Establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (PML) expression

Abstract

Previous studies have suggested that most papillomaviruses enter the host cell via clathrin-dependent receptor-mediated endocytosis but have not addressed later steps in viral entry. To examine these events, we followed the localization of L2 and packaged DNA after entry of infectious virions or L1͞L2 pseudovirions. Confocal microscopic analyses of HeLa cells showed a time-dependent uncoating of capsids in cytoplasmic vesicles and the accumulation of both L2 and viral DNA at distinct nuclear domains identified as nuclear domain 10 (ND10). Both L2 and the pseudogenome had a punctate distribution and localized to ND10 in promyelocytic leukemia protein (PML)-expressing cells, whereas L2 had a diffuse nuclear distribution in PML؊͞؊ cells. The number of pseudovirus-infected cells was an order of magnitude higher in the PML؉ cells compared with the PML؊͞؊ cells, and viral genome transcription after infection with authentic bovine papillomavirus virions was similarly elevated in PML؉ cells. The results identify a role for PML in the enhancement of viral infectivity in the early part of the life cycle. We propose a model in which L2 chaperones the viral genome to ND10 to efficiently initiate viral transcription.

Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection

“…It has been demonstrated that HPV33 capsids undergo a subtle conformational change after cell attachment (32), raising the possibility of cell-surface cleavage due to exposure of the N terminus of L2. Alternatively, cleavage might occur in an endocytic compartment, because the capsids clearly undergo further conformational changes, as shown by the exposure of the genome and to antibodies (5).…”

“…Construction of CHA-L2 is described in ref. 5. To create NHA-L2, the previously described vector PMLH containing the BPV codon-modified L2 sequence was digested with the restriction endonucleases KpnI and BssHI and ligated with the annealed and likewise digested primers (5Ј-CACGCGTGGTACCGCCATGTACCCATAC-GATGTTCCAGATTACGCTAGCGCCCGCAAGAGAGT-GAAGCGCGCCAGCGCC-3Ј and its reverse complement).…”

“…The PV entry process has extremely slow kinetics, with disassembly being initially detected 6-8 h after entry (5). By 24 h after entry, the C-terminally tagged L2 protein was predominantly detected in the nucleus, along with the pseudoviral genome (Fig.…”

“…The low number of L2 proteins per virion and the lack of synchronicity during the viral entry process prevented us from performing in vivo biochemical analysis. However, it was possible to follow the trafficking of L2 proteins microscopically during infection by using an assay we recently developed to detect uncoated pseudovirus (5). In this assay, the pseudoviral genome is labeled with 5-bromodeoxyuridine (BrdUrd) during pseudovirus production.…”

“…When viral entry is studied with these BrdUrd-labeled pseudovirions, the encapsidated genomes become detectable with anti-BrdUrd antibodies only after disassembly of the viral particles in the endosome. The CHA also becomes detectable only after uncoating (5). To detect L2 cleavage in vivo, we also examined the NHA, whose tag would be predicted to be lost during infection.…”