The role of putative DNA-binding domains of human papillomavirus (HPV) capsid proteins for DNA encapsidation in vivo is still unknown. We have now analyzed mutants of the major capsid protein L1 of HPV type 33, which are defective for DNA binding, for their ability to encapsidate DNA using an in vivo packaging approach. Since the DNA-binding domain and the nuclear localization signal (NLS) of L1 overlap, both a carboxy-terminal deletion mutant (L1-1/470) and a substitution mutant (L1-1/477M9) were analyzed. L1-1/477M9 has the classical NLS replaced by a noncanonical NLS taken from the human hnRNP protein A1. The mutant proteins were defective for DNA binding in contrast to wild-type (wt) L1 proteins of several HPV types. L1-1/470 localized to the cytoplasm when expressed in human cell lines, whereas L1-1/477M9 was found mainly in the nucleus. When coexpressed with wt L2, mutant L1 proteins colocalized with L2 in nuclear domains 10 (ND10) similar to wt L1 and assembled into virus-like particles incorporating L2. Wt L1 and L2 but not mutant L1 proteins and wt L2 efficiently encapsidated DNA and transferred it into cells, suggesting that physical interaction of L1 with DNA is essential for DNA inclusion into VLPs. We also compared the infectivity of histone-free DNA-containing pseudovirions generated in vitro with the infectivity of pseudovirions generated in vivo, which most likely harbor DNA in the form of chromatin. Pseudovirions generated in vivo were, on a per genome basis, four to five times more infectious than pseudovirions generated in vitro, even though no structural or biochemical difference was obvious. This indicates that chromatin may enhance the infectivity of pseudovirions.