It is generally believed that cellular chaperones facilitate the folding of virus capsid proteins, or that capsid proteins fold spontaneously. Here we show that p73, the major capsid protein of African swine fever virus (ASFV) failed to fold and aggregated when expressed alone in cells. This demonstrated that cellular chaperones were unable to aid the folding of p73 and suggested that ASFV may encode a chaperone. An 80-kDa protein encoded by ASFV, termed the capsid-associated protein (CAP) 80, bound to the newly synthesized capsid protein in infected cells. The 80-kDa protein was released following conformational maturation of p73 and dissociated before capsid assembly. Coexpression of the 80-kDa protein with p73 prevented aggregation and allowed the capsid protein to fold with kinetics identical to those seen in infected cells. CAP80 is, therefore, a virally encoded chaperone that facilitates capsid protein folding by masking domains exposed by the newly synthesized capsid protein, which are susceptible to aggregation, but cannot be accommodated by host chaperones. It is likely that these domains are ultimately buried when newly synthesized capsid proteins are added to the growing capsid shell.Chaperones prevent the irreversible aggregation of proteins in cells (15,16). The Hsp70/DnaK chaperones bind short stretches of hydrophobic amino acids exposed in nascent chains emerging from ribosomes. Proteins released from Hsp70 attempt to fold and bury hydrophobic domains, but if unsuccessful they rebind or are transferred to ring chaperonins such as GroEL and GroES or the TCP1 (TriC/CCT) chaperonin. A broad spectrum of newly synthesized polypeptides associate with these chaperones "in vivo," and the chaperone pathway is viewed as one of broad specificity and high capacity (15,16,19,37). The folding of some proteins, however, requires specialized chaperones, and these may be needed to coordinate protein folding with subunit assembly (18). The PapD chaperones of Escherichia coli, for example, reduce nonproductive interactions between pilin subunits before they are assembled into the base of the growing pilus (3).The careful coordination of protein folding and subunit assembly are important during the assembly of icosahedral viruses. Icosahedral capsids contain an exact number of protein subunits assembled into an ordered lattice; the simplest ones contain 60 identical subunits, while the largest ones contain several thousand. Capsid subunits are synthesized as monomers in the cytosol and expose domains that are ultimately buried during capsid assembly. In order to prevent nonproductive capsid aggregation, it is important that inappropriate interactions between these domains are minimized before delivery of the capsid subunit onto the growing capsid shell. For some viruses aggregation may be prevented by host chaperones (14,17,24,25) or through assembly with scaffold proteins (22,29).The Iridoviridae and African swine fever virus (ASFV) virus are a group of cytoplasmic DNA viruses with very large capsids. ASFV shares th...