Recent outbreaks of Zika virus (ZIKV) highlight an urgent need for therapeutics.The protease complex NS2B-NS3 plays essential roles during flaviviral polyprotein processing, and thus represents an attractive drug target. Here, we developed a split luciferase complementation-based high-throughput screening assay to identify orthosteric inhibitors that directly target flavivirus NS2B-NS3 interactions. By screening a total of 2 816 approved and investigational drugs, we identified three potent candidates, temoporfin, niclosamide, and nitazoxanide, as flavivirus NS2B-NS3 interaction inhibitors with nanomolar potencies. Significantly, the most potent compound, temoporfin, not only inhibited ZIKV replication in human placental and neural progenitor cells, but also prevented ZIKV-induced viremia and mortality in mouse models. Structural docking suggests that temoporfin potentially binds NS3 pockets that hold critical NS2B residues, thus inhibiting flaviviral polyprotein processing in a non-competitive manner. As these drugs have already been approved for clinical use in other indications either in the USA or other countries, they represent promising and easily developed therapies for the management of infections by ZIKV and other flaviviruses.

No abstract

The genetic characterization of a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHIV) is described. The mutant, Albany 4 (Alb4), is both temperature sensitive and thermolabile. Analysis of the progeny of a mixed infection showed that the defective Alb4 allele is recessive to wild type, and its gene product is diffusible. The N protein of Alb4 was found to be smaller than its wild-type counterpart, and sequence analysis of the Alb4 N gene revealed that it contains an internal deletion of 87 nucleotides, producing an in-frame deletion of 29 amino acids. All of these properties of Alb4 made it ideal for use as a recipient in a targeted RNA recombination experiment in which the deletion in Alb4 was repaired by recombination with synthetic RNA7, the smallest MHV subgenomic mRNA. Progeny from a cotransfection of Alb4 genomic RNA and synthetic RNA7 were selected for thermal stability. Polymerase chain reaction analysis of candidate recombinants showed that they had regained the material that is deleted in the Alb4 mutant. They also had acquired a five-nucleotide insertion in the 3' untranslated region, which had been incorporated into the synthetic RNA7 as a molecular tag. The presence of the tag was directly verified, as well, by sequencing the genomic RNA of purified recombinant viruses. This provided a clear genetic proof that the Alb4 phenotype was due to the observed deletion in the N gene. In addition, these results demonstrated that it is possible to obtain stable, independently replicating progeny from recombination between coronavirus genomic RNA and a tailored, synthetic RNA species.

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.

The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus ( The assembly of coronaviruses is driven principally by homotypic and heterotypic interactions between the two most abundant virion proteins, the membrane protein (M) and the nucleocapsid protein (N) (14, 32). The M protein is a triplespanning transmembrane protein residing in the virion envelope, which is derived from the cellular budding site, the endoplasmic reticulum-Golgi intermediate compartment. More than half of the M molecule, its carboxy-terminal endodomain, is situated in the interior of the virion, where it contacts the nucleocapsid (46, 50). Also found in the virion envelope is the spike protein (S), which, although crucial for viral infectivity, is not an essential participant in assembly. The other canonical component of the coronavirus envelope is the small envelope protein (E), the function of which is enigmatic. Some evidence suggests that the E protein does not make sequence-specific contacts with other viral proteins (27) but instead functions by modifying the budding compartment, perhaps as an ion channel (56, 57). Alternatively, or additionally, E could act in a chaperone-like fashion to facilitate homotypic interactions between M protein monomers or oligomers (4).The N protein is the only protein constituent of the helically symmetric nucleocapsid, which is located in the interior of the virion. Coronavirus N proteins are largely basic phosphoproteins that share a moderate degree of homology across all three of the phylogenetic groups within the family (29). Some time ago, we proposed a model that pictured the N protein as comprising three domains separated by two spacers (A and B) (40). This arrangement was originally inferred from a sequence comparison of the N genes of multiple strains of the prototypical group 2 coronavirus, mouse hepatitis virus (MHV), and its validity seemed to be reinforced by numerous sequences that later became available. Part of this model, the delineation of spacer B and the acidic, carboxy-terminal domain 3, has been well supported by subsequent work (22,25,41,42). However, a wealth of recent, detailed structural studies of bacterially expressed domains of the N proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV) and of infectious bronchitis virus (IBV) has much more precisely mapped boundaries within the remainder of the N molecule (8,16,21,23,47,51,60). The latter studies have shown that the N protein contains two independently folding domains, designated the N-terminal domain (NTD) and the C-terminal domain (CTD). It should be pointed out that this nomenclature can be misleading: the NTD does not contain the amino terminus of ...

Coronavirus assembly results from an accumulation of interactions among four structural proteins, the positive-sense RNA genome, and a host membrane envelope obtained from the site of budding, which is the endoplasmic reticulum-Golgi intermediate compartment. Three of the four structural proteins are embedded in the virion envelope: the spike protein (S), the membrane protein (M), and the small envelope protein (E). The fourth, the nucleocapsid protein (N), resides in the virion interior, wrapping the positive-strand RNA genome into a helical nucleocapsid.The most abundant viral constituent is M, a 25-kDa protein containing three transmembrane segments flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. The M protein is the central organizer of assembly, in that it self-associates (8, 10, 21), captures S for virion incorporation (36, 37), and selectively packages the fraction of N protein that is bound to genomic RNA (32-34). The S protein is responsible for viral attachment to host cell receptors and for the membrane fusion event that initiates infection. This type I membrane protein, shortly after its synthesis, folding, and oligomerization, forms complexes with M protein in the endoplasmic reticulum (36, 37). The M-interacting domain of the S protein was localized to the transmembrane-endodomain region of the molecule, based on the assembly of chimeric S proteins into virus-like particles (VLPs) (16) or virions (18, 23). More recently, this determinant was further localized to the endodomain of S, which could confer virion assembly competence if transferred to a heterologous transmembrane protein (48).The general (1, 3, 4, 31, 45), but not universal (20), consensus from studies of VLPs is that formation of coronaviruses is mediated by just the M and the E proteins, and that neither S protein nor the nucleocapsid plays an obligate role in virion morphogenesis. The precise role of E protein in this process is enigmatic, with some evidence pointing to a direct interaction between E and M (1, 5) and other observations suggesting that E acts independently of M in the budding compartment (4,26,41). A very recent finding that may shed light on the workings of the E protein is the demonstration that the E protein of severe acute respiratory syndrome coronavirus has the properties of a cation-selective ion channel (47). The construction of E protein mutants of mouse hepatitis virus (MHV) has confirmed the critical role of E in viral assembly (14). Surprisingly, however, MHV remains viable, although severely impaired, following deletion of the E gene (25), whereas disruption of the E gene of porcine transmissible gastroenteritis virus (TGEV) is lethal (6,38).The interaction between M protein and N protein has been previously explored by biochemical and molecular biological methods for both MHV (33,34, 44) and TGEV (12). In a genetic approach, we constructed and analyzed a highly defective MHV mutant with a carboxy-terminal truncated M protein, M⌬2, and we identified suppressors of this...

We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Masters, J. Virol. 66:1841-1848. By using a thermolabile N protein mutant of MHV (Alb4) as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, we selected engineered recombinant viruses as heat-stable progeny resulting from cotransfection. We have now been able to greatly increase the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that, with Alb4 as the recipient, recombinants can be directly identified without using thermal selection. The synthetic DI RNA has been used to demonstrate that the lesion in another temperature-sensitive and thermolabile MHV mutant, Albl, maps to the N gene. Sequencing of the Albl N gene revealed two closely linked point mutations that fall in a region of the N molecule previously noted as being the most highly conserved region among all of the coronavirus N proteins. Analysis of revertants of the Albl mutant revealed that one of the two mutations is critical for the temperature-sensitive phenotype; the second mutation is phenotypically silent.

C oronaviruses (CoVs) are a family of enveloped positive-strand RNA viruses that cause disease in numerous mammalian and avian hosts (1, 2). Of the six coronaviruses that can infect humans, the two of greatest current concern are the etiologic agents of severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV). Virions of coronaviruses contain a canonical set of four structural proteins. The most numerous constituent, the membrane (M) protein, makes up a lattice in the viral envelope that associates with the other components. Trimers of spike (S) protein form projections on the virion surface responsible for attachment to host cell receptors, and minor amounts of the small envelope (E) protein also appear in the viral membrane. In the virion interior, the nucleocapsid (N) protein encloses the ϳ30-kb viral genome into a helically symmetric ribonucleoprotein.Much of our knowledge of coronavirus assembly has been worked out through studies with the prototype coronavirus mouse hepatitis virus (MHV). MHV falls into the betacoronaviruses, the second of the four genera of the family and the one which also includes SARS-CoV and MERS-CoV. Key contributions to understanding virion morphogenesis have also been made through analyses of the gammacoronavirus infectious bronchitis virus (IBV) and the alphacoronavirus transmissible gastroenteritis virus (TGEV). A large body of work points to M protein as the major player in virion assembly. Coexpression of subsets of viral proteins revealed that just M protein and E protein are sufficient for the formation of virus-like particles (VLPs) (3-5). The inclusion of N protein, although it is not strictly required, greatly enhances the efficiency of VLP formation (6, 7). The critical role of E protein is carried out at the site of budding, the endoplasmic reticulum-Golgi intermediate compartment, with very little E being carried over into assembled virions (8). Additionally, M protein captures S protein for incorporation into virions or VLPs (9, 10), but S is an optional participant in virus formation (11,12), even though it is essential for infectivity.Thus, extensive networks of protein-protein interactions in coronavirus assembly involve one or both of the most abundant virion components, M and N. The N protein is a highly basic phosphoprotein containing the structurally distinct amino-terminal RNA-binding domain (NTD) and the carboxy-terminal RNAbinding domain (CTD) (13), which we have previously called domains N1b and N2b, respectively (14-16) (Fig. 1A). In MHV N