An infectious and full-length molecular clone of genomic human spumaretrovirus (HSRV) DNA was constructed. The infectivity of the pHSRV13 clone was demonstrated after transfection into susceptible cells by passage of HSRV-specific cytopathic effects as a cell-free culture supernatant, by electron microscopy of HSRV particles in pHSRV13 DNA-transfected cells, by detection of HSRV transcripts, and by identification of HSRV-encoded proteins with Env- and Bel-specific antisera in indirect immunofluorescence assays and in protein blotting. The predominant HSRV protein detected in immunoblots by both Bel 1- and Bel 2-specific antisera had an apparent molecular weight of 56 kDa and corresponds to Bet. The amino-terminus of Bet is encoded by part of a Bel 1-specific RNA and the larger Bet domain by an RNA species from the bel 2 gene (Muranyi, W., and Flügel, R. M. J. Virology 65, 727-735, 1991). HSRV-specific proteins of 36 and 43 kDa reacted with Bel 1 and Bel 2 antisera, consistent with the values calculated for the bel 1 and bel 2 gene products, respectively. Deletion mutagenesis of the transcriptional HSRV-specific trans-activator bel 1 and the bet genes completely abolished the infectivity of the pHSRV13 clone. The defect in RNA, protein, and virion synthesis was trans-complemented by cotransfection of an expression clone harboring the complete bel coding region. This result demonstrates that the bel 1 gene is required for viral replication. It remains to be determined whether other HSRV gene products, like bet that share a common region with bel 1, contributed to the defect observed.