Microtubule-dependent Organization of Vaccinia Virus Core–derivd Early mRNAs into Distinct Cytoplasmic Structures

Mallardo, Massimo; Schleich, Sibylle; Locker, Jacomine Krijnse

doi:10.1091/mbc.12.12.3875

Cited by 60 publications

(72 citation statements)

References 53 publications

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“…The existence of some kind of fenestration(s) in the VV core wall was postulated in previous studies that focused on the early viral RNA transcription, demonstrated to happen within apparently completely encapsulated cores (44). It was also possible to reconstitute the early transcription in vitro with apparently intact cores after depletion of the outer membrane (45,46).…”

Section: Discussionmentioning

confidence: 95%

Cryo-electron tomography of vaccinia virus

Cyrklaff

Risco²,

Fernández³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

184

150

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The combination of cryo-microscopy and electron tomographic reconstruction has allowed us to determine the structure of one of the more complex viruses, intracellular mature vaccinia virus, at a resolution of 4 -6 nm. The tomographic reconstruction allows us to dissect the different structural components of the viral particle, avoiding projection artifacts derived from previous microscopic observations. A surface-rendering representation revealed brickshaped viral particles with slightly rounded edges and dimensions of Ϸ360 ؋ 270 ؋ 250 nm. The outer layer was consistent with a lipid membrane (5-6 nm thick), below which usually two lateral bodies were found, built up by a heterogeneous material without apparent ordering or repetitive features. The internal core presented an inner cavity with electron dense coils of presumptive DNA-protein complexes, together with areas of very low density. The core was surrounded by two layers comprising an overall thickness of Ϸ18 -19 nm; the inner layer was consistent with a lipid membrane. The outer layer was discontinuous, formed by a periodic palisade built by the side interaction of T-shaped protein spikes that were anchored in the lower membrane and were arranged into small hexagonal crystallites. It was possible to detect a few pore-like structures that communicated the inner side of the core with the region outside the layer built by the T-shaped spike palisade.cryo-EM ͉ virus structure ͉ 3D reconstruction

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Section: Discussionmentioning

confidence: 95%

Cryo-electron tomography of vaccinia virus

Cyrklaff

Risco²,

Fernández³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

184

150

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“…An antibody to VV cores was generated that recognizes incoming cores, but not intact particles, by immunofluorescence microscopy (12). Furthermore, an assay to follow the fate of the viral early mRNAs once released from the core was previously established (14).…”

Section: Resultsmentioning

confidence: 99%

“…BrUTP transfection to visualize viral early mRNAs was done as described previously (14). Briefly, cells were infected for 15 min in the presence of 1 g of actinomycin D/ml and then were transfected with BrUTP (Sigma) for 1 h in the presence of the drug.…”

Section: Methodsmentioning

confidence: 99%

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The Vaccinia Virus I3L Gene Product Is Localized to a Complex Endoplasmic Reticulum-Associated Structure That Contains the Viral Parental DNA

Welsch

Doglio

Schleich

et al. 2003

J Virol

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The vaccinia virus (VV) I3L gene product is a single-stranded DNA-binding protein made early in infection that localizes to the cytoplasmic sites of viral DNA replication (S. C. Rochester and P. Traktman, J. Virol. 72:2917-2926, 1998). Surprisingly, when replication was blocked, the protein localized to distinct cytoplasmic spots (A. Domi and G. Beaud, J. Gen. Virol. 81:1231-1235, 2000). Here these I3L-positive spots were characterized in more detail. By using an anti-I3L peptide antibody we confirmed that the protein localized to the cytoplasmic sites of viral DNA replication by both immunofluorescence and electron microscopy (EM). Before replication had started or when replication was inhibited with hydroxyurea or cytosine arabinoside, I3L localized to distinct cytoplasmic punctate structures of homogenous size. We show that these structures are not incoming cores or cytoplasmic sites of VV early mRNA accumulation. Instead, morphological and quantitative data indicate that they are specialized sites where the parental DNA accumulates after its release from incoming viral cores. By EM, these sites appeared as complex, electron-dense structures that were intimately associated with the cellular endoplasmic reticulum (ER). By double labeling of cryosections we show that they contain DNA and a viral early protein, the gene product of E8R. Since E8R is a membrane protein that is able to bind to DNA, the localization of this protein to the I3L puncta suggests that they are composed of membranes. The results are discussed in relation to our previous data showing that the process of viral DNA replication also occurs in close association with the ER.Vaccinia virus (VV) is the prototype member of the Poxviridae, a family of large DNA viruses. They are characterized by a complex life cycle that occurs entirely in the cytoplasm of the infected host cell, including viral transcription and DNA replication (15). Infection is initiated upon entry and delivery of the core into the cytoplasm. From this core, which contains the viral genome and enzymes to carry out the process of early transcription, a set of early mRNAs is transcribed (11,16). The translation of the early messengers is required to uncoat the core and to release the parental DNA from the core and for the subsequent process of DNA replication. The latter process initiates the transcription of late genes, which encode for the late proteins that are required for the assembly of new virions (15). Virion assembly results in the production of two infectious forms, the intracellular mature virus that obtains its membranes from the intermediate compartment or smooth ER and the extracellular enveloped virus, which acquires an additional membrane from the trans-Golgi network or from endosomes (21, 26; reviewed in reference 23).A number of recent studies were aimed at understanding how some of the early steps of viral morphogenesis relate to each other and to determine if and how these steps interact with the host cell. We could show, for instance, that the viral early mRNA...

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“…In contrast to all other DNA viruses, the infection cycles of poxviruses (2,3,(20)(21)(22) and Mimivirus are entirely cytoplasmic. Early transcription in both poxviruses (20,23) and Mimivirus is initiated within intact cores after their delivery to the host cytoplasm through the previously described (10-12) stargate portal. As is the case for poxviruses (2,20), Mimivirus mRNAs accumulate at sites that were distinct from those where viral replication is initiated.…”

Section: Discussionmentioning

confidence: 99%

Vaccinia-like cytoplasmic replication of the giant Mimivirus

Mutsafi¹,

Zauberman²,

Sabanay

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

111

127

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Poxviruses are considered to be unique among all DNA viruses, because their infection cycle is carried out exclusively in the host cytoplasm. Such an infection strategy is of interest, because it necessitates generation of elaborate factories in which viral replication and assembly are promoted. By using diverse imaging techniques, we show that the infection cycle of the largest virus currently identified, the Acanthamoeba polyphaga Mimivirus, similarly occurs exclusively in the host cytoplasm. We further show that newly synthesized mRNAs accumulate at discrete cytoplasmic sites that are distinct from the sites where viral replication occurs, and this is observed in vaccinia infection. By revealing substantial physiologic similarity between poxviruses and Mimivirus and thus, implying that an entirely cytoplasmic viral replication might be more common than generally considered, these findings underscore the ability of DNA viruses to generate large and elaborate replication factories.electron tomography | nucleocytoplasmic large DNA viruses | poxviruses | viral factories W ith the single exception of poxviruses, all currently known DNA viruses carry out replication and transcription either entirely or partially within host nuclei. The overwhelming bias to nucleus-centered viral transactions is rationalized by the fact that the nucleus provides the elaborate machinery required for these processes. Moreover, by concentrating essential factors into particular intranuclear sites as well as by enabling spatiotemporal regulation of viral replication and transcription, the nuclear environment considerably enhances the efficiency of these processes (1). Such underlying benefits render the entirely cytoplasmic infection cycle of poxviruses all of the more intriguing (2, 3). Nucleus-centered viral transactions present, however, remarkable hurdles associated with the prerequisite of transporting viral genomes from their entry sites at the cell periphery to and into the nucleus. Specifically, the cellular milieu is refractory to motion of DNA molecules because of the high viscosity of the cytosol and the dense molecular sieve generated by the cytoskeleton (4). Moreover, the nuclear envelope imposes a severe barrier for both entry and exit of long DNA molecules (1).The hurdles associated with genome trafficking are particularly high for nucleocytoplasmic large DNA viruses (NCLDV), which include the eukaryote-infecting families Poxviridae, Phycodnaviridae, Iridoviridae, and Asfarviridae, all characterized by DNA genomes longer than 100 kbp (5). After entry, genomes of phycodnaviruses (6), iridoviruses (7), and the African swine fever virus (8) are released into the cytoplasm at the host cell periphery and then, are shuttled toward and transported into the nucleus where initial replication cycles occur. Viral DNA is subsequently delivered to specific cytoplasmic factories in which all transactions required for viral assembly take place. The factors that attenuate the barriers imposed by the constrained DNA motion and thus, enabl...

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Microtubule-dependent Organization of Vaccinia Virus Core–derivd Early mRNAs into Distinct Cytoplasmic Structures

Cited by 60 publications

References 53 publications

Cryo-electron tomography of vaccinia virus

Cryo-electron tomography of vaccinia virus

The Vaccinia Virus I3L Gene Product Is Localized to a Complex Endoplasmic Reticulum-Associated Structure That Contains the Viral Parental DNA

Vaccinia-like cytoplasmic replication of the giant Mimivirus

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