mRNAs is critical for understanding viral replication. Furthermore, cellular mechanisms
controlling translation are often elucidated by studying translation during viral infection.
During VSV infection, host gene expression is rapidly inhibited by the matrix (M) protein. The M protein inhibits host
gene expression at multiple levels, including transcription (1, 2,
4, 13), transport of mRNA to the cytoplasm (12, 17, 37, 38),
and translation (2, 27, 32, 43). Previous experiments have
shown that host translation is inhibited at the initiation step (7,
27) and is likely due to modification of the cap-binding eukaryotic initiation factor 4F (eIF4F) (8, 9, 11). However, it seems
paradoxical that translation of host mRNAs would be inhibited
while translation of viral mRNAs proceeds, since VSV mRNAs
are structurally similar to host mRNAs. Yet in cells infected
with VSV, as host protein synthesis is inhibited, viral protein
synthesis becomes predominant (8, 9, 29, 32, 43, 45). The goal
of the experiments presented here was to determine why viral
mRNAs are translated during the time that translation of host
mRNAs is inhibited.
Several viruses have been shown to allow preferential translation of viral mRNAs through the use of cis-acting sequences.
For example, picornavirus mRNAs contain cis-acting internal
ribosome entry sites that recruit the translation machinery (21,
34, 35). We showed previously that the preferential translation
of VSV mRNAs is independent of cis-acting sequences in viral
mRNA (46). The data presented here show that the timing of
mRNA synthesis relative to infection is involved in controlling
translation in VSV-infected cells. We found that in vitro-transcribed mRNA introduced into VSV-infected cells by transfection after infection was resistant to VSV-induced inhibition of
translation, while translation of mRNA transfected into cells
Vesicular stomatitis virus (VSV) is a member of the rhabdovirus family and is widely studied as a model of negativesense single-stranded RNA viruses. Like many negative-strand
viruses, VSV replicates in the cytoplasm of infected cells, and
viral mRNAs are transcribed from the viral genome by the
viral RNA-dependent RNA polymerase (RDRP). VSV transcription produces five mRNAs that encode the five major viral
proteins. These mRNAs are similar in structure to host
mRNAs. Their 5⬘ ends contain 2⬘-O-methylated adenosine
capped by 7-methyl guanosine linked by 5⬘-5⬘ triphosphate (22,
30, 31, 39, 40, 44). VSV mRNAs also have a 3⬘ poly(A) tail that
is similar in length to that of cellular mRNAs (16, 19, 20). The
synthesis of VSV mRNAs, including the 5⬘ and 3⬘ end modifications, is accomplished entirely in the cytoplasm by the viral
RDRP (23).
Translation of VSV mRNAs, and of all viral mRNAs, is
dependent on the host cell translation machinery. During virus
infection, the cellular translation machinery is often modified,
leading to a decrease in synthesis of host proteins, while viral
protein synthesis increases. Many viruses, such as picornaviruses, influenza viruses, and VSV, are thought to inhibit host
protein synthesis in order to suppress cellular antiviral responses (28). Viruses have developed a variety of mechanisms
to inhibit host protein synthesis while viral mRNAs are pref-
* Corresponding author. Mailing address: Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC
27157. Phone: (336) 716-4237. Fax: (336) 716-7671. E-mail: dlyles
@wfubmc.edu.
† Present address: Laboratory of Viral Diseases, National Institute
of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
‡ Present address: Department of Microbiology, Boston University
School of Medicine, Boston, MA 02118.
䌤
Published ahead of print on 19 December 2007.
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During vesicular stomatitis virus (VSV) infection, host protein synthesis is inhibited, while synthesis of viral
proteins increases. VSV infection causes inhibition of host transcription and RNA transport. Therefore, most
host mRNAs in the cytoplasm of infected cells were synthesized before infection. However, viral mRNAs are
synthesized throughout infection and are newer than preexisting host mRNAs. To determine if the timing of
appearance of mRNAs in the cytoplasm affected their translation during VSV infection, we transfected reporter
mRNAs into cells at various times relative to the time of infection and measured their rate of translation in
mock- and VSV-infected cells. We found that translation of mRNAs transfected during infection was not
inhibited but that translation of mRNAs transfected prior to infection was inhibited during VSV infection.
Based on these data, we conclude that the timing of viral mRNA appearance in the cytoplasm is responsible,
at least in part, for the preferential translation of VSV mRNAs. A time course measuring translation efficiencies of viral and host mRNAs showed that the translation efficiencies of viral mRNAs increased between 4 and
8 h postinfection, while translation efficiencies of host mRNAs decreased. The increased translation efficiency
of viral mRNAs occurred in cells infected with an M protein mutant virus that is defective in host shutoff,
demonstrating that the enhanced translation of viral mRNA is genetically separable from inhibition of
translation of host mRNA.
VOL. 82, 2008
PREFERENTIAL TRANSLATION OF VSV mRNAs
MATERIALS AND METHODS
Cells and viruses. HeLa cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 7.5% fetal bovine serum (FBS). HeLa cells stably
expressing enhanced green fluorescent protein (HeLa-EGFP) were generated by
transfection with EGFP-N1 plasmid (Clontech) and selection in medium containing 600 ␮g/ml G418. Recombinant wild-type VSV (rwt virus), recombinant
VSV expressing EGFP as a foreign gene (rwt-EGFP virus), and recombinant
M51R-M protein mutant VSV (rM51R-M virus) were described previously (46).
Recombinant M51R-M protein mutant VSV expressing EGFP as a foreign gene
(rM51R-M-EGFP) was recovered from plasmid DNA, similar to rwt-EGFP virus
(46). Virus stocks were prepared as described previously (24). Cells were infected
in DMEM with 2% FBS at a multiplicity of infection of 10 PFU/cell to ensure
synchronous infection.
Metabolic labeling. Approximately 7 ⫻ 105 cells grown in six-well dishes were
washed twice with DMEM without methionine and then incubated in DMEM
without methionine for 0.5 h. Cells were pulse labeled for 1 hour in DMEM
containing 200 ␮Ci/ml [35S]methionine. Cells were then harvested for immunoprecipitation by adding 500 ␮l radioimmunoprecipitation assay (RIPA) buffer
(0.15 M NaCl, 1% deoxycholic acid, 1% Triton X-100, 10 mM Tris-Cl, pH 7.4,
and 0.1% sodium dodecyl sulfate [SDS]) with 1 mg/ml bovine serum albumin
(BSA), 10 mM benzamidine, and 10 mM phenylmethylsulfonyl fluoride to each
well. Plates containing RIPA buffer were rocked gently until cells were visibly
lifted from the dish. Lysates were then centrifuged at 20,000 ⫻ g for 15 min at
4°C. For analysis of total protein synthesis, cells were harvested following pulse
labeling, using 500 ␮l RIPA buffer without BSA, and 360 ␮l of cell extract was
added to 40 ␮l of 10⫻ SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
sample loading buffer. For analysis of total protein synthesis, 10 ␮l of lysate was
electrophoresed in a 10 or 12% SDS-PAGE gel. Gels were dried and analyzed
by phosphorimaging (Molecular Dynamics). Quantitation was performed using
ImageQuant 5.2 (Molecular Dynamics).
Immunoprecipitation. Immunoprecipitation of EGFP was performed by adding 3.8 ␮g goat anti-GFP (RDI) to 100 ␮l of cell lysate. Samples were incubated
overnight at 4°C. Twenty microliters of protein G-Sepharose (Sigma) in NETN
buffer (20 mM Tris-Cl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.5% NP-40, and
4% BSA) was added and incubated for 1 h. Samples were centrifuged at 500 ⫻
g at 4°C, and pellets were washed five times with 400 ␮l of RIPA buffer with high
SDS (1% SDS). Five microliters of SDS loading buffer was added to final pellets,
and samples were heated to ⬃95°C, separated in 10 or 12% SDS-PAGE gels, and
analyzed as described above.
Northern blotting. RNAs were harvested from 6 ⫻ 106 HeLa cells by using 3
ml of Trizol (Invitrogen) according to the manufacturer’s specifications. Five
micrograms of RNA harvested from stably transfected cells or 0.5 ␮g of RNA
harvested from HeLa cells infected with rVSV-EGFP or rM51R-M-EGFP was
run in a 1.2% glyoxal agarose gel. The gel was incubated in 800 ml of transfer
buffer (0.01 M NaOH, 3 M NaCl) for 20 min and then transferred to a GeneScreen Plus (Perkin-Elmer) hybridization transfer membrane by upward capillary transfer (42). An [␣-32P]dCTP-labeled EGFP probe was prepared using a
Prime-a-Gene kit (Promega). Membranes were probed using ExpressHyb hybridization solution (BD Biosciences Clontech) according to the manufacturer’s
specifications. Membranes were analyzed by phosphorimaging as described
above.
In vitro translation. RNAs harvested at 8 h postinfection by using Trizol
(Invitrogen) were used to direct translation in rabbit reticulocyte lysates (Promega). Reaction mixtures had a 0.1-ml total volume, with 27 ␮g total RNA or 1.6
␮g of poly(A) RNA added. Poly(A) RNA was isolated from total RNA by using
oligo(dT)-cellulose columns (Amersham Biosciences) and two rounds of purifi-
cation. Translation reactions were carried out in a water bath at 30°C for 2 h and
stopped by incubation on ice. Three microliters of each reaction mix was removed for analysis of all products of protein synthesis. Remaining volumes were
diluted in 1.2 ml RIPA buffer plus 1 mg/ml BSA, 10 mM benzamidine, and 10
mM phenylmethylsulfonyl fluoride. EGFP was immunoprecipitated as described
above.
In vitro transcription. EGFP mRNA was transcribed in vitro using an mMessage mMachine SP6 kit (Ambion) according to the manufacturer’s instructions,
using the EGFP template plasmid pSD4.EGFP. Template DNA was linearized
by cleavage with SalI restriction endonuclease and ethanol precipitated prior to
transcription. The pSD4.EGFP plasmid directed transcription of EGFP mRNA
with a 36-nucleotide (nt) 5⬘ untranslated region (UTR) and a 124-nt 3⬘ UTR that
contained 66 nt of poly(A) sequence. Most of the mRNA produced by this
transcription reaction is capped, with half of the 7mG(5⬘)ppp(5⬘)G cap structures in the proper orientation (25). The final RNA product was precipitated
from the transcription reaction solution by using lithium chloride according to
the manufacturer’s instructions.
mRNA transfection. mRNA was transfected into HeLa cells in six-well dishes.
Transfections were done when the cell density was between 40 and 70% confluence, using a transit-mRNA transfection kit (Mirus Bio Corporation). Transfection mixtures were prepared with mRNA as described by the manufacturer and
were added to cells over 1 ml of fresh growth medium (DMEM with 7.5% FBS).
Infections of transfected cells were done by removing the transfection medium
and immediately adding virus, suspended in DMEM with 7.5% FBS, to the wells.
RESULTS
During VSV infection, the inhibition of host transcription
and mRNA transport prevents new host mRNAs from reaching the cytoplasm. During this time, viral transcription is the
primary source of newly synthesized mRNAs. These events
also coincide with the inhibition of host translation and the
predominance of viral protein synthesis. These observations
suggested that the timing of transcription may be involved in
controlling translation in VSV-infected cells; specifically, the
most recently synthesized mRNAs may be translated preferentially. We tested whether the time of appearance of mRNAs
in the cytoplasm relative to the time of infection was involved
in controlling translation by transfecting cells with in vitrotranscribed EGFP reporter mRNAs at various times relative to
the time of infection.
The purpose of the experiments shown in Fig. 1 was to
determine the time required after transfection for mRNA to
enter the cytoplasm and direct translation. mRNA was synthesized in vitro, using SP6 RNA polymerase (SP6-EGFP) in the
presence of a cap analog, from a vector that directed transcription of an mRNA with an EGFP open reading frame flanked
by a short vector-derived 5⬘ UTR and a 3⬘ UTR containing a
poly(A) tract. HeLa cells were transfected with SP6-EGFP
mRNA, and translation rates at various times after transfection were determined by pulse labeling with [35S]methionine.
EGFP synthesis was then analyzed by immunoprecipitating
and measuring labeled EGFP by SDS-PAGE and phosphorimaging. Figure 1A shows a representative phosphorimage of
EGFP synthesized at various times posttransfection. Figure 1B
shows results from four experiments normalized to EGFP
translation at 8 h posttransfection. At 2 h posttransfection,
EGFP synthesis was already detectable. By 4 h posttransfection, EGFP synthesis had almost reached a maximum level,
which was maintained throughout the time course. These results indicate that the minimum time required for transfected
SP6-EGFP mRNA to be translated optimally is around 4 h.
To determine if newly transfected mRNAs were resistant to
VSV-induced inhibition of translation, HeLa cells were trans-
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before infection was inhibited. This result indicates that the
inhibition of host translation reflects the inhibition of translation of preexisting mRNAs together with the lack of production of new host mRNAs due to the inhibition of transcription
and transport by M protein. In contrast to that of host mRNAs,
the translation efficiency of mRNAs generated by the viral
RDRP increased between 4 and 8 h postinfection. This increase in translation efficiency of viral mRNA also occurred in
cells infected with an M protein mutant virus that is defective
in the inhibition of host translation. This result indicates that
inhibition of host translation is not essential for the efficient
translation of viral mRNAs.
2287
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WHITLOW ET AL.
fected with SP6-EGFP mRNA at three different times relative
to the time of infection. Figure 2A shows a timeline of the
experiment. The time of infection or mock infection is the zero
time point, and transfection times are shown relative to this
time point. Cells were transfected with SP6-EGFP mRNA 22 h
before infection, 4 h before infection, or 1 h after infection with
a recombinant VSV encoding a wild-type M protein (rwt virus). At 5.5 h postinfection, cells were pulse labeled with
[35S]methionine for 1 h. The time of the pulse was short relative to the rate of EGFP turnover, so the incorporation of the
label reflected the rate of EGFP synthesis. The three time
points for transfection were chosen based on the data in Fig. 1.
mRNAs transfected 22 h before infection would have been in
the translating polysomes for 14 to 18 h at the time of infection,
similar to host mRNAs that are turned over slowly (15).
mRNAs transfected 4 h before infection would be entering
polysomes from about the time of infection until about 4 h
postinfection. mRNAs transfected at 1 h postinfection would
be entering polysomes from approximately 5 to 9 h postinfection, when viral protein synthesis is optimal. We did not attempt to transfect cells at later times postinfection due to
concern that the viral cytopathic effects beginning around 4 h
postinfection would alter the transfection efficiency.
Translation rates of transfected SP6-EGFP mRNAs were
determined by immunoprecipitating EGFP from cell lysates
FIG. 2. EGFP mRNA transfected concurrent with infection is translated preferentially. (A) Experimental design for analyzing EGFP synthesis
in HeLa cells transfected with SP6-EGFP mRNA at three different times relative to the time of infection, set as time zero. SP6-EGFP mRNA was
transfected 22 h before infection, 4 h before infection, or 1 h after infection with rwt virus. (B) Phosphorimages of immunoprecipitated EGFP from
cells transfected 22 h before infection, 4 h before infection, or 1 h after infection. Each time of transfection was performed as a separate
experiment. (C) Phosphorimage of total cell lysates of cells that were transfected 22 h before infection or of untransfected cells that were
subsequently mock infected or infected with rwt virus. Inhibition of total protein synthesis by rwt virus was similar for all three times of transfection.
(D) EGFP synthesis or total host protein synthesis in rwt virus-infected cells relative to that in mock-infected cells that were transfected with
SP6-EGFP at different times relative to the time of infection. Data shown are means ⫾ SE for experiments performed on at least three different
days for each time of transfection (⫺22 h, n ⫽ 4; ⫺4 h, n ⫽ 6; 1 h, n ⫽ 5).
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FIG. 1. Time course of translation of transfected SP6-EGFP
mRNA. (A) Phosphorimage of immunoprecipitated EGFP, analyzed
by SDS-PAGE, from HeLa cells that were transfected with in vitrotranscribed EGFP mRNA (SP6-EGFP) and pulse labeled with
[35S]methionine at various times after transfection. Control cells
(C) were not transfected. (B) Quantification of EGFP labeling at
various times after transfection of SP6-EGFP mRNA into HeLa cells.
Data are expressed as fractions of the maximum at 8 h posttransfection
and are means for four experiments (⫾ standard errors [SE]).
J. VIROL.
VOL. 82, 2008
2289
translation rate of SP6-EGFP mRNA transfected 1 h after
infection into rwt virus-infected cells relative to mock-infected
cells was significantly different from that of overall protein
synthesis (P ⫽ 0.007, using a two-sample t test). These data
show that mRNAs introduced into the cytoplasm during infection or close to the time of infection are resistant to VSVinduced inhibition of translation.
Because VSV mRNAs turn over more rapidly than do host
mRNAs (15, 36), there may be a continuous supply of newly
synthesized viral mRNAs appearing in the cytoplasm of infected cells, which is resistant to the inhibition of translation.
Alternatively, translation of VSV mRNAs may become inhibited at later times postinfection as VSV mRNAs become progressively older. To distinguish these possibilities, the translation efficiencies of host- and virus-derived mRNAs were
measured over the period when translation of most host
mRNAs is increasingly inhibited. Furthermore, we also analyzed cells infected with a recombinant M protein mutant virus
(rM51R-M virus) defective in inhibiting host gene expression
(2, 8). For these experiments, we used HeLa cells that stably
express EGFP from transfected EGFP plasmid DNA (HeLaEGFP cells) and recombinant viruses that express EGFP as a
foreign gene: rwt-EGFP virus has a wild-type M protein, and
rM51R-M-EGFP virus has a mutant M protein and is defective
in inhibiting host gene expression.
Figure 3A shows the rates of EGFP synthesis in HeLaEGFP cells infected with rwt or rM51R-M virus and in HeLa
cells infected with rwt-EGFP or rM51R-M-EGFP virus expressing EGFP as a foreign gene. EGFP synthesis was measured by pulse labeling with [35S]methionine at 4, 8, or 12 h
postinfection, followed by immunoprecipitation, SDS-PAGE,
and phosphorimaging. Extracts containing virus-derived EGFP
were diluted 1:20 prior to immunoprecipitation. A representative image obtained from duplicate cell cultures at 8 h postinfection is shown. Data from three separate experiments were
quantified and are shown relative to the rate for mock-infected
HeLa-EGFP control cells. EGFP translation in HeLa-EGFP
cells was inhibited to a greater extent following infection with
rwt virus than with rM51R-M virus (Fig. 3A, left panel), as
expected based on previous studies with these viruses (2, 8).
While translation of host-derived EGFP mRNA was reduced during VSV infection, translation of virus-derived
mRNA occurred at high levels (notice the difference in scale
for the two graphs in Fig. 3A). In HeLa cells infected with
rwt-EGFP virus, EGFP synthesis increased between 4 and 8 h
postinfection but decreased by 12 h postinfection (Fig. 3A,
right panel). In HeLa cells infected with rM51R-M-EGFP
virus, synthesis of EGFP was similar to that in HeLa cells
infected with rwt-EGFP virus at 4 and 8 h postinfection. However, at 12 h postinfection, EGFP synthesis remained high in
cells infected with the recombinant M protein mutant virus
expressing EGFP, in contrast to cells infected with rwt-EGFP
virus (Fig. 3A, right panel).
EGFP mRNA levels were determined by Northern blotting.
A representative Northern blot obtained from duplicate cultures at 8 h postinfection is shown in Fig. 3B. Extracts containing virus-derived EGFP mRNA were diluted 1:10 prior to
Northern analysis. Data from three separate experiments were
quantified and are shown relative to the mRNA levels in mockinfected HeLa-EGFP control cells and also expressed as ratios
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and analyzing it by SDS-PAGE and phosphorimaging (Fig.
2B). In mock-infected cells, EGFP synthesis was easily detected. However, in rwt virus-infected cells that were transfected 22 or 4 h before infection, EGFP was synthesized at
much lower rates. The intense band for rwt virus-infected cell
lysates that migrates slightly slower than EGFP is nonspecifically immunoprecipitated VSV M protein. This was shown
previously by its precipitation in control samples, using an
irrelevant antibody (46).
The key result in Fig. 2 is that when SP6-EGFP mRNA was
transfected at 1 h postinfection, EGFP was synthesized to
higher levels in rwt virus-infected cells than in mock-infected
cells (Fig. 2B). These results show that mRNA transfected
during infection is resistant to VSV-induced translation inhibition, while mRNA transfected before infection is translationally inhibited. In controls for the specificity of immunoprecipitation (Fig. 2B), there was a faint band from a nonspecific host
protein that migrated similarly to EGFP in the untransfected
cells that were mock infected. This band did not appear in the
immunoprecipitation product from rwt virus-infected cells. Because of the nonspecifically immunoprecipitated protein,
EGFP labeling for transfected cells that were mock infected
was likely to be overestimated, while quantitation of the EGFP
labeling intensity for rwt virus-infected cells transfected at 1 h
postinfection was likely to be more accurate because the nonspecific protein was not labeled in these cells, while EGFP was
labeled. Therefore, these results showing that SP6-EGFP
mRNA is resistant to VSV-induced inhibition of translation
when transfected around the time of infection are a conservative underestimate of this resistance.
As controls for the effect of transfection on the rates of
translation of host and viral proteins, total cellular protein
synthesis was also analyzed by SDS-PAGE and phosphorimaging (Fig. 2C). Transfection of SP6-EGFP mRNA slightly
reduced levels of total protein synthesis compared to those of
untransfected controls, independent of infection. Consequently, overall host translation was not reduced as dramatically during rwt virus infection of transfected cells as during
infection of untransfected cells (Fig. 2C). Quantitation of host
protein synthesis indicated that in transfected cells, rwt virus
infection reduced host protein synthesis by around 60%,
whereas in untransfected cells, rwt virus infection reduced
translation by around 80%. Results for total host protein synthesis were similar for each transfection time. Transfection had
little, if any, effect on viral protein synthesis.
Rates of EGFP synthesis from transfected SP6-EGFP
mRNA, as well as total host protein synthesis, relative to those
in mock-infected cells were determined by quantifying results
from multiple experiments similar to the experiments shown in
Fig. 2B and C (Fig. 2D). When cells were transfected 22 h
before infection, translation of SP6-EGFP mRNA was reduced
by rwt virus infection to less than half of the level in mockinfected cells, similar to the reduction of overall translation in
these cells (Fig. 2D). When cells were transfected 4 h before
infection, translation of SP6-EGFP mRNA was not inhibited
as much as it was for mRNA transfected 22 h before infection
or as much as inhibition of overall translation in these cells
(Fig. 2D). However, when SP6-EGFP mRNA was transfected
1 h after infection, EGFP was translated almost twofold better
in rwt virus-infected cells than in mock-infected cells. The
PREFERENTIAL TRANSLATION OF VSV mRNAs
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WHITLOW ET AL.
J. VIROL.
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FIG. 3. Determination of translation efficiencies of host-derived and virus-derived EGFP mRNAs at different times after infection with virus
containing wild-type or mutant M protein. (A) Rate of EGFP synthesis from host versus viral mRNA. HeLa-EGFP cells were mock infected or
infected with rwt or M51R-M virus, and HeLa cells were infected with rwt-EGFP or M51R-M-EGFP virus. Cells were radiolabeled at 4, 8, or 12 h
postinfection, and EGFP was immunoprecipitated and analyzed by SDS-PAGE and phosphorimaging. Extracts containing virus-derived EGFP
were diluted 1:20 prior to immunoprecipitation. Each condition was analyzed in duplicate cell cultures. A representative image obtained at 8 h
postinfection is shown. Data from three separate experiments were quantified and are shown relative to the rate for HeLa-EGFP mock-infected
control cells (means ⫾ SE) for HeLa-EGFP cells infected with rwt or M51R-M virus (left) and for HeLa cells infected with rwt-EGFP or
rM51R-M-EGFP virus (right). (B) EGFP mRNA levels in HeLa-EGFP cells that were mock infected or infected with rwt or M51R-M virus and
in HeLa cells infected with rwt-EGFP or M51R-M-EGFP virus. A representative Northern blot obtained from duplicate cell cultures at 8 h
postinfection is shown. Extracts containing virus-derived EGFP mRNA were diluted 1:10 prior to Northern analysis. Data from three separate
experiments were quantified and are shown relative to the mRNA level in HeLa-EGFP mock-infected control cells (means ⫾ SE) for HeLa-EGFP
cells infected with rwt or M51R-M virus (left) and for HeLa cells infected with rwt-EGFP or rM51R-M-EGFP virus (right). (C) EGFP translation
efficiencies were calculated as protein synthesis (A) divided by the mRNA level (B) and are given relative to the efficiency in mock-infected
HeLa-EGFP control cells.
VOL. 82, 2008
2291
FIG. 4. Virus-derived EGFP mRNA harvested at various times
postinfection directs translation in vitro with similar efficiencies.
(A) Phosphorimage of immunoprecipitated EGFP synthesized in rabbit reticulocytes programmed with total RNA harvested from HeLa
cells infected with recombinant rwt-EGFP or M51R-M-EGFP virus at
4, 8, or 12 h postinfection (hpi). Translation reaction mixes also contained total RNA harvested from HeLa cells so that each reaction mix
contained the same amount of total RNA. (B) Phosphorimage of
Northern blot of EGFP mRNA samples like those used for in vitro
translation reactions. (C) In vitro translation efficiencies of EGFP
mRNAs relative to the efficiency of translation of EGFP mRNA harvested from HeLa cells infected with rwt-EGFP virus at 4 h postinfection.
EGFP mRNAs harvested from infected cells at various
times postinfection were all translated with similar efficiencies
in vitro. Based on these data, we concluded that the increase in
translation efficiencies of virally derived EGFP mRNAs from 4
to 8 h postinfection was not due to differences in the mRNAs.
Instead, these differences were the result of changes in the
translation program of infected cells.
DISCUSSION
The predominance of viral protein synthesis in cells where
host translation is inhibited is an important feature of infection
displayed by many viruses. VSV infections are particularly
striking in the extent to which viral protein synthesis predominates over the synthesis of host proteins. Mechanisms that
viruses use to achieve preferential translation of viral mRNAs
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to the levels in mock-infected HeLa-EGFP cells (Fig. 3B). For
HeLa-EGFP cells infected with rwt virus, the level of EGFP
mRNA was not significantly different from the level observed
in mock-infected cells until 12 h postinfection. At this time, the
EGFP mRNA level in rwt virus-infected cells was reduced to
0.28 relative to that in mock-infected HeLa-EGFP control cells
(Fig. 3B, left panel), likely due to decay of EGFP mRNA
following inhibition of its synthesis by wt M protein. In HeLaEGFP cells infected with rM51R-M virus, EGFP mRNA levels
were not significantly different from the control level at any
time point tested (Fig. 3B, left panel). In HeLa cells infected
with recombinant viruses expressing EGFP as a foreign gene,
EGFP mRNA levels were consistent throughout the time
course, at levels 10- to 12-fold above the level in mock-infected
HeLa-EGFP control cells (Fig. 3B, right panel).
Translation efficiencies were determined by dividing the
EGFP translation rates (Fig. 3A) by the EGFP mRNA levels
(Fig. 3B) and are shown in Fig. 3C as ratios to the translation
efficiency in mock-infected HeLa-EGFP cells. Infection of
HeLa-EGFP cells by rwt virus dramatically reduced the translation efficiency of EGFP, to 0.10 relative to the control, by
12 h postinfection. Infection by rM51R-M virus also reduced
the translation efficiency of EGFP mRNA in HeLa-EGFP
cells, but to a lesser extent, as expected based on previously
reported data regarding translation in cells infected with these
viruses (8, 9).
In contrast to host-derived EGFP mRNA, translation efficiencies of virus-derived EGFP mRNA increased during infection with both rwt-EGFP and rM51R-EGFP viruses (Fig. 3C).
Translation efficiencies of EGFP mRNAs from the recombinant viruses at 4 h postinfection were similar to the translation
efficiency of host-derived EGFP mRNA. However, the translation efficiencies of virus-derived EGFP mRNAs increased
between 4 and 8 h postinfection, to around 2.0. Twelve hours
after infection with rwt-EGFP virus, the translation efficiency
of EGFP mRNA declined to 1.2 relative to that of the control
(Fig. 3C). The EGFP translation efficiency in HeLa cells infected with rM51R-M-EGFP remained high throughout the
time course (Fig. 3C). These results indicate that inhibition of
host translation does not affect translation efficiency of viral
mRNAs before 8 h postinfection and therefore separate the
ability of VSV to inhibit host translation from its ability to
promote translation of viral mRNAs.
In vitro translation assays were used to determine whether
the increase in translation efficiencies of virus-derived mRNAs
from 4 to 8 h postinfection was due to differences in viral
mRNAs synthesized at different times postinfection (e.g.,
changes in cap structure, etc.). Total RNA from HeLa cells
infected with EGFP-expressing viruses was added to rabbit
reticulocyte lysate translation reaction mixtures so that the
levels of EGFP mRNA were similar. Translation reactions
were carried out in the presence of [35S]methionine, and
EGFP was immunoprecipitated and analyzed by SDS-PAGE
and phosphorimaging. A representative gel is shown in Fig.
4A. The EGFP mRNA level in each reaction mix was determined by Northern blotting (Fig. 4B), and relative translation
efficiencies of EGFP mRNA in vitro (Fig. 4C) were determined by dividing the relative intensities of EGFP immunoprecipitated from in vitro translation reactions by the relative
intensities of EGFP mRNA levels from Northern blotting.
PREFERENTIAL TRANSLATION OF VSV mRNAs
2292
WHITLOW ET AL.
FIG. 5. Model for preferential translation of newly appearing
mRNAs during VSV infection. (A and B) Modification of translation
machinery during VSV infection, indicated by dephosphorylation of
eIF4E (A), leads to inhibition of translation of preexisting host
mRNAs (B). (C) Reinitiation of translation is prevented by shuttling of
these mRNAs into translationally inactive mRNPs. (D) Viral transcription provides a continuous supply of newly synthesized viral
mRNAs, which are translated by the reprogrammed translation apparatus. (E) M protein enhances translation of viral mRNAs.
difference between host and viral mRNAs in their dependence
on eIF4F.
Our results also show that not only are viral mRNAs resistant to inhibition, but there is actually an increase in the translation efficiency of viral mRNAs between 4 and 8 h postinfection, the same time when translation of host mRNAs is
inhibited (Fig. 3). Our results also showed an increased translation of SP6-EGFP mRNA transfected 1 h after VSV infection relative to that in mock-infected cells (Fig. 2), at a time
when translation of host mRNAs is reduced. For virally derived mRNA, this increase in translation efficiency was seen
with a virus containing wt M protein as well as an M protein
mutant virus defective in inhibiting host gene expression.
These results show that efficient translation of viral mRNAs or
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are varied but often rely on cis-acting sequences, such as the
internal ribosome entry site sequences used by picornaviruses
and the tripartite leader sequences of adenovirus late mRNAs.
However, we showed previously that VSV mRNAs are preferentially translated through a mechanism that does not appear
to involve cis-acting sequences (46). Instead, as shown here,
the translation program of infected cells is altered such that the
timing of transcription and appearance in the cytoplasm of
VSV mRNAs allows them to be translated preferentially. Furthermore, the data presented here as well as data previously
reported (10) demonstrate that the inhibition of host translation is genetically separable from the promotion of translation
of viral mRNAs.
The resistance of newly appearing mRNAs to VSV-induced
inhibition of translation was shown by transfecting mRNAs
into cells at various times relative to the time of infection (Fig.
2). The resistance of SP6-EGFP mRNA transfected after infection to VSV-induced translation inhibition also confirms
the sequence independence of efficient translation of VSV
mRNAs. Because VSV mRNAs are also new to infected cells,
these results imply that viral mRNAs are resistant to VSVinduced inhibition of translation. This resistance can account
in part for the efficient translation of viral mRNAs relative to
that of host mRNAs. Furthermore, viral mRNAs are turned
over much more rapidly than most host mRNAs (half-life of
1.5 to 2 h versus 6 to 12 h) (15, 36) and are continuously
replenished by the viral transcriptase throughout the time
course of infection. Thus, there is a continuous supply of “new”
mRNAs that are resistant to the mechanism of host shutoff.
These data also imply that the inhibition of host translation is
dependent on the inhibition of host transcription and mRNA
transport by the VSV M protein. The ability of VSV to inhibit
host mRNA transcription and transport has never been separated genetically from its ability to inhibit host translation.
During infection with M protein mutant viruses defective in
shutoff of host gene expression, the continued translation of
host-derived mRNAs (Fig. 3) may be due to the failure to shut
off host transcription and transport.
Figure 5 shows a model by which the translation program
may be altered in infected cells, resulting in selective translation of newly introduced mRNAs. Previous data have shown
that inhibition of translation of host mRNAs is due at least in
part to alterations in the eIF4F cap-binding complex, reflected
in the dephosphorylation of the cap-binding subunit eIF4E (8,
9, 11). According to this model, dephosphorylation of eIF4E
(Fig. 5A) leads to inhibition of translation of preexisting host
mRNAs (Fig. 5B). Reinitiation of translation is prevented by
shuttling of these mRNAs into translationally inactive mRNPs
(41, 46), where they are stably maintained in infected cells (Fig.
5C). While VSV M protein inhibits the production of newly
synthesized host mRNA, viral transcription provides a continuous supply of newly synthesized viral mRNAs, which are
translated by the reprogrammed translation apparatus (Fig.
5D). It might be expected that viral mRNAs would be less
sensitive than host mRNAs to alterations in the eIF4F capbinding complex because of their relatively short and unstructured 5⬘ UTRs. We have shown previously that extending the
5⬘ UTR of virally derived EGFP mRNA to a length typical of
host mRNAs has little, if any, effect on translation (46). Nonetheless, it will be important to determine whether there is a
J. VIROL.
VOL. 82, 2008
2293
that the introduction of mRNA into the cytoplasm allowed
preferential translation during influenza virus infection. In
their study, the timing of introduction of mRNA into the cytoplasm was not addressed specifically. However, the timing of
mRNA introduction into influenza virus-infected cells may be
critical to its resistance to inhibition of translation.
The differential translation of newly appearing versus preexisting mRNAs also likely plays a role in the poliovirus-induced inhibition of host translation. Expression of the poliovirus 2A protease results in cleavage of the scaffolding subunit
of the eIF4F complex (eIF4G) at different rates, depending
on the eIF4G isoform present in the complex (eIF4GI ⬎
eIF4GII). Cleavage of eIF4GI preferentially inhibits translation of newly synthesized mRNAs, while cleavage of eIF4GII is
required to inhibit translation of preexisting mRNAs (6). Likewise, cleavage of poly(A)-binding protein by poliovirus 3C
protease in vitro preferentially inhibits the translation of newly
initiated mRNAs but has little effect on ribosome recycling of
mRNAs whose translation was initiated prior to cleavage (26).
The preference for newly synthesized mRNAs by the translation machinery is likely to be a normal phenomenon that is
exploited by VSV and perhaps other viruses. For example, the
shuttling of preexisting mRNAs to inactive mRNPs (41, 46)
and the preferential translation of newly synthesized mRNAs
(shown here) are reminiscent of the changes in mRNA distribution during certain types of stress responses, in which preexisting mRNAs are shuttled into stress granules while newly
synthesized mRNAs are preferentially translated (3, 18). Addressing these similarities would help to define the mechanism
that leads to preferential translation of newly transcribed
mRNAs in VSV-infected cells.
ACKNOWLEDGMENTS
We thank Maryam Ahmed and Elizabeth Pettit-Kneller for helpful
advice and comments on the manuscript.
This work was supported by NIH grant RO1AI052304.
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