Characterization of the Human Herpesvirus 6 U69 Gene Product and Identification of Its Nuclear Localization Signal

Abstract

To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminal deletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescent protein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into the nucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. The nuclear transport of the GST-GFP fusion protein containing the N-terminal NLS of U69 was inhibited by wheat germ agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into the nucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that the nuclear transport of the U69 protein was mediated by importin ␣/␤ in conjunction with the small GTPase Ran. When the import assay was performed with a low concentration of each importin-␣ subtype, NPI2/importin-␣7 elicited more efficient transport activity than did Rch1/importin-␣1 or Qip1/importin-␣3. These results suggest a relationship between the localization of NPI2/importin-␣7 and the cell tropism of HHV-6.

Viral Serine/Threonine Protein Kinases

“…Thus far, for all CHPK, a fraction has been described to be present in the nucleus of infected cells (47,49,71,72,84,143,169,226), indicating that the kinase may play important, conserved roles in the nuclear phase of infection.…”

Characterization of the Nucleocytoplasmic Transport Mechanisms of Epstein-Barr Virus BFLF2

“…pBFLF2(NLS)-EYFP-BKRF2 was yielded by inserting the oligonucleotides of BFLF2 NLS into the BglII and HindⅢ digested pEYFP-BKRF2. TAP, dominant negative (DN) mutant RanGTP (Ran-Q69L) [25], DN kα1 (importin α5) [26] and DN kβ1 (importin β1) [27] were subcloned into pmCherry-N1 to yield pTAPmCherry, pRan-Q69L-mCherry, pDN kα1-mCherry and pDN kβ1-mCherry, respectively. All constructs were confirmed by DNA sequencing, and primers used in this study are available upon request.…”

Characterization of nuclear import and export signals determining the subcellular localization of WD repeat‐containing protein 42A (WDR42A)

“…The Ran protein has been verified to be required for classic NLSdependent nuclear transport [12]. To further explore the nuclear import mechanism of WDR42A, a dominant negative Ran protein, Ran-GTP Q69L, which is deficient in GTP hydrolysis was introduced to determine whether Ran is indispensable for the nuclear transport of WDR42A.…”

“…For expression of HA/Flag-WDR42A, we subcloned the WDR42A from pWDR42A-EYFP to the vector pCMV-N-HA/Flag. The plasmid pGEX6p-1Q69L Ran was a generous gift from Dr. Yoshinari Yasuda and Q69L Ran was then sub-cloned into pECFP-N1 (Clontech) to yield a dominant negative mutant pRan-Q69L-ECFP [12]. In addition, the plasmid pRev-NES-EGFP was a generous gift from Dr. Gillian Elliott, and Rev-NES was sub-cloned into pEYFP-N1 to yield pRev-NES-EYFP.…”