Serotype-specific structural differences in the protease-cofactor complexes of the dengue virus family.

With an estimated 40% of the world population at risk, dengue poses a significant threat to human health, especially in tropical and subtropical regions. Preventative and curative efforts, such as vaccine development and drug discovery, face additional challenges due to the occurrence of four antigenically distinct serotypes of the causative dengue virus (DEN1 to -4). Complex immune responses resulting from repeat assaults by the different serotypes necessitate simultaneous targeting of all forms of the virus. One of the promising targets for drug development is the highly conserved two-component viral protease NS2B-NS3, which plays an essential role in viral replication by processing the viral precursor polyprotein into functional proteins. In this paper, we report the 2.1-A crystal structure of the DEN1 NS2B hydrophilic core (residues 49 to 95) in complex with the NS3 protease domain (residues 1 to 186) carrying an internal deletion in the N terminus (residues 11 to 20). While the overall folds within the protease core are similar to those of DEN2 and DEN4 proteases, the conformation of the cofactor NS2B is dramatically different from those of other flaviviral apoprotease structures. The differences are especially apparent within its C-terminal region, implicated in substrate binding. The structure reveals for the first time serotype-specific structural elements in the dengue virus family, with the reported alternate conformation resulting from a unique metal-binding site within the DEN1 sequence. We also report the identification of a 10-residue stretch within NS3pro that separates the substrate-binding function from the catalytic turnover rate of the enzyme. Implications for broad-spectrum drug discovery are discussed.

Severe acute respiratory syndrome coronavirus nonstructural protein 2 interacts with a host protein complex involved in mitochondrial biogenesis and intracellular signaling.

The severe acute respiratory syndrome coronavirus (SARS-CoV) generates 16 nonstructural proteins (nsp's) through proteolytic cleavage of a large precursor protein. Although several nsp's exhibit catalytic activities that are important for viral replication and transcription, other nsp's have less clearly defined roles during an infection. In order to gain a better understanding of their functions, we attempted to identify host proteins that interact with nsp's during SARS-CoV infections. For nsp2, we identified an interaction with two host proteins, prohibitin 1 (PHB1) and PHB2. Our results suggest that nsp2 may be involved in the disruption of intracellular host signaling during SARS-CoV infections.

The intracellular cargo receptor ERGIC-53 is required for the production of infectious arenavirus, coronavirus, and filovirus particles.

Arenaviruses and hantaviruses cause severe human disease. Little is known regarding host proteins required for their propagation. We identified human proteins that interact with the glycoproteins (GPs) of a prototypic arenavirus and hantavirus and show that the lectin endoplasmic reticulum (ER)-Golgi intermediate compartment 53 kDa protein (ERGIC-53), a cargo receptor required for glycoprotein trafficking within the early exocytic pathway, associates with arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus GPs. ERGIC-53 binds to arenavirus GPs through a lectin-independent mechanism, traffics to arenavirus budding sites, and is incorporated into virions. ERGIC-53 is required for arenavirus, coronavirus, and filovirus propagation; in its absence, GP-containing virus particles form but are noninfectious, due in part to their inability to attach to host cells. Thus, we have identified a class of pathogen-derived ERGIC-53 ligands, a lectin-independent basis for their association with ERGIC-53, and a role for ERGIC-53 in the propagation of several highly pathogenic RNA virus families.

By inhibiting replication, the large hepatitis delta antigen can indirectly regulate amber/W editing and its own expression.

Hepatitis delta virus (HDV) expresses two essential proteins with distinct functions. The small hepatitis delta antigen (HDAg-S) is expressed throughout replication and is needed to promote that process. The large form (HDAg-L) is farnesylated, is expressed only at later times via RNA editing of the amber/W site, and is required for virion assembly. When HDAg-L is artificially expressed at the onset of replication, it strongly inhibits replication. However, there is controversy concerning whether HDAg-L expressed naturally at later times as a consequence of editing and replication can similarly inhibit replication. Here, by stabilizing the predicted secondary structure downstream from the amber/W site, a replication-competent HDV mutant that exhibited levels of editing higher than those of the wild type was created. This mutant expressed elevated levels of HDAg-L early during replication, and at later times, its replication aborted prematurely. No further increase in amber/W editing was observed following the cessation of replication, indicating that editing was coupled to replication. A mutation in HDAg-L and a farnesyl transferase inhibitor were both used to abolish the ability of HDAg-L to inhibit replication. Such treatments rescued the replication defect of the overediting mutant, and even higher levels of amber/W editing resulted. It was concluded that when expressed naturally during replication, HDAg-L is able to inhibit replication and thereby inhibit amber/W editing and its own synthesis. In addition, the structure adjacent to the amber/W site is suboptimal for editing, and this creates a window of time in which replication can occur in the absence of HDAg-L.

Determination of the multimerization state of the hepatitis delta virus antigens in vivo.

Hepatitis delta virus expresses two essential proteins, the small and large delta antigens, and both are required for viral propagation. Proper function of each protein depends on the presence of a common amino-terminal multimerization domain. A crystal structure, solved using a peptide fragment that contained residues 12 to 60, depicts the formation of an octameric ring composed of antiparallel coiled-coil dimers. Because this crystal structure was solved for only a fragment of the delta antigens, it is unknown whether octamers actually form in vivo at physiological protein concentrations and in the context of either intact delta antigen. To test the relevance of the octameric structure, we developed a new method to probe coiled-coil structures in vivo. We generated a panel of mutants containing cysteine substitutions at strategic locations within the predicted monomer-monomer interface and the dimer-dimer interface. Since the small delta antigen contains no cysteine residues, treatment of cell extracts with a mild oxidizing reagent was expected to induce disulfide bond formation only when the appropriate pairs of cysteine substitution mutants were coexpressed. We indeed found that, in vivo, both the small and large delta antigens assembled as antiparallel coiled-coil dimers. Likewise, we found that both proteins could assume an octameric quaternary structure in vivo. Finally, during the course of these experiments, we found that unprenylated large delta antigen molecules could be disulfide cross-linked via the sole cysteine residue located within the carboxy terminus. Therefore, in vivo, the C terminus likely provides an additional site of protein-protein interaction for the large delta antigen.