Cryo-Electron Microscopy Structure of Adenovirus Type 2 Temperature-Sensitive Mutant 1 Reveals Insight into the Cell Entry Defect

Abstract

The structure of the adenovirus type 2 temperature-sensitive mutant 1 (Ad2ts1) was determined to a resolution of 10 Å by cryo-electron microscopy single-particle reconstruction. Ad2ts1 was prepared at a nonpermissive temperature and contains the precursor forms of the capsid proteins IIIa, VI, and VIII; the core proteins VII, X (mu), and terminal protein (TP); and the L1-52K protein. Cell entry studies have shown that although Ad2ts1 can bind the coxsackievirus and Ad receptor and undergo internalization via ␣v integrins, this mutant does not escape from the early endosome and is targeted for degradation. Comparison of the Ad2ts1 structure to that of mature Ad indicates that Ad2ts1 has a different core architecture. The Ad2ts1 core is closely associated with the icosahedral capsid, a connection which may be mediated by preproteins IIIa and VI. Density within hexon cavities is assigned to preprotein VI, and membrane disruption assays show that hexon shields the lytic activity of both the mature and precursor forms of protein VI. The internal surface of the penton base in Ad2ts1 appears to be anchored to the core by interactions with preprotein IIIa. Our structural analyses suggest that these connections to the core inhibit the release of the vertex proteins and lead to the cell entry defect of Ad2ts1.Cryo-electron microscopy (cryo-EM) studies of adenovirus (Ad) combined with atomic resolution structures of component proteins (hexon, penton base, fiber, and protease) have led to a detailed structural model for the mature Ad virion (31). While the Ad protein capsid is icosahedral, the core does not follow the overall symmetry of the particle, and thus the core is not well represented in cryo-EM structures (43). The core is composed of the 36-kb double-stranded DNA (dsDNA) genome complexed with four viral proteins (V, VII, mu, and terminal protein [TP]) and the virally encoded cysteine protease. The core of the mature virion may also contain a few copies of the L1-52K protein (7), a possible scaffolding protein that is present in higher copy numbers in assembling virions (18).The capsid contains the major capsid proteins, hexon, penton base, and fiber, together with four minor capsid proteins (IIIa, VI, VIII, and IX). Cryo-EM difference mapping analyses have led to revised assignments for the locations of the minor capsid proteins, with protein IX on the exterior and the other three proteins on the inner capsid surface (9, 38). A scanning transmission EM study indicated that four trimers of protein IX stabilize the group of nine hexons in the center of each facet (11). However, more recent cryo-EM studies indicated that only the N-terminal domain of protein IX forms these trimeric assemblies (37, 38), while the C-terminal domain, which has a long predicted ␣-helix with strong propensity for coiled coil formation, associates in helical bundles at the facet edges (38). Two cryo-EM studies support the assignment of the tetrameric helical bundle on the capsid exterior to the C-terminal domain of protein IX (10, 23). Cur...

The Role of Capsid Maturation on Adenovirus Priming for Sequential Uncoating

“…Loss of peripheral core material is consistent with previous observations indicating release of some internal components in the early endosome, such as core polypeptide V and, more critically, the membrane disrupting polypeptide VI (12,13). Both V and VI are thought to occupy positions bridging the internal capsid surface to the core (4,5,62). Other candidates to be released from slightly disrupted WT capsids are the small peptides resulting from AVP cleavages.…”

“…In both cases, large conformational rearrangements accompany a progressive stabilization of the virus particle. In contrast, adenovirus maturation is not accompanied by large structural rearrangements (4,5). Now we show that, in contrast to the bacteriophage case, the immature adenovirus particle is more stable than the mature one under a variety of stress conditions.…”

“…First, the virus core is more compact and stable due to the condensing action of unprocessed polypeptides pVII and pre-; second, a "molecular stitch" formed by pIIIa and pVIII underpins the boundary between GOS and GONs (5,26); and third, there is a more ordered interaction between pVI and the inner cavity of each hexon in the capsid (4,5). To further investigate how the presence of uncleaved precursors translates into a deficient uncoating phenotype, we have compared the in vitro stability and disassembly of ts1 and WT virions under different types of stress: thermal, chemical, or mechanical.…”

“…Finally, IIIa and VIII cooperate to bind each GOS to its five surrounding GONs. The remaining minor capsid protein, polypeptide VI, could not be traced in this study, but it has been assigned to density within the internal cavity of each hexon trimer (4,5). The adenovirus core contains a single copy of the dsDNA genome and over 25 MDa of protein, including four DNA-binding viral proteins: polypeptides V, VII, , and the terminal protein.…”

Direct Evidence from Single-Cell Analysis that Human α-Defensins Block Adenovirus Uncoating To Neutralize Infection

“…Having established conditions to produce labeled virus particles, we next sought to ensure that the immunofluorescence signal from BrdU was specific to uncoated virus in infected cells and that the labeling procedure did not alter the entry phenotype of the virus. One tool to aid these studies was a well-characterized HAdV-2 temperature sensitive mutant (HAdV-2ts1) that encodes a mutation in the viral protease gene (P137L) that greatly reduces the infectivity of virus grown at the nonpermissive temperature due to a failure to uncoat and escape the endosome (12,21,25). HAdV-2ts1 was produced under the same labeling conditions (4 g/ml BrdU) as wild-type HAdV-2 and was also found to contain labeled genomic DNA.…”