The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.When expressed in cells, the precursor Gag (PrGag) proteins of retroviruses such as human immunodeficiency virus (HIV), Rous sarcoma virus (RSV), and murine leukemia viruses are sufficient for the production of virus-like particles (VLP) (36). These proteins encode N-terminal matrix (MA) domains involved in membrane-binding (M) functions; variably located late (L) domains, which are important for VLP budding; and protein-protein interaction (I) regions, composed of the capsid (CA) and nucleocapsid (NC) domains (36). The NC domains bind viral RNA, facilitating its encapsidation, and also have an assembly function (3,5,6,7,10,11,25,26,32,33,35,37,41,42). Results from investigations on NC mutants are consistent with the notion that NC-RNA binding is essential for efficient VLP assembly (3,5,6,10,11,32,33,35,41,42).Previously, we showed that the assembly function of HIV type 1 (HIV-1) NC could be replaced by heterologous dimerization domains (42), an observation that was confirmed by others (1, 19), and suggested that the assembly role of the NC-RNA interaction is to foster dimerization of PrGag proteins. In vitro investigations on RSV Gag proteins carrying the CA N-terminal domains (NTDs), C-terminal domains (CTDs), and NC domains have supported this view (7,25,26) and led to a dimerization model for VLP assembly. As shown in Fig. 1A, Gag proteins composed of the CA NTD, CA CTD, and NC domains concentrate on RNA by virtue of the NC-RNA interaction. Close juxtaposition of the proteins leads to dimerization, which then triggers the assembly of higher-order oligomers.Assuming that the assembly role of the NC-RNA interaction for all retroviruses is to usher the formation of assembly-competent Gag dimers, then other mechanisms for pairing...

The matrix (MA) domain of the HIV-1 structural precursor Gag (PrGag) protein targets PrGag proteins to membrane assembly sites, and facilitates incorporation of envelope proteins into virions. To evaluate the specific requirements for the MA membrane-binding domain (MBD) in HIV-1 assembly and replication, we examined viruses in which MA was replaced by alternative MBDs. Results demonstrated that the pleckstrin homology domains of AKT protein kinase and phospholipase C delta1 efficiently directed the assembly and release of virus-like particles (VLPs) from cells expressing chimeric proteins. VLP assembly and release also were mediated in a phorbol ester-dependent fashion by the cysteine-rich binding domain of phosphokinase Cgamma. Although alternative MBDs promoted VLP assembly and release, the viruses were not infectious. Notably, PrGag processing was reduced, while cleavage of GagPol precursors resulted in the accumulation of Pol-derived intermediates within virions. Our results indicate that the HIV-1 assembly machinery is flexible with regard to its means of membrane association, but that alternative MBDs can interfere with the elaboration of infectious virus cores.

We have identified sultam thioureas as novel inhibitors of West Nile virus (WNV) replication. One such compound inhibited WNV, with a 50% effective concentration of 0.7 M, and reduced reporter expression from cells that harbored a WNV-based replicon. Our results demonstrate that sultam thioureas can block a postentry, preassembly step of WNV replication.West Nile virus (WNV) and Japanese encephalitis virus (JEV) are members of the Flavivirus genus of the Flaviviridae family of viruses (9, 13). These viruses are considered emerging human pathogens (11,12,19,29,32,37). They are closely related to the yellow fever and dengue flaviviruses, and together, these four pathogens are responsible for a significant percentage of virally induced human encephalitis cases worldwide (10-12, 19, 29, 32, 37). One line of defense against flaviviruses is the formulation of vaccines, usually directed against the viral surface envelope (E) proteins (12, 37). Another possible option is the intravenous administration of antiviral antibodies (25,35). A complementary approach has been the development of small-molecule flavivirus inhibitors (7,9,15,17,20,26,27,29,32,36,38,39).To assay for novel WNV inhibitors, we screened a diverse library of approximately 3,500 members for compounds that protected Vero cells from WNV-induced cytopathic effects (CPE). Cells were exposed continuously to a compound concentration of 10 g/ml (10 to 50 M) along with a 1% dimethyl sulfoxide (DMSO) carrier, infected with WNV (NY 1999) (19, 24) at a multiplicity of infection (MOI) of 0.2, and monitored for CPE at 3 to 5 days postinfection (p.i.). Of the candidate WNV inhibitors identified, the sultam thiourea TYT-1 (Fig. 1) appeared the most potent in replicate screens. TYT-1's anti-WNV effects were confirmed in virus yield reduction assays (19,29). Mock-treated and TYT-1-treated Vero cells were infected for 24 h, after which virus-containing medium samples were titrated by limiting dilution on fresh cells in the absence of new compound. An example of our results is shown in Fig. 2. As illustrated and expected, medium from mock-treated, mock-infected ("no virus") cells yielded no deleterious effects on new cells. In contrast, dilutions of Ն10 5 from mock-treated infected ("no TYT-1") cells generated virus sufficient to lyse new cell monolayers completely. However, treatment of cells with 2.3 or 23 M TYT-1 reduced 24-h virus yields Ն100-fold (Fig. 2), substantiating the initial screen results.Determination of the TYT-1 concentration needed to reduce WNV titers twofold (50% effective concentration [EC 50 ]) followed the virus yield reduction regimen described above. As illustrated in Fig. 3 (black bars), the EC 50 of TYT-1 against WNV was approximately 0.7 M. Since our original screening protocol scored for protection of cells from virus-induced CPE, it appeared that TYT-1 was not toxic to cells, at least at 23 M. However, to test this directly, cells were treated with increasing concentrations of TYT-1 and assayed after 48 h for dehydrogenase levels in metabol...