Combined results from solution studies on intact influenza virus M1 protein and from a new crystal form of its N-terminal domain show that M1 is an elongated monomer

The amino-terminal domain of influenza A virus matrix protein (residues 1-164) was crystallized at pH 7 into a new crystal form in space group P1. This packing of the protein implies that M1(1-164) was monomeric in solution when it crystallized. Otherwise, the structure of the M1 fragment in the pH 7 crystals was the same as the monomers in crystals formed at pH 4 where crystal packing resulted in dimer formation [B. Sha and M. Luo, 1997, Nature Struct. Biol. 4, 239-244]. Analysis of intact M1 protein, the N-terminal domain, and the remaining C-terminal fragment (residues 165-252) in solution also showed that the N-terminal domain was monomeric with the same dimensions as determined from the crystal structure. Intact M1 protein was also monomeric but with an elongated shape due to the presence of the C-terminal part. Circular dichroism showed that the C-terminal part of M1 contained helical structure. A model for soluble M1 is presented, based on the assumption that the C-terminal domain is spherical, in which the N- and C-terminal domains are connected by a linker sequence which is available for proteolytic attack.     

A crucial role of N-terminal domain of influenza A virus M1 protein in interaction with swine importin α1 protein

The matrix 1 (M1) protein is a multifunctional protein in the life cycle of influenza virus. It plays an important role in virus budding and intracellular trafficking of viral ribonucleoproteins (vRNPs). The M1 protein consists of three domains based on the structure: N-terminal domain, Middle domain, and C-terminal domain. However, the functions of different domains of the M1 protein remain largely unclear. In this study, using bimolecular fluorescence complementation assays (BIFC) we demonstrated that swine importin α1 interacts with the M1 protein and transports it to the nucleus. Interestingly, M1 with mutated nuclear localization signal (NLS; 101-RKLKR-105 to 101-AALAA-105) still interacts with swine importin α1 and is localized in the nucleus, suggesting that the NLS located at residues 101–105 is not the only NLS within M1 recombinant protein containing 1–160 residues of M1 with mutated nuclear localization signal is able to interact with swine importin α1, but M1/60-252 domains cannot bind importin α1. Further mapping showed that the deletion of residues 1–20 impaired the interaction between N terminus of M1 and importin α1. Collectively, our data suggested that the N-terminal domain of M1 protein is critical for binding swine importin α1 and for nuclear localization.     

Evidence for two nonoverlapping functional domains in the potato virus X 25K movement protein.

To study subdomain organization of the potato virus X (PVX) movement protein (MP) encoded by the first gene in the triple gene block (TGB), we mutated the 25-kDa TGBp1 protein. The N-terminal deletion of the helicase motifs I, IA, and II resulted in loss of the ATPase activity and RNA binding. A frameshift mutation truncating the C-terminal motifs V and VI gave rise to increase of the TGBp1 ATPase activity and had little effect on RNA binding in vitro. Fusions of the green fluorescent protein with 25-kDa MP and its derivative lacking motifs V-VI exhibited similar fluorescence patterns in epidermal cells of Nicotiana benthamiana leaves. Cell-to-cell movement of the 25K-deficient PVX genome was not complemented by the TGBp1 of Plantago asiatica mosaic potexvirus (PlAMV) but was efficiently complemented by a chimeric TGBp1 consisting of the N-terminal part of PlAMV protein (motifs I-IV) and the PVX-specific C-terminal part (motifs V-VI). These results suggest that NTP hydrolysis, RNA binding, and targeting to the specific cellular compartment(s) are associated with the N-terminal domain of the TGBp1 including the helicase motifs I-IV and that the C-terminal domain is involved in specific interactions with other virus proteins.     

Replication in vitro of the influenza virus genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein complexes

Summary Replication of the influenza virus genome involves two discrete step reactions: vRNA-directed primer-independent (unprimed) synthesis of cRNA; and cRNA-directed unprimed synthesis of vRNA. Nuclear extracts from both MDCK and HeLa cells infected with influenza virus A/PR8/34 exhibited unprimed synthesis of both cRNA and vRNA strands (a parameter of RNA replication). Ribonucleoprotein (RNP) complexes with the replication activity were isolated from these nuclear extracts by glycerol gradient centrifugation in the presence of 0.1 M KCl. At 0.5 M KCl, however, these complexes were dissociated into stripped RNP and soluble protein fractions. The soluble fraction contained the activity of exogenous template-dependent unprimed RNA synthesis, indicating that the RNA replicase is dissociated from RNP upon exposure to high salt concentrations. On the other hand, the high salt-treated RNP catalyzed only primer-dependent RNA synthesis, but regained a low level activity of exogenous template-dependent unprimed RNA synthesis by adding nuclear extracts from uninfected cells, suggesting that host factor(s) is involved in the functional interconversion of influenza virus RNA polymerase.     

N-terminal region of the PB1-F2 protein is responsible for increased expression of influenza A viral protein PB1

Influenza A virus (IAV) PB1-F2 protein is encoded by an alternative reading frame (+1) within the PB1 gene. PB1-F2 has been shown to contribute to the pathogenesis of influenza virus infection as well as to the secondary bacterial infection. More recently has been shown that PB1-F2 protein may regulate a viral RNA (vRNA) polymerase activity by the interaction with PB1 protein. We proved that PB1-F2 protein increased the level of expression of PB1 protein and vRNA in the infected cells. Moreover, we demonstrated that a higher level of vRNA expression resulted in the increase of expression of multiple viral proteins, including NP, M1, and NS1. Finally, we used plasmids expressing N-terminal (1-50 aa) or C-terminal (51-87 aa) region of the PB1-F2 molecule for transfection of MDCK cells co-infected with influenza A/Puerto Rico/8/34 (H1N1) virus deficient in the PB1-F2 protein expression (PR8ΔPB1-F2). These experiments clearly showed that N-terminal region of PB1-F2 protein was responsible for the increase in PB1 protein expression. C-terminal region of PB1-F2 protein had no effect. Thus, we have identified the important function for N-terminal region of PB1-F2 protein.     

Comparing the antibody responses against recombinant hemagglutinin proteins of avian influenza A (H5N1) virus expressed in insect cells and bacteria

The hemagglutinin (HA) of influenza A virus plays an essential role in mediating the entry of the virus into host cells. Here, recombinant full-length HA5 protein from a H5N1 isolate (A/chicken/hatay/2004(H5N1)) was expressed and purified from the baculovirus-insect cell system. As expected, full-length HA5 elicits strong neutralizing antibodies, as evaluated in micro-neutralization tests using HA5 pseudotyped lentiviral particles. In addition, two fragments of HA5 were expressed in bacteria and the N-terminal fragment, covering the ectodomain before the HA1/HA2 polybasic cleavage site, was found to elicit neutralizing antibodies. But the C-terminal fragment, which covers the remaining portion of the ectodomain, did not. Neutralizing titer of the anti-serum against the N-terminal fragment is only four times lower than the anti-serum against the full-length HA5 protein. Using a novel membrane fusion assay, the abilities of these antibodies to block membrane fusion were found to correlate well with the neutralization activities.