|Journal of Virology Structure and Assembly|
Rotavirus (RV) nonstructural protein 4 (NSP4) is a virulence factor that disrupts cellular Ca2+ 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 Ca2+ at its core, the CCD of human strain ST3 forms a pentamer lacking the bound Ca2+ despite the residues (E120 and Q123) that coordinate Ca2+ 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 Ca2+ binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca2+ or pH. Biochemical, biophysical, and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high-affinity binding to Ca2+ at neutral pH and forms a tetramer, it does not bind Ca2+ at low pH and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca2+ 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 Ca2+ 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 Ca2+-bound tetrameric state and a Ca2+-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 Ca2+-bound tetramer to a Ca2+-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.
Herpes simplex virus 1 (HSV-1) and HSV-2 are among the most prevalent human pathogens. Both viruses can recognize, via the surface envelope glycoprotein D (gD), human nectin-1 as a functional receptor. Previous studies have successfully elucidated the molecular basis of the binding between HSV-1 gD and nectin-1 by cocrystallography. Despite a high sequence identity between HSV-1 and HSV-2 gDs, the atomic intermolecule details for the HSV-2-gD/nectin-1 interaction remain elusive. Here, we report the crystal structures of both the unbound and the nectin-1-bound HSV-2 gDs. The free-gD structure expectedly comprises an IgV-like core and the surface-exposed terminal extensions as observed in its HSV-1 counterpart but lacks traceable electron densities for a large portion of the terminal elements. These terminal residues were clearly traced in the complex structure as a definitive loop in the N terminus and an aalpha;-helix in the C terminus, thereby showing a conserved nectin-1-binding mode as reported for HSV-1 gD. The interface residues in nectin-1 were further mutated and tested for the gD interaction by surface plasmon resonance. The resultant binding patterns were similar for HSV-1 and HSV-2 gDs, further supporting a homologous receptor-binding basis by the two viruses for nectin-1. These data, together with a cell-based fusion assay showing a cross-inhibition of the gD/nectin-1-mediated cell-cell fusion by soluble HSV-1 and HSV-2 gDs, provided solid structural and functional evidence that HSV-1 and HSV-2 recognize nectin-1 via the same binding mode. Finally, we also demonstrated that nectin-1 I80 is an important residue involved in gD interaction.
IMPORTANCE Despite intensified studies, a detailed picture of the molecular features in the HSV-2-gD/nectin-1 interaction remains unavailable. Previous work focused on HSV-1 gD, which folds into an IgV-like core with large terminal extensions and utilizes the extension elements to engage nectin-1. Here, we report the crystal structures of HSV-2 gD in both the free and the nectin-1-bound forms. The atomic intermolecule details for HSV-2-gD/nectin-1 interaction are clearly presented. The observed binding mode is identical to that reported for its HSV-1 counterpart. This structural observation was further supported by our comparative functional assays showing that nectin-1 mutations similarly affect the ligand-receptor interaction of both virus gDs. Taken together, we provide comprehensive structural and functional data demonstrating a conserved receptor-binding mode between HSV-1 and HSV-2 for nectin-1. Our results also indicate that the tropism difference between the two viruses likely arises from aspects other than the gD/nectin-1 binding features.
HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virionmmdash;termed maturationmmdash;that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal half-life (t1/2) of ~5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity.
IMPORTANCE Cleavage of the Gag and Gag-Pol HIV-1 polyproteins into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release, and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here, we developed an inhibitor washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted, and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing.