Hence, the accessibility of the antibody epitopes may be affected by differences in intertrimer contacts around the head of the VP6 molecule (13)

Hence, the accessibility of the antibody epitopes may be affected by differences in intertrimer contacts around the head of the VP6 molecule (13). epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a VH1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibodydouble-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or PDE-9 inhibitor accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding KSR2 antibody of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore. == INTRODUCTION == Rotaviruses (RVs) are nonenveloped, triple-layered icosahedral viruses that belong to theReoviridaefamily, and they are the leading cause of severe diarrheal illness in infants and young children worldwide (1). The inner capsid layer of virus protein 2 (VP2) encloses 11 segments of double-stranded RNA that each encode a single protein, except for segment 11, which codes for two proteins. The VP2 layer is surrounded by 780 molecules of virus protein 6 (VP6). VP6 is arranged into 260 trimers and forms the intermediate viral capsid layer. The VP2 and VP6 layers form the transcriptionally active double-layered particle (DLP). The outer capsid layer is composed of virus protein 7 (VP7) with spikes of virus protein 4 (VP4) and forms the transcriptionally inactive mature infectious virion particle (28). RV VP6 is folded into two distinct domains: an -helical base domain with a triangular cross section and PDE-9 inhibitor a jelly-roll head domain that forms a roughly hexagonal cross section. The T=13 icosahedral symmetry of the VP6 and VP7 layers defines three types of channels that exist within the viral architecture: type I, II, and III channels. Twelve type I channels are located down the icosahedral 5-fold axes and serve as egress points of nascent viral mRNA during viral transcription (6). Sixty type II channels are arranged to surround the type I channels, and 60 type III channels are positioned PDE-9 inhibitor around the icosahedral 3-fold axes (5,912). While the VP6 layer has a T=13 symmetry, the inner VP2 layer of the DLP has a T=1 architecture. This mismatch in symmetry between the VP2 and VP6 layers leads to five distinct VP6 trimer positions on the DLP outer layer, designated P (pentad), P (peripentonal), T (triad), T (adjacent to triad), or D (dyad) (4,13). The P trimers surround the PDE-9 inhibitor icosahedral 5-fold axes, the T trimers are located directly on the 3-fold axes, and the D trimers are adjacent to the 2-fold axes. The P trimers are the only trimers that contact the pentad trimers, and the T trimers are the only ones to contact the triad trimers. Antibody repertoire studies in our laboratory identified VH1-46 as the dominant antibody heavy chain variable gene segment used for the natural human B cell response to RV VP6 in infants and adults (1417). Representative antibodies encoded by the VH1-46 gene segment were expressed and characterized (18). For example, the monoclonal antibody (MAb) RV6-26 was shown to inhibit virus replication when introduced into PDE-9 inhibitor cells, whereas MAb RV6-25, another human VP6-specific antibody encoded by VH1-46, was unable to do so. Previously, we elucidated the structural basis for viral inhibition by RV6-26 (19). In the studies presented here, we sought to understand why the closely related MAb RV6-25 was unable to inhibit virus, even though it was derived from the same donor, bound VP6, and also was encoded by the identical VHgene segment. We employed a hybrid method approach for epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange mass spectrometry (DXMS) techniques. The data revealed that antibody bound to the location of the RV6-25 epitope on the apical surface of the RV VP6 head domain and does not obstruct the.