Virus-like particles (VLPs) produced by recombinant expression of the major viral structural proteins could be an attractive method for severe acute respiratory syndrome (SARS) control. In this study, using the baculovirus system, we generated recombinant viruses that expressed S, E, M and N structural proteins of SARS-CoV either individually or simultaneously. The expression level, size and authenticity of each recombinant SARS-CoV protein were determined. In addition, immunofluorescence and FACS analysis confirmed the cell surface expression of the S protein. Co-infections of insect cells with two recombinant viruses demonstrated that M and E could assemble readily to form smooth surfaced VLPs. On the other hand, simultaneous high level expression of S, E and M by a single recombinant virus allowed the very efficient assembly and release of VLPs. These data demonstrate that the VLPs are morphological mimics of virion particles. The high level expression of VLPs with correct S protein conformation by a single recombinant baculovirus offers a potential candidate vaccine for SARS.

Bluetongue virus (BTV) is transmitted by Culicoides sp. biting midges to livestock, causing severe hemorrhagic disease in sheep, but is asymptomatic in the insect host. Similarly, BTV causes rapid cell death in infected mammalian cells in culture, whereas infections of insect cells are long-term and unapparent, despite productive virus replication. To assess whether apoptosis plays any role in these two distinct cell responses, we have investigated apoptosis in cultured insect and mammalian cells. Three different mammalian cell lines and three different insect cell lines including Culicoides variipennis (KC) cells were infected with BTV serotype 10, and the key apoptosis indicators of cell morphology, chromosomal DNA fragmentation, and caspase-3 activation were monitored. BTV infection induced apoptosis with the activation of the transcription factor nuclear factor B (NF-B) in all three mammalian cell lines. In contrast, no evidence for apoptosis was detected in any of the three insect cell lines in response to BTV infection. Using inhibitors of endosomal acidification and UV-inactivated virus, we established that virus uncoating, but not productive virus replication, is necessary for BTV to trigger apoptosis in mammalian cells. Intracellular expression of the viral outer capsid proteins VP2 and VP5 or the two major nonstructural proteins NS1 and NS2 was not sufficient to trigger an apoptotic response. However, extracellular treatment with a combination of purified recombinant VP2 and VP5, but not with each protein used separately, resulted in an apoptotic response. Virus-and VP2-VP5-stimulated apoptotic responses were both inhibited by inhibitors of endosomal acidification. Thus, for BTV the viral outer capsid proteins alone are sufficient to trigger apoptosis.

Page 2881, Fig. 8. Panels A and B are duplicates of panel C and should be deleted. Panel C now comprises the entire Fig. 8. The revised figure legend should read: FIG. 8. Translocation of NF-B to the nucleus in HeLa cells infected with BTV-10 or treated with a mixture of purified VP2 and VP5 (VP2/5). Nuclear extracts were prepared from infected/treated cells at 12 h after infection or treatment, and both the p65 and p50 subunits of NF-B were detected by Western blot analysis.

ABSTRACT. Infection of feline immunodeficiency virus (FIV) has been shown to induce apoptosis that might be associated with the lymphocyte depletion in the infected cats. To investigate the inhibitory effect of antioxidants on FIV-induced apoptosis, we examined the effect of N-acetylcysteine (NAC) and ascorbic acid (AA) on apoptosis and virus replication in feline lymphoblastoid (Fel-039) and fibroblastoid (CRFK) cell lines infected with FIV. The treatment with NAC or AA induced a significant inhibition of viral replication and apoptosis in Fel-039 cells and tumor necrosis factor α (TNF-α) -treated CRFK cells infected with FIV. Both cell lines in the presence of noncytotoxic concentrations of NAC or AA showed an increase of intracellular glutathione (GSH) level, which might protect the cells against oxidative stresses exerted by FIV infection and TNF-α treatment. On the basis of these in vitro results, we suggest that antioxidant therapies aimed at restoring depleted GSH level might be effective for inhibition of viral replication and cell death associated with the development of immunodeficiency. -KEY WORDS: apoptosis, ascorbic acid, FIV, N-acetylcysteine.

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An in vitro model of acute and chronic infections with feline immunodeficiency virus (FIV) was used to examine the effect of two immunosuppressive agents, cyclosporin A (CsA) and tacrolimus (also known as FK506), on the inhibition of the replication of the virus and of apoptosis. Both drugs significantly suppressed virus production in a dose-dependent manner in acutely and chronically infected cells. The ability of FK506 to inhibit virus replication was much lower than that of CsA, and was accompanied by marked antiproliferative activity. Treatment of infected cells with either CsA or FK506 did not affect the rise of free intracellular Ca2+ but did protect the cells against apoptosis. Thus, the antiviral activity of CsA and FK506 makes these compounds promising candidates for the development of drugs suitable for the treatment of AIDS.