Virulent Avian Virus Research Articles

Newcastle disease virus Malaysian strain AF2240 induces apoptosis in MCF-7 human breast carcinoma cells at an early stage of the virus life cycle

Newcastle disease virus (NDV) AF2240 Malaysian strain is a very virulent avian virus. NDV strain AF2240 was previously demonstrated to induce apoptosis in human breast carcinoma MCF-7 cells. However, at which stage of the NDV life cycle apoptosis is induced and whether NDV replication and protein synthesis are involved in apoptosis induction have yet to be determined. In the present study, we investigated the time course of NDV strain AF2240 nucleoprotein (NP) gene expression and the early apoptotic signs in the form of activation of caspase-8 and mitochondrial transition pore opening. In addition, the induction of apoptosis by both ultraviolet-inactivated and cycloheximide-treated NDV-infected MCF-7 cells were examined. Our findings showed that NDV strain AF2240 induced apoptosis at 1h post-infection (pi) through activation of mitochondrial transition pore opening and at 2h through activation of caspase-8, while the NP gene was expressed at 6h pi. The induced apoptosis was independent of both virus replication and protein synthesis. In conclusion, NDV strain AF2240 induces apoptosis at an early stage of its life cycle, possibly during virus binding or fusion with the cell membrane. The mitochondrial-related pathway may be the central activator in NDV strain AF2240-induced apoptosis.

Virulent influenza A viruses induce apoptosis in chickens

Virulent avian influenza A viruses produce lethal disease in chickens. Since cell death can be caused by either necrosis or apoptosis, we investigated the types of cell death that occur in natural hosts, chickens, infected with virulent avian viruses. Using biochemical methods, we demonstrate that virulent avian influenza viruses induce apoptosis of vascular endothelial cells in liver, kidney, and brain. Viral antigens were also detected in these organs, suggesting that viral replication induces apoptosis in infected chickens. These results indicate that apoptosis does occur in virulent avian influenza virus infection in a natural host, and may contribute to the lethality of the virus.

Structure of the Hemagglutinin Precursor Cleavage Site, a Determinant of Influenza Pathogenicity and the Origin of the Labile Conformation

The membrane fusion potential of influenza HA, like many viral membrane-fusion glycoproteins, is generated by proteolytic cleavage of a biosynthetic precursor. The three-dimensional structure of ectodomain of the precursor HA0 has been determined and compared with that of cleaved HA. The cleavage site is a prominent surface loop adjacent to a novel cavity; cleavage results in structural rearrangements in which the nonpolar amino acids near the new amino terminus bury ionizable residues in the cavity that are implicated in the low-pH-induced conformational change. Amino acid insertions at the cleavage site in HAs of virulent avian viruses and those of viruses isolated from the recent severe outbreak of influenza in humans in Hong Kong would extend this surface loop, facilitating intracellular cleavage.

Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses.

Among the proprotein-processing subtilisin-related endoproteases, furin has been a leading candidate for the enzyme that activates the hemagglutinin (HA) of virulent avian influenza viruses. In the present study, we examined the cleavage activity of two other recently isolated ubiquitous subtilisin-related proteases, PACE4 and PC6, using wild-type HA of A/turkey/Ireland/1378/83 (H5N8) and a series of its mutant HAs. Vaccinia virus-expressed wild-type HA was not cleaved in human colon adenocarcinoma LoVo cells, which lack active furin. This processing defect was corrected by the expression of furin and PC6 but not of PACE4 and a control wild-type vaccinia virus. PC6 showed a sequence specificity similar to that with the endogenous proteases in cultured cells. When LoVo cells were infected with a virulent avian virus, A/turkey/Ontario/7732/66 (H5N9), only noninfectious virions were produced because of the lack of HA cleavage. However, when the cells were coinfected with vaccinia virus that expressed either furin or PC6, the avian virus underwent multiple cycles of replication, indicating that both furin and PC6 specifically cleave the virulent virus HA at the authentic site. These data suggest that PC6, as well as furin, can activate virulent avian influenza viruses in vivo, implying the presence of multiple HA cleavage enzymes in animals.

Location and character of the cellular enzyme that cleaves the hemagglutinin of a virulent avian influenza virus

H.-D. Klenk, W. Garten, and R. Rott (1984, EMBO J. 3, 2911–2915) have reported that hemagglutinin (HA) cleavage of virulent avian influenza viruses occurs in later steps of its intracellular transport and that the cleavage enzyme is calcium dependent and has a neutral pH optimum. The precise intracellular location of the HA cleavage, however, has never been established. Furthermore, because Klenk et al. used the whole cell lysate to examine the cleavage activity and the amino acid sequencing of the cleaved product was not done, the identity of the cleavage enzyme remains to be established. We therefore attempted to systematically characterize the HA cleavage of the virulent avian virus A/tern/South Africa/61 (H5N3). Using an inhibitor of glycoprotein transport (Brefeldin A) and temporal markers of glycoprotein processing, we found that the endoprotease responsible for the HA cleavage acts after the acquisition of endo- N-acetylglucosaminidase H resistance but before the addition of galactose to the molecule, and thus is located in the medial and/or trans Golgi. This observation was directly confirmed by in vitro experiments using rat liver subcellular membrane fractions containing Golgi complex. A fraction rich in galactosyltransferase (a trans Golgi marker) demonstrated the highest HA cleavage activity. The endoprotease in this fraction cleaved only the HA of the virulent avian influenza virus but not that of an avirulent virus. Through amino-terminal sequencing of the HA2 produced by digestion with the endoprotease in the rat Golgi fraction, we established that HA cleavage by the protease occurs at the authentic site. Further studies using the rat Golgi fraction showed that the HA cleavage enzyme is calcium dependent and has a low pH (6.0) optimum. Thus, the pH optimum of the enzyme in the Golgi fraction differs from that in whole cell lysate reported previously.