Sequence-Independent Targeting of Transmembrane Proteins Synthesized within Vaccinia Virus Factories to Nascent Viral Membranes

Husain, Matloob; Weisberg, Andrea S.; Moss, Bernard

doi:10.1128/jvi.02631-06

Cited by 19 publications

(18 citation statements)

References 39 publications

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“…The A14 and A17 proteins, which are major components of this membrane and are essential for its formation (13)(14)(15)(16)(17)(18), are thought to be synthesized in the ER despite the absence of signal sequences. They, along with components of the ER membrane, are diverted to sites of membrane biogenesis; indeed, it is the absence of specific trafficking signals that enables their inclusion in the virion membrane as opposed to their progression through the secretory pathway (21,22). Membrane biogenesis is also dependent upon the viral F10 kinase (7,(10)(11)(12)23), although how F10 mediates the early stages of assembly remains unknown.…”

Section: Discussionmentioning

confidence: 99%

“…Although A14 and A17 do not possess any recognizable signal sequences, they are thought to be synthesized in the ER (21,22,37,38), after which they can be detected in crescents and in the membranes of immature and mature virions. As an additional means of addressing membrane association, we utilized in vitro transcription-translation reactions (IVTT) to test whether A14 and A17 could undergo co-and/or posttranslational insertion into microsomal membranes (MM).…”

Section: Repressionmentioning

confidence: 99%

“…Neither the origin of the seed membrane nor the mechanism by which the membrane is diverted for maturation into the virion membrane is understood. However, both of the A14 and A17 proteins are thought to be synthesized in the endoplasmic reticulum (ER), despite lacking conventional signal sequences (21,22). Inducible recombinants that express A14 in a tetracycline (TET)-dependent and A17 in an isopropyl-␤-D-thiogalactopyranoside (IPTG)-dependent manner have been invaluable in studying the role of these proteins in membrane biogenesis.…”

mentioning

confidence: 99%

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Biogenesis of the Vaccinia Virus Membrane: Genetic and Ultrastructural Analysis of the Contributions of the A14 and A17 Proteins

Unger

Mercer

Boyle

et al. 2013

J Virol

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Vaccinia virus membrane biogenesis requires the A14 and A17 proteins. We show here that both proteins can associate with membranes co-but not posttranslationally, and we perform a structure function analysis of A14 and A17 using inducible recombinants. In the absence of A14, electron-dense virosomes and distinct clusters of small vesicles accumulate; in the absence of A17, small vesicles form a corona around the virosomes. When the proteins are induced at 12 h postinfection (hpi), crescents appear at the periphery of the electron-dense virosomes, with the accumulated vesicles likely contributing to their formation. A variety of mutant alleles of A14 and A17 were tested for their ability to support virion assembly. For A14, biologically important motifs within the N-terminal or central loop region affected crescent maturation and the immature virion (IV)¡mature virion (MV) transition. For A17, truncation or mutation of the N terminus of A17 engendered a phenotype consistent with the N terminus of A17 recruiting the D13 scaffold protein to nascent membranes. When N-terminal processing was abrogated, virions attempted to undergo the IV-to-MV transition without removing the D13 scaffold and were therefore noninfectious and structurally aberrant. Finally, we show that A17 is phosphorylated exclusively within the C-terminal tail and that this region is a direct substrate of the viral F10 kinase. In vivo, the biological competency of A17 was reduced by mutations that prevented its serine-threonine phosphorylation and restored by phosphomimetic substitutions. Precleavage of the C terminus or abrogation of its phosphorylation diminished the IV¡MV maturation; a block to cleavage spared virion maturation but compromised the yield of infectious virus. P oxviruses are complex DNA viruses that are ubiquitous in nature; the orthopoxvirus family includes variola virus, the etiologic agent of smallpox, and the closely related monkeypox virus, which is endemic in Africa and also causes a severe, acute febrile illness (1, 2). The prototypic poxvirus for experimental study is the closely related vaccinia virus, which has played an eminent role in public health as the vaccine strain used in the successful eradication of smallpox. Poxviruses are unique among DNA viruses in that they replicate exclusively in the cytoplasm of infected cells (2). This physical autonomy is enabled by the presence of ϳ200 genes in the viral genome that encode the machinery for virion entry, gene expression, genome replication and maturation, virion assembly and maturation, and virion egress. The assembly of nascent virions is one of the most unique facets of poxviral infection (3). Unlike most other enveloped viruses, vaccinia virus does not acquire its delimiting membrane by budding into an organelle or through the plasma membrane. Instead, the membrane develops within the cytoplasm, seeded by small patches of membrane that grow into characteristic crescents. As these nascent membrane sheets develop, they engulf virosomal proteins destined for encapsidat...

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Section: Discussionmentioning

confidence: 99%

Section: Repressionmentioning

confidence: 99%

mentioning

confidence: 99%

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Biogenesis of the Vaccinia Virus Membrane: Genetic and Ultrastructural Analysis of the Contributions of the A14 and A17 Proteins

Unger

Mercer

Boyle

et al. 2013

J Virol

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“…Such observations are consistent with the identification of ER-and ER-Golgi-intermediate compartment-derived membrane tubules and vesicles near VV crescents. Interestingly, recent work shows that if the VV A9 membrane protein is targeted to the ER, it will subsequently become incorporated into the inner envelope of the virus (17,18). This suggests that even though a direct continuity of membranes between cellular compartments and the viral envelopes has been difficult to demonstrate (22), there is communication between the ER and the lipid envelope of VV.…”

Section: Vol 82 2008 Asfv Inner Envelope Is a Single Membrane 7909mentioning

confidence: 99%

The Envelope of Intracellular African Swine Fever Virus Is Composed of a Single Lipid Bilayer

Hawes

Netherton

Wileman

et al. 2008

J Virol

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African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.The assembly and envelopment of the large nucleocytoplasmic DNA viruses such as African swine fever virus (ASFV) and vaccinia virus (VV) have been the subject of many studies, but the mechanisms and pathways that govern the morphogenesis of each virus still remain controversial (1,11,12,23,24). ASFV virions have a classic icosahedral structure that is similar to that of the iridoviruses, whereas VV immature virions (IV) are spherical. Despite the obvious differences in appearance between intracellular ASFV virions and VV IV, the structural organizations are similar. The center of each virus consists of the genome surrounded by a layer of protein, which in the case of ASFV is called the matrix or inner core shell. This is in turn enclosed by a lipid envelope which lies beneath the capsid (2, 3, 6, 7, 10). The immature VV matures into the brick-shaped intracellular mature virus. ASFV gains a further outer envelope as it buds through the plasma membrane on exit (21). The following experiments refer to the intracellular form of ASFV only.Considerable analysis of the envelopes of the spherical immature and brick-shaped intracellular mature forms of VV suggests that the envelope originates from membrane crescents formed in the cytoplasm near assembly sites. Early models of VV assembly proposed that the crescents and IV envelopes were single lipid bilayers formed by de novo membrane biogenesis (8). However, sequencing of the poxvirus genome did not support this initial hypothesis, as genes encoding lipid synthesis enzymes were not found (25). In addition, intracellular membranes are usually present as double-membrane cisternae, and it was unclear how the virus could stabilize single bilayers within the cellular environment. In an ...

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“…Subsequently, the intermediate compartment between the endoplasmic reticulum (ER) and Golgi membrane was considered the source of the crescent membrane based on the localization of certain viral proteins (9)(10)(11)(12). Further studies, however, showed that proteins could traffic from the ER to the crescent membrane and that blockade of the secretory pathway from the ER to the Golgi apparatus did not perturb either crescent formation or their progression to immature virions (IVs) and mature virions (MVs) (13)(14)(15). The findings that the VACV L2 membrane protein is required for IV formation and that it is located at the edges of the crescents and the ER further support an ER origin of the viral membrane (16,17).…”

mentioning

confidence: 99%

Direct Formation of Vaccinia Virus Membranes from the Endoplasmic Reticulum in the Absence of the Newly Characterized L2-Interacting Protein A30.5

Maruri-Avidal

Weisberg

Moss

2013

J Virol

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Sequence-Independent Targeting of Transmembrane Proteins Synthesized within Vaccinia Virus Factories to Nascent Viral Membranes

Cited by 19 publications

References 39 publications

Biogenesis of the Vaccinia Virus Membrane: Genetic and Ultrastructural Analysis of the Contributions of the A14 and A17 Proteins

Biogenesis of the Vaccinia Virus Membrane: Genetic and Ultrastructural Analysis of the Contributions of the A14 and A17 Proteins

The Envelope of Intracellular African Swine Fever Virus Is Composed of a Single Lipid Bilayer

Direct Formation of Vaccinia Virus Membranes from the Endoplasmic Reticulum in the Absence of the Newly Characterized L2-Interacting Protein A30.5

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