Atomic Structure of Human Adenovirus by Cryo-EM Reveals Interactions Among Protein Networks

Liu, Hongrong; Jin, Lei; Koh, Sok Boon S.; Atanasov, Ivo; Schein, Stan; Wu, Lily; Zhou, Z. Hong

doi:10.1126/science.1187433

Cited by 341 publications

(459 citation statements)

References 39 publications

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“…Full atomic models by the cryoEM method were previously only achieved for single particles with icosahedral symmetry (18,19). Reaching such resolution for helical objects opens the door for atomic-resolution structural determinations of important systems such as amyloid fibers, flagellar filaments, and actin.…”

Section: Discussionmentioning

confidence: 99%

“…On the one hand, magnification variability due to nonparallel illumination and phase errors due to beam tilt can severely limit the resolution of the cryoEM structures obtained. Recently, techniques such as parallel illumination and coma-free alignment have been used in cryoEM (18,19), eliminating many of the resolution barriers imposed by previous imaging systems. On the other hand, previous helical reconstruction methods have demanded "perfect" helical symmetry for higher-resolution reconstructions, a condition that cannot be met by helical assemblies in reality.…”

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confidence: 99%

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Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches

Zhou

2011

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

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116

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Helical assemblies such as filamentous viruses, flagella, and F-actin represent an important category of structures in biology. As the first discovered virus, tobacco mosaic virus (TMV) was at the center of virus research. Previously, the structure of TMV was solved at atomic detail by X-ray fiber diffraction but only for its dormant or high-calcium-concentration state, not its low-calcium-concentration state, which is relevant to viral assembly and disassembly inside host cells. Here we report a helical reconstruction of TMV in its calcium-free, metastable assembling state at 3.3 Å resolution by cryo electron microscopy, revealing both protein side chains and RNA bases. An atomic model was built de novo showing marked differences from the high-calcium, dormant-state structure. Although it could be argued that there might be inaccuracies in the latter structure derived from X-ray fiber diffraction, these differences can be interpreted as conformational changes effected by calcium-driven switches, a common regulatory mechanism in plant viruses. Our comparisons of the structures of the low-and highcalcium states indicate that hydrogen bonds formed by Asp116 and Arg92 in the place of the calcium ion of the dormant (highcalcium) state might trigger allosteric changes in the RNA basebinding pockets of the coat protein. In turn, the coat protein-RNA interactions in our structure favor an adenine-X-guanine (A*G) motif over the G*A motif of the dormant state, thus offering an explanation underlying viral assembly initiation by an AAG motif.H elical assemblies represent a very important class of biological structures, performing various tasks for the proper functions of life or executing malicious functions in disease. For example, protein helical assemblies such as cytoskeletal networks and muscle fibers are essential components in our bodies (1, 2). Other helical assemblies include bacterial flagella and secretion systems. Helical viruses, such as tobacco mosaic virus (TMV), account for a major fraction of the virus kingdom. Such helical structures, bearing a special kind of 2D periodicity and potential flexibility, are difficult for structural determination to atomic resolution by conventional methods including X-ray crystallography and NMR. Thus, the molecular mechanisms of action in many of these systems are not understood to atomic detail. Even for TMV, the first virus ever isolated and extensively studied (3) by both X-ray diffraction (e.g., 4-8) and cryo electron microscopy (cryoEM) (9, 10), we still do not know its structure at atomic detail in a state that is relevant to its assembly and disassembly processes, and the underlying mechanisms of these processes remain unknown.The viral RNA of TMV is infectious by itself, but RNA on its own is very unstable. The addition of a 17-kDa coat protein (CP) around the RNA protects the RNA from degrading and makes TMV very stable. The assembly of a rod-shaped virion begins at a two-turn helical CP complex, called the 20S aggregate (11), which recognizes the initiation moti...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches

Zhou

2011

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

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116

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“…It has been suggested that VCP may be required for initial unfolding of tightly folded substrates that lack an intrinsically unstructured region as initiation site for the proteasome (20). The AdV capsid may qualify as such a substrate (8,30). Alternatively, the direct but highly transient interaction between VCP and the proteasome (31) may be specifically stabilized when the proteasome faces a particularly challenging substrate or when it is otherwise impaired.…”

Section: Discussionmentioning

confidence: 99%

AAA ATPase p97/VCP is essential for TRIM21-mediated virus neutralization

Hauler

Mallery

McEwan

et al. 2012

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

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Tripartite motif-containing 21 (TRIM21) is a cytosolic IgG receptor that mediates intracellular virus neutralization by antibody. TRIM21 targets virions for destruction in the proteasome, but it is unclear how a substrate as large as a viral capsid is degraded. Here, we identify the ATPase p97/valosin-containing protein (VCP), an enzyme with segregase and unfoldase activity, as a key player in this process. Depletion or catalytic inhibition of VCP prevents capsid degradation and reduces neutralization. VCP is required concurrently with the proteasome, as addition of inhibitor after proteasomal degradation has no effect. Moreover, our results suggest that it is the challenging nature of virus as a substrate that necessitates involvement of VCP, since intracellularly expressed IgG Fc is degraded in a VCP-independent manner. These results implicate VCP as an important host factor in antiviral immunity.A ntibody neutralization is a key component of the antiviral response and provides protective immunity. Our recent work has shown that neutralization can occur inside cells, in an effector-driven process mediated by tripartite motif-containing 21 (TRIM21). TRIM21, a cytosolic antibody receptor of ultrahigh affinity to IgG Fc (1, 2), is recruited to antibody-bound virus and targets the complex to the proteasome for degradation (3). This process potently neutralizes viral infection and has been termed antibody-dependent intracellular neutralization (ADIN) (4). ADIN is dependent upon the E3 ubiquitin ligase activity of TRIM21 and can be abrogated by chemical inhibition of the proteasome.Although both proteasomal activity and ubiquitination are necessary for ADIN, the exact mechanism of virus degradation is poorly understood. Specifically, it is not clear how the proteasome can degrade a virion, a compact proteinaceous particle much larger than the proteasome itself. The 26S proteasome has a mass of ∼2.5 MDa (5), and the pore through which substrates must pass to access the proteolytic chamber is no greater than 2 nm in diameter (6, 7). In contrast, human adenovirus (AdV), a virus potently neutralized by TRIM21, has a diameter of ∼100 nm and a mass of 150 MDa (8).Although there are ATPases in the 19S regulatory subunit of the 26S proteasome that may unfold substrates and allow them to enter through the pore (5, 6), AdV virions are much larger than any of the proteasome's known cellular substrates. Because ADIN has been shown to be independent of autophagy but dependent on proteasomal degradation (3), we hypothesized that an additional energydependent step of AdV capsid disassembly and/or unfolding might precede proteasomal degradation of the virus.In recent studies, the ATPase p97/VCP of the AAA (ATPases associated with diverse cellular activities) family has been implicated in the proteasomal degradation of certain cytosolic substrates (9-13). VCP is capable of dissociating proteins from large cellular structures such as the endoplasmic reticulum (14), the mitotic spindle (12), the nuclear envelope (15), and chromatin (1...

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“…Cryo-EM reconstructions of "single particles"-macromolecules or assemblies that do not form higher-order arrays-have been increasing in resolution over the last several years and have recently yielded the first few atomic and near-atomic resolution structures (Cong et al, 2010;Liu et al, 2010;Ludtke et al, 2008;Yu et al, 2008;Zhang et al, 2010;Zhang et al, 2008). However, these successes are confined mainly to samples with long histories as benchmarks in the field or to those exhibiting a large degree of internal symmetry (such as icosahedral viruses).…”

Section: Introductionmentioning

confidence: 99%

Validation of the orthogonal tilt reconstruction method with a biological test sample

Chandramouli

Hernández-López

Wang

et al. 2011

Journal of Structural Biology

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Atomic Structure of Human Adenovirus by Cryo-EM Reveals Interactions Among Protein Networks

Cited by 341 publications

References 39 publications

Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches

Hydrogen-bonding networks and RNA bases revealed by cryo electron microscopy suggest a triggering mechanism for calcium switches

AAA ATPase p97/VCP is essential for TRIM21-mediated virus neutralization

Validation of the orthogonal tilt reconstruction method with a biological test sample

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