TY - JOUR
T1 - The high risk HPV16 L2 minor capsid protein has multiple transport signals that mediate its nucleocytoplasmic traffic
AU - Mamoor, Shahan
AU - Onder, Zeynep
AU - Karanam, Balasubramanyam
AU - Kwak, Kihyuck
AU - Bordeaux, Jennifer
AU - Crosby, Lauren
AU - Roden, Richard B.S.
AU - Moroianu, Junona
N1 - Funding Information:
We thank Kathy Bockstall for initial transfection assays with EGFP–16L2 and ΔN, ΔC and ΔNΔC mutants. We thank Courtney Halista for technical assistance with plasmid purifications and initial transfections and localization studies for EGFP–16L2ms5 and EGFP–16L2ms5t1. We also thank Dr. Joshua Rosenberg for excellent technical assistance with confocal fluorescence microscopy. This work was supported by a grant from the National Institutes of Health ( R01 CA94898 ) to Junona Moroianu and CA133749, CA118790 and P50 CA098252 and RC2 CA148499 to RBSR.
PY - 2012/1/20
Y1 - 2012/1/20
N2 - In this study we examined the transport signals contributing to HPV16 L2 nucleocytoplasmic traffic using confocal microscopy analysis of enhanced green fluorescent protein-L2 (EGFP-L2) fusions expressed in HeLa cells. We confirmed that both nuclear localization signals (NLSs), the nNLS (1MRHKRSAKRTKR12) and cNLS (456RKRRKR461), previously characterized in vitro (Darshan et al., 2004), function independently in vivo. We discovered that a middle region rich in arginine residues (296SRRTGIRYSRIGNKQTLRTRS316) functions as a nuclear retention sequence (NRS), as mutagenesis of critical arginine residues within this NRS reduced the fraction of L2 in the nucleus despite the presence of both NLSs. Significantly, the infectivity of HPV16 pseudoviruses containing either RR297AA or RR297EE within the L2 NRS was strongly reduced both in HaCaT cells and in a murine challenge model. Experiments using Ratjadone A nuclear export inhibitor and mutation-localization analysis lead to the discovery of a leucine-rich nuclear export signal ( 462LPYFFSDVSL) mediating 16L2 nuclear export. These data indicate that HPV16 L2 nucleocytoplasmic traffic is dependent on multiple functional transport signals.
AB - In this study we examined the transport signals contributing to HPV16 L2 nucleocytoplasmic traffic using confocal microscopy analysis of enhanced green fluorescent protein-L2 (EGFP-L2) fusions expressed in HeLa cells. We confirmed that both nuclear localization signals (NLSs), the nNLS (1MRHKRSAKRTKR12) and cNLS (456RKRRKR461), previously characterized in vitro (Darshan et al., 2004), function independently in vivo. We discovered that a middle region rich in arginine residues (296SRRTGIRYSRIGNKQTLRTRS316) functions as a nuclear retention sequence (NRS), as mutagenesis of critical arginine residues within this NRS reduced the fraction of L2 in the nucleus despite the presence of both NLSs. Significantly, the infectivity of HPV16 pseudoviruses containing either RR297AA or RR297EE within the L2 NRS was strongly reduced both in HaCaT cells and in a murine challenge model. Experiments using Ratjadone A nuclear export inhibitor and mutation-localization analysis lead to the discovery of a leucine-rich nuclear export signal ( 462LPYFFSDVSL) mediating 16L2 nuclear export. These data indicate that HPV16 L2 nucleocytoplasmic traffic is dependent on multiple functional transport signals.
KW - Human papillomaviruses
KW - L2 minor capsid protein
KW - Nuclear export signal
KW - Nuclear localization signal
KW - Nuclear retention sequence
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U2 - 10.1016/j.virol.2011.11.007
DO - 10.1016/j.virol.2011.11.007
M3 - Article
C2 - 22154072
AN - SCOPUS:84855205694
SN - 0042-6822
VL - 422
SP - 413
EP - 424
JO - Virology
JF - Virology
IS - 2
ER -