Structural basis for norovirus neutralization by an HBGA blocking human IgA antibody.

Human noroviruses (HuNoVs) cause sporadic and epidemic gastroenteritis worldwide. They are classified into two major genogroups (GI and GII), with each genogroup further divided into multiple genotypes. Susceptibility to these viruses is influenced by genetically determined histo-blood group antigen (HBGA) expression. HBGAs function as cell attachment factors by binding to a surface-exposed region in the protruding (P) domain of the capsid protein. Sequence variations in this region that result in differential HBGA binding patterns and antigenicity are suggested to form a basis for strain diversification. Recent studies show that serum antibodies that block HBGA binding correlate with protection against illness. Although genogroup-dependent variation in HBGA binding specificity is structurally well characterized, an understanding of how antibodies block HBGA binding and how genotypic variations affect such blockade is lacking. Our crystallographic studies of the GI.1 P domain in complex with the Fab fragment of a human IgA monoclonal antibody (IgA 5I2) with HBGA blocking activity show that the antibody recognizes a conformational epitope formed by two surface-exposed loop clusters in the P domain. The antibody engulfs the HBGA binding site but does not affect its structural integrity. An unusual feature of the antigen recognition by IgA 5I2 is the predominant involvement of the CDR light chain 1 in contrast to the commonly observed CDR heavy chain 3, providing a unique perspective into antibody diversity in antigen recognition. Identification of the antigenic site in the P domain shows how genotypic variations might allow escape from antibody neutralization and exemplifies the interplay between antigenicity and HBGA specificity in HuNoV evolution.

Structural plasticity of the coiled-coil domain of rotavirus NSP4.

UNLABELLED: Rotavirus (RV) nonstructural protein 4 (NSP4) is a virulence factor that disrupts cellular Ca(2+) homeostasis and plays multiple roles regulating RV replication and the pathophysiology of RV-induced diarrhea. Although its native oligomeric state is unclear, crystallographic studies of the coiled-coil domain (CCD) of NSP4 from two different strains suggest that it functions as a tetramer or a pentamer. While the CCD of simian strain SA11 NSP4 forms a tetramer that binds Ca(2+) at its core, the CCD of human strain ST3 forms a pentamer lacking the bound Ca(2+) despite the residues (E120 and Q123) that coordinate Ca(2+) binding being conserved. In these previous studies, while the tetramer crystallized at neutral pH, the pentamer crystallized at low pH, suggesting that preference for a particular oligomeric state is pH dependent and that pH could influence Ca(2+) binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca(2+) or pH. Biochemical, biophysical, and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high-affinity binding to Ca(2+) at neutral pH and forms a tetramer, it does not bind Ca(2+) at low pH and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca(2+) binding is necessary for the tetramer formation, as an E120A mutant forms a pentamer. We propose that the structural plasticity of NSP4 regulated by pH and Ca(2+) may form a basis for its pleiotropic functions during RV replication. IMPORTANCE: The nonstructural protein NSP4 of rotavirus is a multifunctional protein that plays an important role in virus replication, morphogenesis, and pathogenesis. Previous crystallography studies of the coiled-coil domain (CCD) of NSP4 from two different rotavirus strains showed two distinct oligomeric states, a Ca(2+)-bound tetrameric state and a Ca(2+)-free pentameric state. Whether NSP4 CCD from the same strain can exist in different oligomeric states and what factors might regulate its oligomeric preferences are not known. This study used a combination of biochemical, biophysical, and crystallography techniques and found that the NSP4 CCD can undergo a reversible transition from a Ca(2+)-bound tetramer to a Ca(2+)-free pentamer in response to changes in pH. From these studies, we hypothesize that this remarkable structural adaptability of the CCD forms a basis for the pleiotropic functional properties of NSP4.

Probing the sites of interactions of rotaviral proteins involved in replication.

UNLABELLED: Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms, which form during virus infection. These processes are orchestrated by yet-to-be-understood complex networks of interactions involving nonstructural proteins (NSPs) 2, 5, and 6 and structural proteins (VPs) 1, 2, 3, and 6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner, NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1, and the inner core protein VP2. Using isothermal calorimetry, biolayer interferometry, and peptide array screening, we examined the interactions between NSP2, VP1, VP2, NSP5, and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close proximity to the RNA template entry and double-stranded RNA (dsRNA) exit tunnels of VP1 and near the catalytic cleft and RNA-binding grooves of NSP2; these sites are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1. Peptide screening of VP2 identified NSP2-binding sites in the regions close to the intersubunit junctions, suggesting that NSP2 binding could be a regulatory mechanism for preventing the premature self-assembly of VP2. The binding sites on NSP2 for NSP6 were found to overlap that of VP1, and the NSP5-binding sites overlap those of VP2 and VP1, suggesting that interaction of these proteins with NSP2 is likely spatially and/or temporally regulated. IMPORTANCE: Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection and are orchestrated by complex networks of interactions involving nonstructural proteins (NSPs) and structural proteins (VPs). A multifunctional RNA-binding NSP2 octamer with nucleotidyl phosphatase activity is central to viroplasm formation and RNA replication. Here we provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from peptide arrays are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1 and also point to NSP2's possible role in preventing the premature self-assembly of VP2 cores. Our findings lead us to propose that the NSP2 octamer with multiple enzymatic activities is a principal regulator of viroplasm formation, recruitment of viral proteins into the viroplasms, and possibly genome replication.

Over-pressure suppresses ultrasonic-induced drug uptake.

Ultrasound (US) is used to enhance and target delivery of drugs and genes to cancer tissues. The present study further examines the role of acoustic cavitation in US-induced permeabilization of cell membranes and subsequent drug or gene uptake by the cell. Rat colon cancer cells were exposed to ultrasound at various static pressures to examine the hypothesis that oscillating bubbles, also known as cavitating bubbles, permeabilize cells. Increasing pressure suppresses bubble cavitation activity; thus, if applied pressure were to reduce drug uptake, cell permeabilization would be strongly linked to bubble cavitation activity. Cells were exposed to 476 kHz pulsed ultrasound at average intensities of 2.75 W/cm(2) and 5.5 W/cm(2) at various pressures and times in an isothermal chamber. Cell fractions with reversible membrane damage (calcein uptake) and irreversible damage (propidium iodide uptake) were analyzed by flow cytometry. Pressurization to 3 atm nearly eliminated the biological effect of US in promoting calcein uptake. Data also showed a linear increase in membrane permeability with respect to insonation time and intensity. This research shows that US-mediated cell membrane permeability is likely linked to cavitation bubble activity.