Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis

Sivan, Gilad; Martin, Scott E.; Myers, Timothy G.; Buehler, Eugen; Szymczyk, K. H.; Ormanoglu, Pinar; Moss, Bernard

doi:10.1073/pnas.1300708110

Cited by 88 publications

(103 citation statements)

References 43 publications

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“…A previous comprehensive screen identified siRNAs to over 500 candidate human genes of more than 20,000 interrogated that inhibited the spread of VACV (28). In order to prioritize the hits, we looked for their enrichment in functional groups.…”

Section: Resultsmentioning

confidence: 99%

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Retrograde Transport from Early Endosomes to the trans -Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions

Sivan

Weisberg

Americo

et al. 2016

J Virol

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The anterograde pathway, from the endoplasmic reticulum through the trans-Golgi network to the cell surface, is utilized by trans-membrane and secretory proteins. The retrograde pathway, which directs traffic in the opposite direction, is used following endocytosis of exogenous molecules and recycling of membrane proteins. Microbes exploit both routes: viruses typically use the anterograde pathway for envelope formation prior to exiting the cell, whereas ricin and Shiga-like toxins and some nonenveloped viruses use the retrograde pathway for cell entry. Mining a human genome-wide RNA interference (RNAi) screen revealed a need for multiple retrograde pathway components for cell-to-cell spread of vaccinia virus. We confirmed and extended these results while discovering that retrograde trafficking was required for virus egress rather than entry. Retro-2, a specific retrograde trafficking inhibitor of protein toxins, potently prevented spread of vaccinia virus as well as monkeypox virus, a human pathogen. Electron and confocal microscopy studies revealed that Retro-2 prevented wrapping of virions with an additional double-membrane envelope that enables microtubular transport, exocytosis, and actin polymerization. The viral B5 and F13 protein components of this membrane, which are required for wrapping, normally colocalize in the trans-Golgi network. However, only B5 traffics through the secretory pathway, suggesting that F13 uses another route to the trans-Golgi network. The retrograde route was demonstrated by finding that F13 was largely confined to early endosomes and failed to colocalize with B5 in the presence of Retro-2. Thus, vaccinia virus makes novel use of the retrograde transport system for formation of the viral wrapping membrane. IMPORTANCEEfficient cell-to-cell spread of vaccinia virus and other orthopoxviruses depends on the wrapping of infectious particles with a double membrane that enables microtubular transport, exocytosis, and actin polymerization. Interference with wrapping or subsequent steps results in severe attenuation of the virus. Some previous studies had suggested that the wrapping membrane arises from the trans-Golgi network, whereas others suggested an origin from early endosomes. Some nonenveloped viruses use retrograde trafficking for entry into the cell. In contrast, we provided evidence that retrograde transport from early endosomes to the trans-Golgi network is required for the membrane-wrapping step in morphogenesis of vaccinia virus and egress from the cell. The potent in vitro inhibition of this step by the drug Retro-2 suggests that derivatives with enhanced pharmacological properties might serve as useful antipoxviral agents. V accinia virus (VACV) is the prototype member of the poxvirus family of cytoplasmic double-stranded DNA viruses (1). The first infectious form of VACV, the intracellular mature virion (MV), is a brick-shaped particle consisting of a nucleoprotein core, lateral bodies, and a surrounding lipoprotein membrane (2). A subset of cytoplasmic MVs are wr...

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Section: Resultsmentioning

confidence: 99%

“…Our particular interest in the retrograde pathway arose from a genome-wide small interfering RNA (siRNA) screen in which depletion of some retrograde transport factors significantly decreased spread of VACV (28). In principal, a block at any step from virus entry to release could affect the spread assay.…”

mentioning

confidence: 99%

Retrograde Transport from Early Endosomes to the trans -Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions

Sivan

Weisberg

Americo

et al. 2016

J Virol

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“…Homologs of D10 are encoded by all poxviruses, whereas D9 is encoded by most chordopoxviruses but not entomopoxviruses. A genome-wide RNA interference (RNAi) screen suggested a role for the 5=-to-3= exoribonuclease Xrn1 in VACV replication (41), which could act in conjunction with the VACV decapping enzymes to degrade mRNA.…”

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confidence: 99%

The D10 Decapping Enzyme of Vaccinia Virus Contributes to Decay of Cellular and Viral mRNAs and to Virulence in Mice

Liu

Wyatt

Orandle

et al. 2014

J Virol

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bPosttranscriptional mechanisms are important for regulation of cellular and viral gene expression. The presence of the 5= cap structure m 7 G(5=)ppp(5=)Nm is a general feature of mRNAs that provides protection from exoribonuclease digestion and enhances translation. Vaccinia virus and other poxviruses encode enzymes for both cap synthesis and decapping. Decapping is mediated by two related enzymes, D9 and D10, which are synthesized before and after viral DNA replication, respectively. The timing of D10 synthesis correlates better with the shutdown of host gene expression, and deletion of this gene has been shown to cause persistence of host and viral mRNAs in infected cells. Here, we constructed specific mutant viruses in which translation of D10 was prevented by stop codons or activity of D10 was abrogated by catalytic site mutations, without other genomic alterations. Both mutants formed plaques of normal size and replicated to similar extents as the parental virus in monkey epithelial cells and mouse embryonic fibroblasts. The synthesis of viral proteins was slightly delayed, and cellular and viral mRNAs persisted longer in cells infected with the mutants compared to either the parental virus or clonal revertant. Despite the mild effects in vitro, both mutants were more attenuated than the revertants in intranasal and intraperitoneal mouse models, and less infectious virus was recovered from organs. In addition, there was less lung histopathology following intranasal infection with mutant viruses. These data suggest that the D10 decapping enzyme may help restrict antiviral responses by accelerating host mRNA degradation during poxvirus infection. P osttranscriptional mechanisms are important for the regulation of cellular and viral gene expression at the levels of RNA stability and translation. The presence of a 5= cap structure m 7 G(5=)ppp(5=)Nm is a general feature of eukaryotic mRNAs and many viral mRNAs that provides protection from exonuclease digestion and enhances translation (1-6). In eukaryotic cells, mRNA decay begins with shortening of the poly(A) tail and proceeds in either the 5=-to-3= or 3=-to-5= direction. The latter is mediated by the cytoplasmic RNA exosome (7-9) and a scavenger enzyme that degrades the cap (8). In the 5=-to-3= pathway, removal of the cap (10-13) is followed by exoribonuclease Xrn1 digestion (14,15). Enzymes with nudix hydrolase motifs that decap cytoplasmic mRNA are present in Saccharomyces cerevisiae and mammalian cells and are thought to function in mRNA decay (10-13, 16, 17).Degradation of cellular mRNA may be advantageous to viruses by decreasing competition for the translational machinery and by reducing the synthesis of factors that contribute to the innate and adaptive immune responses to infection. The ability of viruses to accelerate mRNA decay has been intensively studied with members of the herpesvirus family, which replicate in the nucleus and utilize the transcriptional machinery of the cell (18, 19). For alphaherpesviruses, accelerated mRNA turnover is mediat...

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“…Since then, proteins targeting additional host defense pathways have been identified in a similar way or in an ad hoc manner for many viruses. However, we found that large-scale screens in which expression of individual cellular genes is reduced or prevented by RNAi were successful in identifying host proteins that VACV uses for various steps in replication but not for anti-viral genes, probably because poxviruses evolved adequate defenses during their co-evolution with cells (94). In ongoing work, we have shown that this roadblock to discovery could be overcome by screening mutant viruses that have had one or more defense genes disrupted and consequently have lost the ability to replicate in certain non-permissive cells (95).…”

Section: From Biochemistry To Virologymentioning

confidence: 95%

Investigating Viruses during the Transformation of Molecular Biology

Moss

2017

Journal of Biological Chemistry

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Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis

Cited by 88 publications

References 43 publications

Retrograde Transport from Early Endosomes to the trans -Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions

Retrograde Transport from Early Endosomes to the trans -Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions

The D10 Decapping Enzyme of Vaccinia Virus Contributes to Decay of Cellular and Viral mRNAs and to Virulence in Mice

Investigating Viruses during the Transformation of Molecular Biology

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