Identification of a functional nuclear localization signal in 3Dpol/3CD of duck hepatitis A virus 1.

The nuclear localization signals (NLS) were usually composed of basic residues (K and R) and played an important role in delivery of genomes and structural protein into nucleus. In this research, we identified that 3Dpol/3CD entered into nucleus during viral propagation of duck hepatitis A virus type 1 (DHAV-1). To investigate the reason that 3Dpol/3CD entered into nucleus, the amino acid sequence of 3CD was analyzed through NLS Mapper program. The basic region 17PRKTAYMRS25 was subsequently proved to be a functional NLS to guide 3Dpol/3CD into nucleus. 18R, 19K and 24R were found essential for maintaining the nuclear targeting activity, and exchange between 24R and 24K had no impact on cellular localization of 3Dpol. Since the entry of 3Dpol/3CD into nucleus was essential for shutoff of host cell transcription and maintaining the viral propagation of picornavirus numbers, our study provided new insights into the mechanism of DHAV-1 propagation.

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization.

BACKGROUND: The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription. The 3C protease has been regarded as a valid target due to its structural similarity among different picornaviruses and minimal sequence similarity with host proteins; therefore, the development of potent inhibitors against the 3C protease as an antiviral drug is ongoing. Duck hepatitis A virus (DHAV) belongs to the Picornavidea family and is a major threat to the poultry industry. To date, little is known about the roles of the DHAV 3C protease plays during infection. METHODS: In this study, we compared the full-length DHAV 3C protein sequence with other 3C sequences to obtain an alignment for the construction of a phylogenetic tree. Then, we expressed and purified recombinant DHAV 3C protease in the BL21 expression system using nickel-NTA affinity chromatography. The optimization of the cleavage assay conditions and the kinetic analysis for DHAV 3C protease were done by in vitro cleavage assays with a fluorogenic peptide respectively. The inhibitory activity of rupintrivir against the DHAV 3C protease was further evaluated. The localization of the 3C protease in infected and transfected cells was determined using immunofluorescence and confocal microscopy. RESULTS: Under different expression conditions, the 3C protease was found to be highly expressed after induction with 1 mM IPTG at 16 °C for 10 h. We synthesized a fluorogenic peptide derived from the cleavage site of the DHAV polyprotein and evaluated the protease activity of the DHAV 3C protease for the first time. We used fluorimetric based kinetic analysis to determine kinetic parameters, and Vmax and Km values were determined to be 16.52 nmol/min and 50.78 µM, respectively. Rupintrivir was found to exhibit inhibitory activity against the DHAV 3C protease. Using polyclonal antibody and an indirect immunofluorescence microscopy assay (IFA), it was determined that the DHAV 3C protease was found in the nucleus during infection. In addition, the DHAV 3C protease can enter into the nucleus without the cooperation of viral proteins. CONCLUSIONS: This is the first study to examine the activity of the DHAV 3C protease, and the activity of the DHAV 3C protease is temperature-, pH- and NaCl concentration- dependent. The DHAV 3C protease localizes throughout DHAV-infected cells and can enter into the nucleus in the absence of other viral proteins. The kinetic analysis was calculated, and the Vmax and Km values were 16.52 nmol/min and 50.78 µM, respectively, using the Lineweaver-Burk plot.

Cryptic protein priming sites in two different domains of duck hepatitis B virus reverse transcriptase for initiating DNA synthesis in vitro.

Initiation of reverse transcription in hepadnaviruses is accomplished by a unique protein-priming mechanism whereby a specific Y residue in the terminal protein (TP) domain of the viral reverse transcriptase (RT) acts as a primer to initiate DNA synthesis, which is carried out by the RT domain of the same protein. When separate TP and RT domains from the duck hepatitis B virus (DHBV) RT protein were tested in a trans-complementation assay in vitro, the RT domain could also serve, unexpectedly, as a protein primer for DNA synthesis, as could a TP mutant lacking the authentic primer Y (Y96) residue. Priming at these other, so-called cryptic, priming sites in both the RT and TP domains shared the same requirements as those at Y96. A mini RT protein with both the TP and RT domains linked in cis, as well as the full-length RT protein, could also initiate DNA synthesis using cryptic priming sites. The cryptic priming site(s) in TP was found to be S/T, while those in the RT domain were Y and S/T. As with the authentic TP Y96 priming site, the cryptic priming sites in the TP and RT domains could support DNA polymerization subsequent to the initial covalent linkage of the first nucleotide to the priming amino acid residue. These results provide new insights into the complex mechanisms of protein priming in hepadnaviruses, including the selection of the primer residue and the interactions between the TP and RT domains that is essential for protein priming.

Localization of HSP90 binding sites in the human hepatitis B virus polymerase.

The fact that HSP90 proteins and their chaperonin partners play an important role in epsilon RNA binding of duck HBV Pol protein during duck HBV replication has been reported. To elucidate the molecular basis of HBV Pol/HSP90 interaction, we have characterized the HSP90 interaction to HBV Pol. We found that human HBV Pol protein upon synthesis in rabbit reticulocyte lysate formed a complex with HSP90 in vitro as duck HBV Pol did. In addition, HSP90 protein was copurified with MBP/POL protein expressed in HepG2 cells, suggesting that human HBV Pol protein is associated with HSP90 in vivo. To localize the HSP90 interaction site region, several deletion mutants of HBV Pol translated in vitro were immunoprecipitated with anti-HSP90 antibody. The result indicates that C-terminal regions of the TP and RT domains interact with HSP90 independently.