We have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electrondense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.The study of host-virus interactions not only contributes to our basic knowledge of virology and cell biology but also is important in the further development of gene therapy vector systems (31). We (35) and others (reviewed in reference 4) have observed that the nuclear transport of vector DNA is a major barrier in the transfection of nondividing cells and hence in the application of nonviral gene therapy vectors in vivo. Several DNA-viruses have found an effective solution to this problem (33). The nucleocapsids of adenovirus (14, 15) and the enveloped herpes simplex virus (HSV) (1, 27) are actively transported toward the nucleus and subsequently dock at the nuclear pore. This triggers the release and nuclear transport of the viral genome. The mechanism of this process and the proteins involved are not known. In both cases a nucleocapsid residue is observed at the nuclear pore. However, it is likely that viral proteins are associated with the DNA during transport (14). Nuclear transport of the viral genome depends on the previous process of entry, which may include a passage through the acidic endosomal environment. During this process the viral capsid is modified to allow the next step in the infection sequence (15,33). This entry-dependent modification of viral capsids allows the important functional distinction between an infecting capsid coming in and a newly formed capsid going out of the cell. Detailed knowledge of the nuclear transport pr...