Discovery of pentacyclic triterpenoids as potential entry inhibitors of influenza viruses.

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.

Development of bivalent oleanane-type triterpenes as potent HCV entry inhibitors.

The development of entry inhibitors is an emerging approach to the prevention and reduction of HCV infection. Starting from echinocystic acid (EA), a µM HCV entry inhibitor, we have developed a series of bivalent oleanane-type triterpenes which, upon optimization of the length, rigidity and hydrophobicity of the linker, exert dramatically potent enhancement of inhibition with IC50 values extending into the nM level. This study establishes the importance of triterpene natural products as new leads in the development of potential HCV entry inhibitors.

Development of oleanane-type triterpenes as a new class of HCV entry inhibitors.

Development of hepatitis C virus (HCV) entry inhibitors represents an emerging approach that satisfies a tandem mechanism for use with other inhibitors in a multifaceted cocktail. By screening Chinese herbal extracts, oleanolic acid (OA) was found to display weak potency to inhibit HCV entry with an IC50 of 10 µM. Chemical exploration of this triterpene compound revealed its pharmacophore requirement for blocking HCV entry, rings A, B, and E, are conserved while ring D is tolerant of some modifications. Hydroxylation at C-16 significantly enhanced its potency for inhibiting HCV entry with IC50 at 1.4 µM. Further modification by conjugation of this new lead with a disaccharide at 28-COOH removed the undesired hemolytic effect and, more importantly, increased its potency by ~5-fold (54a, IC50 0.3 µM). Formation of a triterpene dimer via a linker bearing triazole (70) dramatically increased its potency with IC50 at ~10 nM. Mechanistically, such functional triterpenes interrupt the interaction between HCV envelope protein E2 and its receptor CD81 via binding to E2, thus blocking virus and host cell recognition. This study establishes the importance of triterpene natural products as new leads for the development of potential HCV entry inhibitors.

Identification and characterization of three novel nuclear export signals in the influenza A virus nucleoprotein.

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.

A nuclear export signal in the matrix protein of Influenza A virus is required for efficient virus replication.

The influenza A virus matrix 1 protein (M1) shuttles between the cytoplasm and the nucleus during the viral life cycle and plays an important role in the replication, assembly, and budding of viruses. Here, a leucine-rich nuclear export signal (NES) was identified specifically for the nuclear export of the M1 protein. The predicted NES, designated the Flu-A-M1 NES, is highly conserved among all sequences from the influenza A virus subtype, but no similar NES motifs are found in the M1 sequences of influenza B or C viruses. The biological function of the Flu-A-M1 NES was demonstrated by its ability to translocate an enhanced green fluorescent protein (EGFP)-NES fusion protein from the nucleus to the cytoplasm in transfected cells, compared to the even nuclear and cytoplasmic distribution of EGFP. The translocation of EGFP-NES from the nucleus to the cytoplasm was not inhibited by leptomycin B. NES mutations in M1 caused a nuclear retention of the protein and an increased nuclear accumulation of NEP during transfection. Indeed, as shown by rescued recombinant viruses, the mutation of the NES impaired the nuclear export of M1 and significantly reduced the virus titer compared to titers of wild-type viruses. The NES-defective M1 protein was retained in the nucleus during infection, accompanied by a lowered efficiency of the nuclear export of viral RNPs (vRNPs). In conclusion, M1 nuclear export was specifically dependent on the Flu-A-M1 NES and critical for influenza A virus replication.

Subcellular localization and topology of porcine reproductive and respiratory syndrome virus E protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) open reading frame (ORF) 2a contains a small internal ORF (2b) capable of encoding a protein of 70 or 73 amino acids (aa), termed E protein. The function and biochemical information of the E protein are currently not clear. In the present investigation, it was shown that the E protein was mainly located in the endoplasmic reticulum (ER) and Golgi complex in MARC-145 cells. Deletion studies identified the N-terminal 15 residues as an ER localization sequence of the E protein, besides two other localization sequences within positions 23-50 and 50-73, and the N-myristoylation site significantly affected the subcellular localization of the N-terminal 15 residues. The membrane association assay demonstrated that the E protein was an integral membrane protein embedded in the phospholipid bilayer. However, neither the N-myristoylation site nor the hydrophilic C-terminal domain was essential to the membrane association of the E protein. The topology analysis revealed that this protein had N-terminus oriented toward the cytoplasm and C-terminus toward the ER lumen. Finally, immunofluorescence assay indicated that the E protein colocalized with GP2, GP3, GP4 and M protein in cotransfected cells, but not N protein.

Cyclophilin A interacts with influenza A virus M1 protein and impairs the early stage of the viral replication.

Influenza A virus matrix protein (M1) is the most abundant conservative protein that regulates the replication, assembly and budding of the viral particles upon infection. Several host cell factors have been determined to interact with M1 possibly in regulating influenza virus replication. By yeast two-hybrid screening, the isomerase cyclophilin A (CypA) was identified to interact with the M1 protein. CypA specifically interacted with M1 both in vitro and in vivo. The mutagenesis results showed CypA bound to the functional middle (M) domain of M1. The depletion of endogenous CypA by RNA interference resulted in the increase of influenza virus infectivity while overexpression of CypA caused decreasing the infectivity in affected cells. The immunofluorescence assays indicated that overexpressed CypA deduced the infectivity and inhibited the translocation of M1 protein into the nucleus while did not affect nucleoprotein entering the nucleus. Further studies indicated that overexpression of CypA significantly increased M1 self-association. Western blot with purified virions confirmed that CypA was encapsidated within the virus particle. These results together indicated that CypA interacted with the M1 protein and affected the early stage of the viral replication.