The nuclear retention signal of HPV16 L2 protein is essential for incoming viral genome to transverse the trans-Golgi network.

The Human papillomavirus (HPV) capsid is composed of the major and minor capsid proteins, L1 and L2, respectively. Infectious entry requires a complex series of conformational changes in both proteins that lead to uptake and allow uncoating to occur. During entry, the capsid is disassembled and host cyclophilins dissociate L1 protein from the L2/DNA complex. Herein, we describe a mutant HPV16 L2 protein (HPV16 L2-R302/5A) that traffics pseudogenome to the trans-Golgi network (TGN) but fails to egress. Our data provide further evidence that HPV16 traffics through the TGN and demonstrates that L2 is essential for TGN egress. Furthermore, we show that cyclophilin activity is required for the L2/DNA complex to be transported to the TGN which is accompanied by a reduced L1 protein levels.

A membrane-destabilizing peptide in capsid protein L2 is required for egress of papillomavirus genomes from endosomes.

Papillomaviruses are internalized via clathrin-dependent endocytosis. However, the mechanism by which viral genomes pass endosomal membranes has not been elucidated. In this report we show that the minor capsid protein L2 is required for egress of viral genomes from endosomes but not for initial uptake and uncoating and that a 23-amino-acid peptide at the C terminus of L2 is necessary for this function. Pseudogenomes encapsidated by L1 and L2 lacking this peptide accumulated in vesicular compartments similar to that observed with L1-only viral particles, and these mutant pseudoviruses were noninfectious. This L2 peptide displayed strong membrane-disrupting activity, induced cytolysis of bacteria and eukaryotic cells in a pH-dependent manner, and permeabilized cells after exogenous addition. Fusions between green fluorescent protein and the L2 peptide integrated into cellular membranes like the wild type but not like C-terminal mutants of L2. Our data indicate that the L2 C terminus facilitates escape of viral genomes from the endocytic compartment and that this feature is conserved among papillomaviruses. Furthermore, the characteristic of this peptide differs from the classical virus-encoded membrane-penetrating peptides.

Minimal Extent of Sequence Homology Required for Homologous Recombination at the psbA Locus in Chlamydomonas reinhardtii Chloroplasts using PCR-generated DNA Fragments.

The Del1 mutant of the green alga Chlamydomonas reinhardtii with a defined deletion in the chloroplast encoded psbA gene is unable to grow photoautotrophically. We show here that this mutant can be transformed with PCR-generated psbA fragments of varying length to yield photosynthetically growing colonies. PCR fragments need not be purified but can be directly precipitated from the amplification reaction onto tungsten particles, allowing fast and efficient mutagenesis experiments. Flanking regions bordering the deletion breakpoints have been systematically shortened from both sides. The shortest fragment giving rise to significant numbers of transformants contains about 50 bp upstream and 120 bp downstream of the deletion breakpoint. This technique greatly simplifies comprehensive structure-function analyses of the D1 protein in Chlamydomonas, but could perhaps be adapted to other chloroplast genes in this or other organisms as well.