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The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. SVSV-Cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and SMHV-TMDCyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. SVSV-TMDCyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that SVSV-TMDCyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.In the winter of 2002 to 2003, a new type of pneumonia, severe acute respiratory syndrome (SARS), emerged in Guangdong province, China. The etiological agent causing this disease was found to be an unknown coronavirus, which was named SARS coronavirus (SARS-CoV) (10,18,23,30).Among the structural proteins of SARS-CoV, the spike (S) protein is the largest, comprising 1,255 amino acids. Research on SARS and other coronavirus S proteins has shown that S is involved in receptor binding and membrane fusion and is a major determinant of the immune response and pathogenesis (12). The spike protein is a type I membrane protein and is anchored in the membrane of the virion. Peplomers, oligomers of two or three spike proteins (9), form the distinctive "corona" on the virus.The main receptor for SARS-CoV has been identified as angiotensin-converting enzyme 2 (ACE-2) (20). ACE-2 is expressed in several tissues, among which are epithelia in the lung and small intestine (15). It has been shown that amino acids 318 to 510 of SARS-CoV S are sufficient to bind to ACE-2 (1, 44, 45). L-SIGN has been shown to function as an alternative receptor, albeit with a significantly lower affinity than ACE-2 (17). Another lectin, DC-SIGN, has been implicated in enhancement of infection by dendritic cell transfer, a process earlier described for other viruses such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV) (16,24,49).In most but not all coronaviruses, S is cleaved during viral maturation by a host cell protease to create the subunits S1 and S2 (11,40). It is unclear at this moment whether this type of...

BackgroundThe spike protein (S) of SARS Coronavirus (SARS-CoV) mediates entry of the virus into target cells, including receptor binding and membrane fusion. Close to or in the viral membrane, the S protein contains three distinct motifs: a juxtamembrane aromatic part, a central highly hydrophobic stretch and a cysteine rich motif. Here, we investigate the role of aromatic and hydrophobic parts of S in the entry of SARS CoV and in cell-cell fusion. This was investigated using the previously described SARS pseudotyped particles system (SARSpp) and by fluorescence-based cell-cell fusion assays.ResultsMutagenesis showed that the aromatic domain was crucial for SARSpp entry into cells, with a likely role in pore enlargement.Introduction of lysine residues in the hydrophobic stretch of S also resulted in a block of entry, suggesting the borders of the actual transmembrane domain. Surprisingly, replacement of a glycine residue, situated close to the aromatic domain, with a lysine residue was tolerated, whereas the introduction of a lysine adjacent to the glycine, was not. In a model, we propose that during fusion, the lateral flexibility of the transmembrane domain plays a critical role, as do the tryptophans and the cysteines.ConclusionsThe aromatic domain plays a crucial role in the entry of SARS CoV into target cells. The positioning of the aromatic domain and the hydrophobic domain relative to each other is another essential characteristic of this membrane fusion process.

The genome of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) was screened for the presence of putative origins of DNA replication (oris). Using a transient DNA replication assay, several fragments were identified that underwent SeMNPV-dependent DNA replication in Spodoptera frugiperda cells (Sf-AE-21). Preliminary sequence data revealed the presence of multiple copies of homologous repeats (hrs). Restriction fragment XbaI-F2 showed a distinct sequence reminiscent of Autographa californica and Orgyia pseudotsugata MNPV (AcMNPV and OpMNPV) non-hr oris. Deletion analysis of this fragment indicated that the essential sequences of this putative non-hr ori mapped within a region of 800 bp. Sequence analysis of this region showed a unique distribution of six different (im)-

To study the function of the envelope glycoprotein gH of pseudorabies virus, a gH null mutant was constructed. A premature translation termination codon was introduced in the gH gene by linker insertion mutagenesis, and a mutant virus was rescued by using a cell line that expresses the wild-type protein. Mutant virus isolated from complementing cells was unable to form plaques on noncomplementing cells, indicating that gH is essential in the life cycle of the virus. Immunological staining and electron microscopy showed that the mutant virus produced noninfectious progeny and was unable to spread from infected to uninfected cells by cell-cell fusion. Thus, similar to gH of herpes simplex virus, gH of pseudorabies virus is required for entry and cell-to-cell spread.