No abstract

Entry inhibitors are of particular importance in current efforts to develop a new generation of anti-influenza virus drugs. Here we report certain pentacyclic triterpenes exhibiting conserved structure features and with in vitro anti-influenza virus activity comparable to and even higher than that of oseltamivir. Mechanistic studies indicated that these lead triterpenoids bind tightly to the viral envelope hemagglutinin (HA), disrupting the interaction of HA with the sialic acid receptor and thus the attachment of viruses to host cells. Docking studies suggest that the binding pocket within HA for sialic acid receptor potentially acts as a targeting domain, and this is supported by structure-activity data, sialic acid competition studies, and broad anti-influenza spectrum as well as less induction of drug resistance. Our study might establish the importance of triterpenoids for development of entry inhibitors of influenza viruses.

The nuclear export of the influenza A virus ribonucleoprotein (vRNP) is crucial for virus replication. As a major component of the vRNP, nucleoprotein (NP) alone can also be shuttled out of the nucleus by interacting with chromosome region maintenance 1 (CRM1) and is therefore hypothesized to promote the nuclear export of the vRNP. In the present study, three novel nuclear export signals (NESs) of the NP-NES1, NES2, and NES3-were identified as being responsible for mediating its nuclear export. The nuclear export of NES3 was CRM1 dependent, whereas that of NES1 or NES2 was CRM1 independent. Inactivation of these NESs led to an overall nuclear accumulation of NP. Mutation of all three NP-NESs significantly impaired viral replication. Based on structures of influenza virus NP oligomers, these three hydrophobic NESs are found present on the surface of oligomeric NPs. Functional studies indicated that oligomerization is also required for nuclear export of NP. Together, these results suggest that the nuclear export of NP is important for virus replication and relies on its NESs and oligomerization.T he influenza A virus genome consists of eight negative-sense single-stranded RNA segments (vRNA) (17). Each vRNA segment is associated with multiple copies of the viral nucleoprotein (NP) and three polymerase subunits (PA, PB1, and PB2), forming the viral ribonucleoprotein (vRNP) complex. During an early stage of infection, the vRNPs are released into the cytoplasm from virions following fusion of the viral membrane and endosomal membrane (41). Subsequently, the incoming vRNPs are transported into the nucleus, where viral genome replication and transcription occur (41).One of the determinants for vRNP nuclear import is NP (8, 28, 44, 49, 52, 53), the major protein in the vRNP structure. Thus far, two nuclear localization signals (NLSs) and a nuclear accumulation signal (NAS) have been identified in NP. The stronger NLS is an unconventional signal located in the N-terminal basic region (between residues 3 and 13) of NP (28, 44), and the weaker signal, a classical bipartite NLS, is located between residues 198 and 216 (49). The NAS-spanning residues (327 to 345) were identified through analyses of mutants that lacked both of the NLSs but still exhibited partial nuclear distribution (9).At a late stage of virus infection, the vRNPs exit the nucleus to assemble and bud from the apical plasma membrane of polarized cells (3). The trafficking of the vRNPs into and out of the nucleus is a tightly regulated process (7). The nuclear export of progeny vRNPs is mediated by the CRM1 cellular export receptor (12,23,29,48). Nuclear export protein (NEP; formerly referred to as the NS2 protein), which possesses a nuclear export signal (NES), and the matrix protein (M1) of influenza A virus are deemed responsible for directing export of the vRNPs (29, 31). This process is regulated by the Raf/MEK/ERK pathway (32), which is stimulated by the membrane association of influenza virus hemagglutinin (HA) (25). Interestingly, exogenously expresse...