1
Inhibition of host translation by virus infection in
vivo
Classification: Biological Sciences
René Toribio and Iván Ventoso*
Departamento de Virología y Microbiología. Centro de Biología Molecular “Severo
Ochoa” (UAM-CSIC). Nicolás Cabrera 1. Universidad Autónoma de Madrid.
Cantoblanco 28049. Madrid. Spain
Keywords: Virus, Translation, eIF2 phosphorylation, PKR.
Abbreviations: SV, Sindbis virus; pfu, plaque-forming unit; PKR, dsRNA-dependent
protein kinase; eIFs, eukaryotic initiation factors, DLP, downstream hairpin loop; IF,
immunofluorescence.
*To whom correspondence should be addressed. E-mail: [email protected]
2
Abstract
Infection of cultured cells with lytic animal viruses often results in the selective
inhibition of host protein synthesis, whereas viral mRNA is efficiently translated under
these circumstances. This phenomenon, called shut off, has been well described at
molecular level for some viruses, but there is not yet any direct or indirect evidences
supporting the idea that it also should operate in animals infected with viruses. To
address this issue, we constructed recombinant Sindbis virus (SV) expressing reporter
mRNA, whose translation is sensitive or resistant to virus-induced shut off. As found in
cultured cells, replication of SV in mouse brain was associated to a strong
phosphorylation of eukaryotic initiation factor eIF2 that prevented translation of
reporter mRNA (luciferase and EGFP). Translation of these reporters was restored in
vitro, in vivo and ex vivo when a viral RNA structure termed DLP, present in viral 26S
mRNA, was placed at the 5´ end of reporter mRNAs. By comparing the expression of
shut off-sensitive and -resistant reporters, we unequivocally concluded that replication
of SV in animal tissues is associated with a profound inhibition of non-viral mRNAs
translation. A strategy as simple as that followed here might be applicable to other
viruses to evaluate their interference on host translation in infected animals.
3
\body
Introduction
The interference of animal viruses with host translation was first documented in
the decade of the 1960s in human fibroblasts infected with poliovirus (1) Further studies
revealed that the halt of host translation (or shut off) was a general phenomenon
observed in cells infected with lytic RNA and DNA viruses (2-6). Of the three steps in
protein synthesis, viruses mainly affect the initiation step by hijacking or modifying the
activity of key initiation factors (eIFs) to ensure an efficient translation of viral mRNAs
and the simultaneous decline of host translation (5-8). The main targets of viruses are
components of the cap-binding complex (eIF4F) that are required for the recruitment of
ribosomes to mRNAs. Thus, some picornaviruses (e.g poliovirus and rhinovirus) and
lentiviruses (e.g HIV-1) express proteases that proteolyze the eIF4G (the largest
component of eIF4F) and PABP (polyA binding protein) to dismantle cellular cap-
dependent translation, whereas viral translation continues by the presence of IRES
elements in viral mRNA that allow the recruitment of ribosomes in a cap-independent
manner (2, 9-14). Other Picornaviruses (e.g EMC), Rhabdoviruses (VSV) and
Adenovirus decrease the activity of eIF4E (the cap-binding protein of eIF4F complex)
by promoting its dephosphorylation or/and the activation of its inhibitors 4E-BP1 and 2
(15-17). In other cases, such as Rotavirus and Influenza, viral proteins hijack eIF4G to
redirect it towards viral factories where viral mRNA is being translated (18, 19).
Another important point of translation control in infected cells relies on the
activity of eIF2 that brings the initiator Met-tRNA to 40S ribosomes (20-23). In
response to viral infection, host dsRNA-activated kinase (PKR) phosphorylates eIF2 in
an attempt to block general translation (both cellular and viral) (24-27). However, most
viruses avoid this by expressing products that prevent the activation of PKR in infected
cells (reviewed in (28)). A remarkable exception to this are the members of alphavirus
group (Sindbis and Semliki forest virus). Thus, complete phosphorylation of eIF2 was
found in cultured mouse fibroblasts infected with these viruses, so that only viral
mRNA is translated under these circumstances (22, 29). The presence of a secondary
structure termed DLP located 27 nts downstream from the initiator AUG in viral 26S
transcripts allows this mRNA to be translated by an eIF2-independent mechanism in
infected cells (22, 30).
4
The shut off phenomenon has been extensively studied in cultured cells, but not
in infected animals, so that evidences for virus-induced shut off in vivo are still lacking.
In vitro experiments often require somewhat artefactual conditions such as the use of
highly susceptible cell lines to virus and high multiplicity of infection, two conditions
that are difficult to find during virus replication in animals. Moreover, the analysis of
virus-induced shut off in vivo raises technical difficulties to measure de novo protein
synthesis in a single animal´s cell infected with the virus. Many viruses also interfere
with other steps of host gene expression such as transcription, mRNA transport or
stability (3, 29, 31-35), making it very difficult to attribute a reduction in the synthesis
of host proteins (or of a given host protein used as reporter) to the sole effect on
translation.
We show here that, as occurs in vitro, replication of Sindbis virus in mouse
brains is linked to a phosphorylation of eIF2 that was detected in infected neurons. We
show indirect, but strong evidences that shut off of host translation also occurs in
animals infected with viruses.
Results
Engineered reporter mRNAs that mimic translation of cellular and viral mRNA in
SV-infected cells. We previously reported that as consequence of PKR-induced eIF2
phosphorylation, only viral 26S mRNA that bears DLP structure is translated in
alphavirus-infected cells (Fig.1A and (22)). Like the rest of cellular mRNAs,
heterologous mRNA expressed from a recombinant SV was no longer translated in
infected mouse fibroblasts due to eIF2 phosphorylation (Fig. 1C, 1F and (22)).
Translation of reporter mRNA was easily restored when viral DLP (90 nts in length)
structure was placed at the 5´ end of a coding sequence of these mRNAs, allowing a
translation as efficient as that of viral 26S mRNA (Fig. 1E). We reasoned that, in terms
of translation in SV-infected cells, these reporter mRNAs lacking or containing DLP
structures might behave as bona fide cellular and viral mRNAs, respectively. Moreover,
since these reporter mRNAs are transcribed from a viral promotor, differential
expression should reflect exclusively differences in the rate of translation. Thus, virus-
induced shut off in vivo could be easily inferred by comparing the expression of reporter
genes in animals infected with these two types of viruses (SV-reporter vs SV-DLP
reporter). To validate our experimental approach, we first carried out a detailed
5
characterization of recombinant viruses in cultured cells (Fig.1B). The synthesis of
luciferase and EFGP was strongly inhibited in 3T3 cells infected with recombinant SV-
Luc and SV-EGFP, respectively, as compared with their counterparts that bear the viral
DLP structure (SV-DLP Luc and SV-DLP EGFP). No differences in expression were
detected in PKRo/o
cells, showing that phosphorylation of eIF2 hampered translation of
non-viral mRNAs as described before (22). Next, we quantified the extent of
translational exclusion of EGFP mRNA in SV-infected cells compared to cellular
mRNAs (e.g. !-actin). Thus, we metabolicaly labeled infected cells with [35
S]-Met/Cys
to analyze de novo protein synthesis of EGFP, virus capsid protein (SV C) and cellular
!-actin. In parallel, we also analyzed the steady state levels of their corresponding
mRNAs by northern-blot (Fig. 1E). Infection with all recombinant SV viruses induced a
strong inhibition of !-actin synthesis (>90%) without affecting the levels of its
corresponding mRNA in a significant way (Fig. 1E). Similar amounts of EGFP and
DLP-EGFP mRNAs acumulated in infected cells, but only DLP-EGFP mRNA was
translated. Thus, translation of both EGFP and !-actin mRNAs was inhibited to a
similar extent (>90%) in infected cells. On the contrary, we estimated that translation of
DLP-EGFP mRNA was comparable to 26S mRNA that encodes the structural proteins
of virus (including C protein). Translation of EGFP, however, was almost completely
abrogated when the DLP structure was disrupted by point mutations that destroyed its
secondary structure in SV-"DLP EGFP virus, showing that DLP was essential for
translational resistance to eIF2 phosphorylation.
Shut off induced by SV infection of mouse brain. We next infected mice with
recombinant viruses to compare the reporter activity in whole organs (luciferase) or in
single-infected cells (EGFP). SV shows a marked neurotropism in mice, infecting
neurons of the neocortex and hippocampal regions of brain (36-38). Inoculation of mice
with recombinant viruses by the intranasal route resulted in a rapid replication in brains
over a period of 4 days postinfection, yielding 106-10
7 pfu per brain. First, we
cryosectioned brains of infected animals and the resulting slices were subjected to IF
with anti-SV C and anti-phospho eIF2#. At 3 dpi, viral antigens were detected in groups
of neurons in anterior ventral regions of brain, as well in basolateral areas
corresponding to the pyriform cortex (Fig. 2A). At 4-6 dpi, viral antigens were detected
in areas of the somatosensorial and motor cortex as well in the hippocampus, suggesting
that virus entered via the olfatory bulb to further spread out to upper regions of the
6
cortex. Interestingly, a prominent label of phospho-eIF2# was detected only in regions
of virus replication in wild type animals. We found that up to 90% of areas expressing
viral antigens immunoreacted to phospho-eIF2# antibodies, showing that replication of
SV in animals is also associated with inactivation of eIF2. No phospho-eIF2# staining
was detected in PKRo/o
-infected animals, showing that PKR quinase is also responsable
for eIF2 phosphorylation during SV replication in vivo.
Replication of SV-Luc and SV-DLP Luc in mice, measured by viral yields, was
identical (Fig. 2B). However, only luciferase activity was detected in wild type mice
infected with SV-DLPLuc. These differences in luciferase activity between SV-luc and
SV-DLP Luc were even more marked than in cultured cells (see Fig. 1F), showing that
translation of non-viral mRNA was severely impaired in mouse brain neurons of wild
type animals. As expected, no differences in luciferase activity were detected among
SV-Luc and SV-DLP Luc viruses in PKRo/o
mice (Fig. 2C). We next analyzed the
expression of EGFP in brain neurons of mice infected with SV-EGFP and SV-DLP
EGFP at the peak of virus replication (3 days). Although replication of SV-EGFP and
SV-DLP EGFP was indistinguishable as judged by IF staining of viral antigens, we
found strong differences in EGFP expression among brains of animals infected with
these two viruses (Fig. 3A). For SV-DLP EGFP, about 50% of cells that immunoreacted
with anti-SV C antibodies expressed EGFP, whereas only 5-10% of cells infected with
SV-EGFP showed detectable EGFP expression (Fig. 3). Moreover, the few cells
expressing EGFP from SV-EGFP showed a fluorescence intensity lower than their
counterparts infected with SV-DLP EGFP.
Shut off also operates ex-vivo. Organotypical explants can be easily derived from rat
hippocampus and maintained in culture for a variety of purposes including
electrophysiological studies (39, 40). Moreover, hippocampal slices can be transduced
with non-replicative derivates of Sindbis and Semliki Forest viruses for the expression
of foreigner genes (41, 42). It was interesting, therefore, to test whether shut off also
happened in explanted brain slices after in vitro infection with SV. Thus, slices were
incubated with preparations of SV-EGFP or SV-DLP EGFP viruses (104 pfu each) and
analyzed by IF one day later. Both viruses spread rapidly throughout the explant
infecting an elevated number of neurons, most of them with a pyramidal shape.
Notably, a dramatic difference in number and fluorescent intensity of neurons
expressing EGFP was found among slices infected with SV-EGFP and SV-DLP EGFP
(Fig. 3B). Virtually all cells infected with SV-DLP EGFP simultaneously expressed
7
EGFP (94%), whereas only a very small proportion of neurons infected with SV-EGFP
virus showed EGFP fluorescence (2%). Slices were also incubated with anti-
phosphoeIF2# antibodies, showing that ex vivo infection with SV also triggered eIF2#
phosphorylation, as occurred in vitro and in vivo (supporting figure S2).
Discussion
We show here for the first time that replication of a virus in an animal tissue
resulted in the inactivation of a translation initiation factor (eIF2). Although we
ourselves, as well as other investigators, had already reported a strong activation of
PKR that led to a complete phosphorylation of eIF2 in cultured cells infected with SV
and Semliki forest virus (SFV), there was no experimental evidence supporting the idea
that such an event happened in infected animals. A detailed examination of brains from
infected animals revealed that virtually all groups of neurons expressing viral antigens
also immunoreacted to anti-phosphoeIF2# antibodies. This finding was notable and
showed that replication of SV in animals is intimately linked to PKR-mediated eIF2
phosphorylation. Accordingly, PKR expression in mouse brain has been found
particulary high in the neocortex and hippocampus (http://www.brain-map.org/), where
SV replication was easily detected. Interestingly, phosphorylation of eIF2 in cortical
neurons has been reported to occur during ischemic stress and other pathological
situations such as Alzheimer and Huntington diseases (43-47)
By means of recombinant viruses expressing engineered reporter mRNAs, we
present indirect but solid evidence that translation of non-viral mRNA is strongly
inhibited in infected mouse brain neurons. Virus expressing reporter genes were used in
earlier studies to track replication and spreading of the virus to different organs of
infected animals (37, 41, 48). However, to date, the shut off phenomenon has not been
addressed in vivo, probably due to the technical difficulties that such a study raises. Our
approach is based on the assumption that translation of reporter mRNAs used here
faithfully reflected translation of host and viral mRNAs in infected cells. All results
obtained supported this. First, translation of EGFP and luciferase mRNA was inhibited
to a similar extent as !-actin and the majority of cellular mRNAs in infected cells.
Second, the placement of a DLP structure at the 5´ end of the EGFP coding sequence
restored translation to a level comparable to that of translation in viral 26S mRNA.
Third, the use of a viral promotor that drives the synthesis of reporter mRNA allowed
direct measurement of the effect of virus replication on translation, obviating the
8
perturbations that Sindbis and other viruses exert on cellular transcription (3, 31-33, 49).
In fact, infection with alphavirus also results in a halt of host transcription that can be
separated from the translation shut off phenomenon (49). Despite this, we found that
the steady state levels of abundant host RNA, such as ribosomal or !-actin mRNA, did
not significantly decrease at 6 hpi, when translation of host mRNA was completely
inhibited (Fig.1).
A similar experimental strategy to that described here might be applicable to the
study of shut off in other viruses where the interference with host translational
machinery has been well clarified. This approach requires, however, a previous
knowledge of molecular tricks that allow the mRNA of a given virus to be translated in
an enviroment of general translational inhibition. This has been well described for
picornavirus and roughly clarified for VSV, Influenza, Adenovirus and Rotavirus but
not for others such as Poxvirus (6, 16, 17, 19, 50, 51). Thus, the low dependence of
VSV, Adenovirus and Influenza for the cap binding protein eIF4E might be used to
create reporter mRNA with different translational capabilities in cells infected with
these viruses (52-55). A limitation of the strategy described here is that reporter genes
should be placed under subgenomic promotors in RNA or DNA viruses to create a
transcriptional independence, which excludes picornavirus and other viruses that
initiates transcription exclusively from the end of the genomic strand.
The demostration that shut off also takes place in vivo, at least for alphavirus
could have profound implications for a better understanding of virus-host interactions in
infected animals. Moreover, the ability of viruses to block host translation might be
critically regulating their pathogenic potential by preventing the synthesis of proteins
with antiviral function such as interferons and other inflammatory cytokines.
Finally, the influence of viral DLP on translation of mRNA in SV-infected cells
could improve the expression of foreigner genes from SV-derived vectors, which are
widely used to transduce primary neurons and organotypical explants of brain animals
(41, 42).
9
Materials and Methods
Animals and cell lines. Wild type (Charles River) and PKR knock-out animals (kindly
provided by J. C. Bell, University of Ottawa, Canada, (56)) from 129sv strain were
used. Four-weeks old females were infected by the intranasal route with 5x106-10
7 pfu
of Sindbis virus. 3T3 cells derived from wild type and PKRo/o
animals (27) and BHK21
were grown in DMEN suplemented with bovine serum (3T3) and fetal serum (PKRo/o
and BHK21) as described previously (22). MEFs derived from 129sv mice were
prepared following standard protocols (23).
Construction of Recombinant viruses. Recombinant viruses expressing luciferase or
EGFP mRNAs were constructed in the pT7SV-2p plasmid, an infectious cDNA clone
of the Sindbis virus which carries a second subgenomic promotor at the 3´ of genomic
mRNA, and which has been designed to express foreigner genes (57). The construction
of SV-EGFP has been described (22). For SV-Luc, the luciferase coding sequence was
amplified by PCR with the following primers: 5´ Luc GGGCGCTAGCGGATCCA
ATGGAAGACGCCAAAAAC and 3´ Luc: CGCCGCTAGCTTACAATTTGGACT
TTCCGCC. PCR products were digested with NheI enzyme and cloned into the XbaI
site of pT7SV-2p. For SV-DLP EGFP, we amplified by PCR a DNA fragment
containing the DLP of SV fused in frame to the EGFP coding sequence from plasmid
p5´CEGFP-N1 (22). The primers used were: 5´C SV GCGCGCTAGCATGAA
TAGAGGATTC and 3´ EGFP CGCGCTCTAGATTACTTGTACAGCTCGTC. The
resulting PCR product was cloned into the XbaI site of pT7SV-2p as described above.
For SV-"DLP EGFP, the template for PCR amplification was p5´C"DLP EGFP-N1
plasmid carrying point mutations in DLP region that disrupted the secondary structure
of RNA as described before (22). For SV-DLP Luc, the PCR fragment of luciferase
coding sequence described above was cloned into p5´CEGFP-N1 plasmid using BamHI
and XbaI enzymes. Then, a PCR amplification was done using 5´C SV and 3´ Luc
primers and the resulting fragment was cloned into pT7SV-2p plasmid as described
before. All constructions were verified by sequencing. Infectious RNAs were generated
in vitro by transcription with RNApol T7 and electropored in BHK21 cells as described
10
previously (22). Viruses were collected 2-3 days later when the cytophatic effect was
massive and purified by ultracentrifugation (29K for 4 h) through a sucrose cushion at
4ºC. The resulting viral preparation showed titres of 5x108-10
9 pfu/mL and a high
degree of genetic homogeneity (see supplementary data).
Immunofluorescence (IF). For IF of tissues, brains of infected mice were extracted 3
dpi and fixed overnight with 4% PFA at 4ºC and then hydrated with 30% sucrose for
48h. Brains were cryosectioned at 15 µm, postfixed with PFA at RT for 15´ and
permeabelized with 0.2 % Triton X-100 in PBS for 30´. Sections were treated with
ammonium chloride, blocked in 5% BSA-PBS and incubated overnight at 4ºC with
primary antibodies: anti-C SV (1:300), anti-phosphoeIF2# (Cell Signaling, 1:200).
Sections were washed three times for 15´ with PBS-0,1 Triton X-100 and incubated
with secondary antibodies coupled to Alexa 555 or Alexa 594 for 1h. Finally, sections
were washed as described above, mounted and photographed in a Leika confocal
microscope. For IF of cultures cells growing on coverslips, the protocol was similar
except that primary antibodies were incubated 2h at RT. Western Blot was carried out
essentially as described previously (22).
Metabolic labeling of proteins. Cells growing in 24-well plates were infected with a
moi of 25 pfu/cell and 5:30 h later labeled with 25 µCi/mL of [35
S]-Met/Cys (20) for
30´ in medium lacking methionine. After washing with cold medium, monolayers were
lysed in a sample buffer, boiled and analyzed in a 12% SDS-PAGE followed by
fluorography with 1M salicylate solution and exposure to X-ray film.
Luciferase assays. Brains of mice infected with luciferase-expressing viruses were
homogenated in PBS and extracted with 1 volumen of 2X luciferase lysis buffer
(KH2PO4 15 mM, MgSO4 15 mM, EGTA 4 mM, DTT 4mM and T-X100 1%). After
centrifugation at 10K for 5´, 20µL of lysates were used to measure luciferase activity.
Infection of organotypic slices from rat hippocampus. Hippocampal slices from 6-
day-old rats were prepared as described before (40) and maintained in culture for 1
week before infection with 104-10
5 pfu of the indicated virus. A 2 µL drop of virus
preparation was applied on slices twice, and the drops were allowed to drain away
11
between applications. Virus replication and spreading were checked every 24h by living
examination of EGFP fluorescence. IF analysis was identical to described above, except
for incubations were done on floating sections.
Acknowledgments. We are in debt to J. Mª Almendral for having allowed us to work
at his laboratory since 2005 and J.J. Berlanga and J.A. Esteban for providing us with
mice and organotypical preparations, respectively. Thanks go to J. C. Bell, B.R.
Williams and M. A. Sanz for PKRo/o
mice, PKRo/o
3T3 cells and pT7SV2p plasmid,
respectively. This work was supported in part by grants from the Ministerio de Ciencia
e Innovación (SAF2006-09810) and the Fundación Mutua Madrileña (FMM 2008).
Support from the VIRHOST programme and the Fundación Ramón Areces is also
acknowledged. R.T. was a recipient of the SAF2006-09810 contract and I.V. is a
researcher of Ramón y Cajal Programme.
References
1. Holland JJ & Peterson JA (1964) Nucleic Acid And Protein Synthesis During
Poliovirus Infection Of Human Cells. J Mol Biol 8:556-575.
2. Etchison D, Milburn SC, Edery I, Sonenberg N, & Hershey JW (1982)
Inhibition of HeLa cell protein synthesis following poliovirus infection
correlates with the proteolysis of a 220,000-dalton polypeptide associated with
eucaryotic initiation factor 3 and a cap binding protein complex. J Biol Chem
257(24):14806-14810.
3. Kaariainen L & Ranki M (1984) Inhibition of cell functions by RNA-virus
infections. Annu Rev Microbiol 38:91-109.
4. Gingras AC, Raught B, & Sonenberg N (1999) eIF4 initiation factors: effectors
of mRNA recruitment to ribosomes and regulators of translation. Annu Rev
Biochem 68:913-963.
5. Schneider RJ & Mohr I (2003) Translation initiation and viral tricks. Trends
Biochem Sci 28(3):130-136.
6. Bushell M & Sarnow P (2002) Hijacking the translation apparatus by RNA
viruses. J Cell Biol 158(3):395-399.
7. Thompson SR & Sarnow P (2000) Regulation of host cell translation by viruses
and effects on cell function. Curr Opin Microbiol 3(4):366-370.
8. Pestova TV, et al. (2001) Molecular mechanisms of translation initiation in
eukaryotes. Proc Natl Acad Sci U S A 98(13):7029-7036.
12
9. Gradi A, Svitkin YV, Imataka H, & Sonenberg N (1998) Proteolysis of human
eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with
the shutoff of host protein synthesis after poliovirus infection. Proc Natl Acad
Sci U S A 95(19):11089-11094.
10. Ventoso I, Blanco R, Perales C, & Carrasco L (2001) HIV-1 protease cleaves
eukaryotic initiation factor 4G and inhibits cap-dependent translation. Proc Natl
Acad Sci U S A 98(23):12966-12971.
11. Pelletier J & Sonenberg N (1988) Internal initiation of translation of eukaryotic
mRNA directed by a sequence derived from poliovirus RNA. Nature
334(6180):320-325.
12. Chen CY & Sarnow P (1995) Initiation of protein synthesis by the eukaryotic
translational apparatus on circular RNAs. Science 268(5209):415-417.
13. Joachims M, Van Breugel PC, & Lloyd RE (1999) Cleavage of poly(A)-binding
protein by enterovirus proteases concurrent with inhibition of translation in
vitro. J Virol 73(1):718-727.
14. Kerekatte V, et al. (1999) Cleavage of Poly(A)-binding protein by
coxsackievirus 2A protease in vitro and in vivo: another mechanism for host
protein synthesis shutoff? J Virol 73(1):709-717.
15. Gingras AC, Svitkin Y, Belsham GJ, Pause A, & Sonenberg N (1996)
Activation of the translational suppressor 4E-BP1 following infection with
encephalomyocarditis virus and poliovirus. Proc Natl Acad Sci U S A
93(11):5578-5583.
16. Connor JH & Lyles DS (2002) Vesicular stomatitis virus infection alters the
eIF4F translation initiation complex and causes dephosphorylation of the eIF4E
binding protein 4E-BP1. J Virol 76(20):10177-10187.
17. Huang JT & Schneider RJ (1991) Adenovirus inhibition of cellular protein
synthesis involves inactivation of cap-binding protein. Cell 65(2):271-280.
18. Aragon T, et al. (2000) Eukaryotic translation initiation factor 4GI is a cellular
target for NS1 protein, a translational activator of influenza virus. Mol Cell Biol
20(17):6259-6268.
19. Piron M, Vende P, Cohen J, & Poncet D (1998) Rotavirus RNA-binding protein
NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F.
Embo J 17(19):5811-5821.
20. Krishnamoorthy T, Pavitt GD, Zhang F, Dever TE, & Hinnebusch AG (2001)
Tight binding of the phosphorylated alpha subunit of initiation factor 2
(eIF2alpha) to the regulatory subunits of guanine nucleotide exchange factor
eIF2B is required for inhibition of translation initiation. Mol Cell Biol
21(15):5018-5030.
21. Dever TE (2002) Gene-specific regulation by general translation factors. Cell
108(4):545-556.
22. Ventoso I, et al. (2006) Translational resistance of late alphavirus mRNA to
eIF2alpha phosphorylation: a strategy to overcome the antiviral effect of protein
kinase PKR. Genes Dev 20(1):87-100.
23. Berlanga JJ, et al. (2006) Antiviral effect of the mammalian translation initiation
factor 2alpha kinase GCN2 against RNA viruses. Embo J 25(8):1730-1740.
24. Balachandran S, et al. (2000) Essential role for the dsRNA-dependent protein
kinase PKR in innate immunity to viral infection. Immunity 13(1):129-141.
25. Meurs E, et al. (1990) Molecular cloning and characterization of the human
double-stranded RNA-activated protein kinase induced by interferon. Cell
62(2):379-390.
13
26. Stojdl DF, et al. (2000) The murine double-stranded RNA-dependent protein
kinase PKR is required for resistance to vesicular stomatitis virus. J Virol
74(20):9580-9585.
27. Yang YL, et al. (1995) Deficient signaling in mice devoid of double-stranded
RNA-dependent protein kinase. Embo J 14(24):6095-6106.
28. Garcia MA, et al. (2006) Impact of protein kinase PKR in cell biology: from
antiviral to antiproliferative action. Microbiol Mol Biol Rev 70(4):1032-1060.
29. Gorchakov R, Frolova E, Williams BR, Rice CM, & Frolov I (2004) PKR-
dependent and -independent mechanisms are involved in translational shutoff
during Sindbis virus infection. J Virol 78(16):8455-8467.
30. Frolov I & Schlesinger S (1996) Translation of Sindbis virus mRNA: analysis of
sequences downstream of the initiating AUG codon that enhance translation. J
Virol 70(2):1182-1190.
31. Katze MG & Agy MB (1990) Regulation of viral and cellular RNA turnover in
cells infected by eukaryotic viruses including HIV-1. Enzyme 44(1-4):332-346.
32. Rice AP & Roberts BE (1983) Vaccinia virus induces cellular mRNA
degradation. J Virol 47(3):529-539.
33. Inglis SC (1982) Inhibition of host protein synthesis and degradation of cellular
mRNAs during infection by influenza and herpes simplex virus. Mol Cell Biol
2(12):1644-1648.
34. Gustin KE & Sarnow P (2001) Effects of poliovirus infection on nucleo-
cytoplasmic trafficking and nuclear pore complex composition. Embo J 20(1-
2):240-249.
35. Her LS, Lund E, & Dahlberg JE (1997) Inhibition of Ran guanosine
triphosphatase-dependent nuclear transport by the matrix protein of vesicular
stomatitis virus. Science 276(5320):1845-1848.
36. Binder GK & Griffin DE (2001) Interferon-gamma-mediated site-specific
clearance of alphavirus from CNS neurons. Science 293(5528):303-306.
37. Cook SH & Griffin DE (2003) Luciferase imaging of a neurotropic viral
infection in intact animals. J Virol 77(9):5333-5338.
38. Lewis J, Wesselingh SL, Griffin DE, & Hardwick JM (1996) Alphavirus-
induced apoptosis in mouse brains correlates with neurovirulence. J Virol
70(3):1828-1835.
39. Gahwiler BH (1981) Organotypic monolayer cultures of nervous tissue. J
Neurosci Methods 4(4):329-342.
40. Correia SS, et al. (2008) Motor protein-dependent transport of AMPA receptors
into spines during long-term potentiation. Nat Neurosci 11(4):457-466.
41. Ehrengruber MU, et al. (1999) Recombinant Semliki Forest virus and Sindbis
virus efficiently infect neurons in hippocampal slice cultures. Proc Natl Acad
Sci U S A 96(12):7041-7046.
42. Xiong C, et al. (1989) Sindbis virus: an efficient, broad host range vector for
gene expression in animal cells. Science 243(4895):1188-1191.
43. Burda J, et al. (1994) Phosphorylation of the alpha subunit of initiation factor 2
correlates with the inhibition of translation following transient cerebral
ischaemia in the rat. Biochem J 302 (Pt 2):335-338.
44. Onuki R, et al. (2004) An RNA-dependent protein kinase is involved in
tunicamycin-induced apoptosis and Alzheimer's disease. Embo J 23(4):959-968.
45. Bando Y, et al. (2005) Double-strand RNA dependent protein kinase (PKR) is
involved in the extrastriatal degeneration in Parkinson's disease and
Huntington's disease. Neurochem Int 46(1):11-18.
14
46. Peel AL (2004) PKR activation in neurodegenerative disease. J Neuropathol
Exp Neurol 63(2):97-105.
47. Paquet C, et al. (2009) Neuronal phosphorylated RNA-dependent protein kinase
in Creutzfeldt-Jakob disease. J Neuropathol Exp Neurol 68(2):190-198.
48. Rodriguez JF, Rodriguez D, Rodriguez JR, McGowan EB, & Esteban M (1988)
Expression of the firefly luciferase gene in vaccinia virus: a highly sensitive
gene marker to follow virus dissemination in tissues of infected animals. Proc
Natl Acad Sci U S A 85(5):1667-1671.
49. Gorchakov R, Frolova E, & Frolov I (2005) Inhibition of transcription and
translation in Sindbis virus-infected cells. J Virol 79(15):9397-9409.
50. Feigenblum D & Schneider RJ (1993) Modification of eukaryotic initiation
factor 4F during infection by influenza virus. J Virol 67(6):3027-3035.
51. Zhang Y, Feigenblum D, & Schneider RJ (1994) A late adenovirus factor
induces eIF-4E dephosphorylation and inhibition of cell protein synthesis. J
Virol 68(11):7040-7050.
52. Berg DT & Grinnell BW (1992) 5' sequence of vesicular stomatitis virus N-gene
confers selective translation of mRNA. Biochem Biophys Res Commun
189(3):1585-1590.
53. Garfinkel MS & Katze MG (1993) Translational control by influenza virus.
Selective translation is mediated by sequences within the viral mRNA 5'-
untranslated region. J Biol Chem 268(30):22223-22226.
54. Burgui I, Yanguez E, Sonenberg N, & Nieto A (2007) Influenza virus mRNA
translation revisited: is the eIF4E cap-binding factor required for viral mRNA
translation? J Virol 81(22):12427-12438.
55. Cuesta R, Xi Q, & Schneider RJ (2000) Adenovirus-specific translation by
displacement of kinase Mnk1 from cap-initiation complex eIF4F. Embo J
19(13):3465-3474.
56. Abraham N, et al. (1999) Characterization of transgenic mice with targeted
disruption of the catalytic domain of the double-stranded RNA-dependent
protein kinase, PKR. J Biol Chem 274(9):5953-5962.
57. Levis R, Schlesinger S, & Huang HV (1990) Promoter for Sindbis virus RNA-
dependent subgenomic RNA transcription. J Virol 64(4):1726-1733.
15
Figure Legends
Fig.1 In vitro characterization of recombinant SV expressing shut off-sensitive and -
resistant reporter mRNAs. (A) Flow chart showing the main translational alteration in
SV-infected cells (see text for details). (B) Schematic diagram of recombinant SV
expressing reporter mRNAs. The genomic organization of SV RNA is shown, including
the natural and duplicate subgenomic promotors (blue) that drive the synthesis of 26S
mRNA encoding the viral structural proteins and the reporter mRNAs (luciferase or
EGFP), respectively. Arrows show the transcription start site from each promotor. A
downstream hairpin loop structure (DLP) included in the first 90 nts of the 26S mRNA
coding sequence was also placed in the indicated reporter mRNAs. In SV-"DLP EGFP
the secondary structure of DLP was disrupted by point mutations as described before
(22). (C) Western-blot analysis of recombinant SV in wild type (PKR+/+
) and PKRo/o
3T3 cells. Cells were infected with the indicated virus at a moi of 25 pfu/cell and
analyzed at 6 hpi by western-blot with the indicated antibodies. Note that the placement
of 90 nts of the coding sequence of the C protein that includes the DLP increased the
size of EGFP and delayed its electrophoretic mobility. (D) IF of SV expressing the
indicated versions of EGFP in wild type 3T3 cells. Micrographs were taken at 6hpi. (E)
De novo translation of cellular and viral-expressing mRNA in infected cells. Cells were
infected with the indicated virus and labeled with [35
S]Met/Cys at 5:30 hpi for 30´.
Proteins were analyzed by SDS-PAGE and autorradiography. Bands corresponding to
!-actin, SV capside (SV C) and EGFP were quantified by densitometry, corrected for
the number of methionines and cysteines and expressed as percentage of control (mock
for !-actin and DLPEGFP for EGFP). Parallel infections were used for extracting total
RNA and a northern-blot analysis was performed against the indicated mRNAs. (F)
Luciferase activiy of PKR+/+
and PKRo/o
cells infected with the indicated viruses.
Samples were analyzed at 6hpi and luciferase activity was measured as described in
16
materials and methods. The standard deviation from three independent experiments is
shown.
Fig.2. Phosphorylation of eIF2 in mice infected with SV and inhibition of non-viral
translation. (A) Representative IF micrographs of coronal brain sections from wild type
and PKRo/o
mice infected with SV at 3dpi. Adjacent sections were incubated with anti-
SV C or anti-phosphoeIF2# antibodies. 214 out of 238 replication foci scored from
three wild type infected animals showed strong staining of phosphoeIF2# (89%) (right
panel), whereas no eIF2phosphorylation associated to SV replication was detected
in PKRo/o
mice. No immunoreaction of anti-phosphoeIF2# antibodies was detected
away from replication foci in any wild type mouse analyzed. (B) Expression of Luc, but
not of DLP-Luc, was inhibited in brains of wild type animals infected with recombinant
viruses. Mice were infected with the indicated viruses and brain homogenates were
prepared at the indicated times to quantify viral yields (left) and luciferase activity
(right). (C) Translation of luc mRNA was restored in PKRo/o
animals infected with
SV-Luc.
Fig. 3 Inhibition of EGFP expression, but not of DLP-EGFP, in single neurons infected
with recombinant virus in vivo and ex-vivo. (A) Brains of infected animals were
analyzed at 3dpi for simultaneous EGFP fluorescence and anti-SV C reactivity.
Representative micrographs with scale bars are shown. 80 neurons expressing viral
antigens from each virus were scored, and 32 of them showed EGFP fluorescence for
SV-DLP EGFP virus (40%), whereas only 4 neurons infected with SV-EGFP showed
green fluorescence (5%) (lower panel). (B) SV replication and EGFP expression in rat
hippocampal slices infected with the indicated virus and analyzed at 1dpi. Samples were
processed as described above. 372 neurons expressing viral antigens from SV- DLP
EGFP and 1098 from SV EGFP were scored for statistical analysis (lower panel).
SV infection
- host translation inhibited
AAA
A
Luc
- translation of viral 26S mRNAs
PKR activation
structural
genomic mRNADLP
non structural
AAA
Lucreporter AAA
reporter
BC
D
E
DLP
Lucreporter AAA
SV-reporter
SV-DLP reporter
SV- DLP reporter
mock
SV-E
GFP
SV-D
LP EG
FP
SV-
DLP E
GFP
100 2 2.5 3 - 7 100 15
actin synthesisEGFP synthesis
mock
SV-E
GFP
SV-D
LP EG
FPmo
ckSV
-EGF
PSV
-DLP
EGF
P
SV-
DLP E
GFP
SV-
DLP E
GFP
eIF2
PKR
EGFP
SV C
eIF2 PeIF2 P
-actin
EGFPSV C
EGFP mRNA
-actin mRNA
49S mRNA26S mRNA
Mr (kDa)
F
5000
15000
25000
35000
45000
SV-L
uc
SV-L
uc
SV-D
LP L
uc
SV-D
LP L
uc
Luc
ifer
ase
activ
ity(R
LU
/g
of p
rote
in)
SV-E
GFP
SV-D
LP
EG
FPSV
-D
LP
EG
FP
anti-SV C EGFP
PKR+/+ PKRo/o
PKR+/+ PKRo/o
FIG.1
merge
0
0.5
1
1.5
2
2.5
3
0 2 3
1
10
102103104105106107
0 2 3
Luci
fera
se a
ctiv
ity (
arbi
trary
uni
ts)
Vi
rus y
ield
(pfu
/bra
in)
dpi dpi
-eIF2 P-SV C
SV-DLP LucSV-Luc
A
B
FIG.2
PKR+/+
PKRo/o
SV C
+ and
eIF
2p+
0
20
40
60
80
100
PKR+/
+
PKRo/o
0
0,5
1
1,5
2
2,5
3
3,5PKRo/o
SV-L
uc
SV-L
uc
SV-D
LP L
uc
SV-D
LP L
uc
PKR+/+
Luci
fera
se a
ctiv
ity (
arbi
trary
uni
ts)
SV-DLP LucSV-Luc
C
A EGFP-SV CSV
-EG
FPSV
-DL
P E
GFP
B
0
20
40
60
80
100
SV C
+ an
d EG
FP+
SV-E
GFP
SV-D
LP EG
FP
-SV C EGFP
SV-E
GFP
SV-D
LP
EG
FP
SV C
+ an
d EG
FP+
SV-E
GFP
SV-D
LP EG
FP0
10
20
30
40
50
FIG.3
20 µm
merge
600 µm
merge