Expression of HPV 58 Long and Short L1 Capsid Proteins in Primary Mouse Keratinocyte Cultures

Abstract

We studied expression of HPV 58 long and short L1 proteins in primary mouse keratinocyte (KC) cultures by transient transfection of the L1 expression constructs. Following transient transfection, long and short L1 open reading frames (ORFs) were transcribed continuously for 9 days; however, no significant difference was detected between the long and short L1 mRNA levels measured by quantitative RT-PCR. Western blot analysis showed that both long and short L1 proteins were continuously detected in L1-transfected KCs for 9 days post-transfection and the significantly increased signals of the L1 proteins over time were associated with KC differentiation. Moreover, L1 protein was more abundant in KCs transfected with the short L1 ORF than the long L1 ORF. In vitro translation of the L1 mRNAs indicated further that the short L1 mRNA had significantly higher translation efficiency than the long L1 mRNA in cell-free lysate system. The L1 proteins expressed from the two L1 mRNAs in KCs were similarly stable. Thus, approximate 40% lower level of expression of the L1 protein in KCs transfected with the long L1 ORF was probably due to a stem-loop structure with high DeltaG value downstream the first AUG codon in its mRNA secondary structure. This stem-loop structure might prevent efficient binding of the ribosome to mRNA and therefore reduced translation.

Human papillomavirus 16-encoded E7 protein inhibits IFN-γ-mediated MHC class I antigen presentation and CTL-induced lysis by blocking IRF-1 expression in mouse keratinocytes

“…Immunoblot assay. Protein samples were extracted from keratinocytes as described previously (Wang et al, 2009). Protein samples (50 mg) were separated by SDS-PAGE and blotted onto PVDF membrane (Millipore.).…”

“…Keratinocyte transfection. As described previously (Wang et al, 2009), buffer A (24 ml lipofectamin in 600 ml serum-free KC medium; Gibco, Invitrogen), and buffer B, containing either 12 mg pCDNA3-IRF-1 plasmid DNA (kindly provided by Dr Pramod Rath, Jawaharlal Nehru University, India) or pCDNA3-GFP DNA, mixed with 9 ml Lipofectamin Plus reagent (Gibco, Invitrogen) in 600 ml serum-free KC medium, were incubated at 25 uC for 20 min. Buffer A was then mixed with buffer B and incubated at 25 uC for 30 min.…”

The Role of Ubiquitin and Ubiquitin-Like Modification Systems in Papillomavirus Biology

“…and Section 3.6. ), while the L1 [ 49 ] and L2 [ 50 ] late proteins are much more stable ( Section 3.7. ).…”

Codon usage roles in human papillomavirus

“…Keratinocytes are the host cells of PV infection. By establishing human and mouse primary keratinocytes in culture, we were able to efficiently express PV major capsid (L1) proteins by transient transfection of authentic or codon‐modified L1 genes from three PVs (BPV‐1; HPV‐6 and HPV‐58) . Both human and mouse primary keratinocytes cultured in vitro can be induced to proceed cell differentiation.…”