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. 2009 Mar;83(5):2099-108.
doi: 10.1128/JVI.01753-08. Epub 2008 Dec 24.

Orthopoxviruses require a functional ubiquitin-proteasome system for productive replication

Alastair Teale  1 , Stephanie Campbell, Nick Van Buuren, Wendy C Magee, Kelly Watmough, Brianne Couturier, Robyn Shipclark, Michele Barry
Affiliations

Orthopoxviruses require a functional ubiquitin-proteasome system for productive replication

Alastair Teale et al. J Virol. 2009 Mar.

Abstract

Cellular homeostasis depends on an intricate balance of protein expression and degradation. The ubiquitin-proteasome pathway plays a crucial role in specifically targeting proteins tagged with ubiquitin for destruction. This degradation can be effectively blocked by both chemically synthesized and natural proteasome inhibitors. Poxviruses encode a number of proteins that exploit the ubiquitin-proteasome system, including virally encoded ubiquitin molecules and ubiquitin ligases, as well as BTB/kelch proteins and F-box proteins, which interact with cellular ubiquitin ligases. Here we show that poxvirus infection was dramatically affected by a range of proteasome inhibitors, including MG132, MG115, lactacystin, and bortezomib (Velcade). Confocal microscopy demonstrated that infected cells treated with MG132 or bortezomib lacked viral replication factories within the cytoplasm. This was accompanied by the absence of late gene expression and DNA replication; however, early gene expression occurred unabated. Proteasomal inhibition with MG132 or bortezomib also had dramatic effects on viral titers, severely blocking viral replication and propagation. The effects of MG132 on poxvirus infection were reversible upon washout, resulting in the production of late genes and viral replication factories. Significantly, the addition of an ubiquitin-activating enzyme (E1) inhibitor had a similar affect on late and early protein expression. Together, our data suggests that a functional ubiquitin-proteasome system is required during poxvirus infection.

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Figures

FIG. 1.
FIG. 1.
MG132 inhibits late protein expression. (A) HeLa cells were mock infected or infected with VV65 at an MOI of 5 in the presence or absence of 10 μM MG132. VV65-infected samples were harvested at 4, 8, 12, 16, and 24 h, and mock-infected samples were harvested at 4 h and Western blotted with anti-I5L, anti-B5R, anti-Mcl-1, and anti-Bak. α, anti. (B) HeLa cells were infected with VV65 in the presence and absence of 40 μg/ml AraC to determine late expression for I5L and B5R. (C) Metabolic labeling of VV65-infected cells. HeLa cells were mock infected or infected with VV65 at an MOI of 5 and treated with either 10 μM MG132 or 40 μg/ml AraC. Cells were pulse-labeled with [35S]methionine/cysteine at 4.5 h postinfection, and samples were analyzed by SDS-PAGE followed by autoradiography. Late proteins are marked with dots in the VV65-infected sample. (D) BGMK cells were infected with VV65 in the presence and absence of MG132, and plaques were visualized by staining with X-Gal.
FIG. 2.
FIG. 2.
Early protein expression is unaffected by MG132. (A) HeLa cells were infected with VV65 at an MOI of 5 in the presence of 10 μM MG132. At the indicated times, samples were harvested and Western blotted with anti-I3L, anti-M2L, and anti-Bak. α, anti. (B) HeLa cells were infected with VV65 in the presence and absence of 40 μg/ml AraC to determine early expression for I3L and M2L.
FIG. 3.
FIG. 3.
MG132 inhibits late protein expression during cowpox virus and ectromelia virus infection. HeLa cells were infected with ectromelia virus Moscow or cowpox virus Brighton Red at an MOI of 5 in the presence of 10 μM MG132. At the indicated times, samples were harvested and Western blotted with anti-I5L, anti-B5R, anti-I3L, anti-Mcl-1, and anti-Bak. EVM, ectromelia virus Moscow; CPV, cowpox virus; α, anti.
FIG. 4.
FIG. 4.
Multiple proteasome inhibitors block late protein expression. HeLa cells were infected with VV65 at an MOI of 5 in the presence of 10 μM MG115 (A), 10 μM lactacystin (B), and 1 μM bortezomib (C). At the indicated times, samples were harvested and Western blotted with anti-I5L, anti-I3L, and anti-Bak. α, anti.
FIG. 5.
FIG. 5.
MG132 and bortezomib block virus factory formation. (A) HeLa cells were infected with VV65 at an MOI of 5 in the presence or absence of 10 μM MG132, 1 μM bortezomib, or 40 μg/ml AraC. Sixteen hours postinfection, cells were fixed and stained with DAPI to visualize nuclei and virus factories and stained with anti-I3L (αI3L). (a to l) HeLa cells infected with VV65 (a to c), with VV65 in the presence of MG132 (d to f), with VV65 in the presence of bortezomib (g to i), or with VV65 in the presence of AraC (j to l). (B) A slot blot assay was used to assess the accumulation of VV65 DNA using a 32P-labeled probe specific for the E9 DNA polymerase gene in the presence and absence of MG132 or AraC.
FIG. 6.
FIG. 6.
The effect of MG132 is reversible. (A) HeLa cells were infected with VV65 at an MOI of 5. Cells were either pretreated (Pre) or treated with MG132 for 12, 8, or 4 h, at which point cells were washed with PBS to remove MG132 and harvested at 16 h postinfection. Additionally, cells were not treated with MG132 [(−)]. I3L, I5L, and Bak expression was monitored by Western blotting. α, anti. (B) HeLa cells were infected with VV65 in the presence and absence of MG132, and at 4, 8, and 12 h, cells were washed with PBS to remove MG132. Cells were fixed at 16 h postinfection, stained with DAPI to visualize nuclei and virus factories, and stained with anti-I3L. (a to o) HeLa cells infected with VV65 (a to c), with VV65 in the presence of MG132 followed by a washout at 4 h (d to f), with VV65 in the presence of MG132 followed by washout at 8 h (g to i), with VV65 in the presence of MG132 followed by washout at 12 h (j to l), or with VV65 in the presence of MG132 (m to o).
FIG. 7.
FIG. 7.
Proteasome inhibitors act at an early step during infection. (A) HeLa cells were infected with VV65 at an MOI of 5. Cells were either pretreated (Pre); treated with MG132 at either 2, 4, 6, or 8 h postinfection; or not treated with MG132 [(−)]. I3L, I5L, and Bak expression was monitored by Western blotting. α, anti. (B) HeLa cells were infected with VV65 at an MOI of 5. Cells were pretreated with bortezomib (Pre); treated with bortezomib at either 2, 4, 6, or 8 h postinfection; or not treated with bortezomib [(−)]. I3L, I5L, and Bak expression was monitored by Western blotting.
FIG. 8.
FIG. 8.
Inhibition of the E1-activating enzyme inhibits late gene expression. HeLa cells were infected with VV65 at an MOI of 5 and pretreated with 25 μM Pyr-41. Total cellular lysates were harvested at the indicated times and blotted for I5L, I3L, and Bak. α, anti.
FIG. 9.
FIG. 9.
Inhibition of the ubiquitin proteasome system dramatically affects poxvirus production. (A) HeLa cells were infected with VV65 at an MOI of 1, and samples were harvested at 4, 8, 12, and 24 h postinfection. Samples were collected in triplicate, and titers were determined on BGMK cells. (B) HeLa cells were infected with cowpox virus Brighton Red (CPV) at an MOI of 1. (C) HeLa cells were infected with ectromelia virus strain Moscow (EVM) at an MOI 1. (D) HeLa cells were infected with VV65 at an MOI 5 for 10 or 24 h or the presence of 10 μM MG132 for 10 h. Additionally, MG132 was removed by washout at 10 h and infection was allowed to progress for a further 8 h.
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References

    1. Afonso, C. L., E. R. Tulman, Z. Lu, L. Zsak, G. F. Kutish, and D. L. Rock. 2000. The genome of fowlpox virus. J. Virol. 743815-3831. - PMC - PubMed
    1. Andrei, G., D. B. Gammon, P. Fiten, E. De Clercq, G. Opdenakker, R. Snoeck, and D. H. Evans. 2006. Cidofovir resistance in vaccinia virus is linked to diminished virulence in mice. J. Virol. 809391-9401. - PMC - PubMed
    1. Babiuk, L. A., B. Meldrum, V. S. Gupta, and B. T. Rouse. 1975. Comparison of the antiviral effects of 5-methoxymethyl-deoxyuridine with 5-iododeoxyuridine, cytosine arabinoside, and adenine arabinoside. Antimicrob. Agents Chemother. 8643-650. - PMC - PubMed
    1. Banks, L., D. Pim, and M. Thomas. 2003. Viruses and the 26S proteasome: hacking into destruction. Trends Biochem. Sci. 28452-459. - PubMed
    1. Barry, M., and K. Fruh. 2006. Viral modulators of cullin RING ubiquitin ligases: culling the host defense. Sci. STKE 2006pe21. - PubMed

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