Poxviruses are large, enveloped viruses that replicate in the cytoplasm and encode proteins for DNA replication and gene expression. Hairpin ends link the two strands of the linear, double-stranded DNA genome. Viral proteins involved in DNA synthesis include a 117-kDa polymerase, a helicase–primase, a uracil DNA glycosylase, a processivity factor, a single-stranded DNA-binding protein, a protein kinase, and a DNA ligase. A viral FEN1 family protein participates in double-strand break repair. The DNA is replicated as long concatemers that are resolved by a viral Holliday junction endonuclease.

The spike protein (S), a membrane component of severe acute respiratory syndrome coronavirus (SARS-CoV) is anticipated to be an important component of candidate vaccines. We constructed recombinant forms of the highly attenuated modified vaccinia virus Ankara (MVA) containing the gene encoding full-length SARS-CoV S with and without a C-terminal epitope tag called MVA/S-HA and MVA/S, respectively. Cells infected with MVA/Sor MVA/S-HA synthesized a 200-kDa protein, which was recognized by antibody raised against a synthetic peptide of SARS-CoV S or the epitope tag in Western blot analyses. Further studies indicated that S was N - glycosylated and migrated in SDS polyacrylamide gels with an apparent mass of ≈160 kDa after treatment with peptide N-glycosidase F. The acquisition of resistance to endoglycosidase H indicated trafficking of S to the medial Golgi compartment, and confocal microscopy showed that S was transported to the cell surface. Intranasal or intramuscular inoculations of BALB/c mice with MVA/S produced serum antibodies that recognized the SARS S in ELISA and neutralized SARS-CoV in vitro . Moreover, MVA/S administered by either route elicited protective immunity, as shown by reduced titers of SARS-CoV in the upper and lower respiratory tracts of mice after challenge. Passive transfer of serum from mice immunized with MVA/S to naïve mice also reduced the replication of SARS-CoV in the respiratory tract after challenge, demonstrating a role for antibody to S in protection. The attenuated nature of MVA and the ability of MVA/S to induce neutralizing antibody that protects mice support further development of this candidate vaccine.

For many viruses, one or two proteins enable cell binding, membrane fusion and entry. The large number of proteins employed by poxviruses is unprecedented and may be related to their ability to infect a wide range of cells. There are two main infectious forms of vaccinia virus, the prototype poxvirus: the mature virion (MV), which has a single membrane, and the extracellular enveloped virion (EV), which has an additional outer membrane that is disrupted prior to fusion. Four viral proteins associated with the MV membrane facilitate attachment by binding to glycosaminoglycans or laminin on the cell surface, whereas EV attachment proteins have not yet been identified. Entry can occur at the plasma membrane or in acidified endosomes following macropinocytosis and involves actin dynamics and cell signaling. Regardless of the pathway or whether the MV or EV mediates infection, fusion is dependent on 11 to 12 non-glycosylated, transmembrane proteins ranging in size from 4- to 43-kDa that are associated in a complex. These proteins are conserved in poxviruses making it likely that a common entry mechanism exists. Biochemical studies support a two-step process in which lipid mixing of viral and cellular membranes is followed by pore expansion and core penetration.