We report that the JNK pathway is involved in lentivirus gene transfer. Specifically, we found that exposure to lentivirus phosphorylated JNK and that blocking JNK with a chemical inhibitor or siRNA decreased gene transfer significantly, indicating that activation of the JNK pathway is necessary for efficient lentiviral transduction. Cell cycle experiments with exponentially growing or confluent cells lacking JNK1/2 suggested that the effects of JNK were not mediated by changes in the cell cycle. Interestingly, while not necessary for virus binding to the cell surface, JNK activation was required for efficient lentivirus entry into target cells. These results shed light into the role of JNK in early steps of the lentivirus life cycle and may suggest strategies for antiretroviral therapies or for promoting gene transfer for gene therapy applications.
Notably, activation of JNK by lentivirus was sustained for only 1 h and decreased sharply by 2 h, despite continued exposure of cells to the virus. Interestingly, the kinetic profile of JNK phosphorylation by lentivirus was similar to that induced by tumor necrosis factor alpha (TNF-α), which was shown to suppress JNK signaling through NF-κB activation (36
). Several viruses, including HIV-1, influenza virus, hepatitis B and C viruses, and herpesvirus, have been shown to activate NF-κB (40
), raising the possibility that lentivirus may also activate NF-κB, which in turn suppresses JNK to prevent apoptosis of infected cells. If this is true, it may be a cellular protective mechanism that evolved to prevent more viral particles from entering an infected cell.
Although lentivirus infection does not require cell division (32
), the growth rate of target cells was shown to affect the transduction efficiency (11
). Since the JNK inhibitor SP600125 changed the cell cycle profile and lowered the growth rate of target cells, we examined whether the effects of JNK inhibition on gene transfer were due to indirect effects on cell proliferation. Surprisingly, JNK knockdown by siRNA had no effect on the cell cycle and even increased target cell proliferation. In addition, JNK inhibition decreased gene transfer to confluent cell monolayers, where most cells were already arrested in G1
phase, and neither SP600125 nor JNK1/2 siRNA had additional effects on cell cycle distribution. Taken together, these experiments suggested that the effect of JNK on gene transfer was not due to indirect effects on the cell cycle. Furthermore, while siRNA knockdown of JNK1 decreased gene transfer, JNK2 knockdown had no effect, clearly implicating only the JNK1 isoform in lentivirus-cell interactions. Interestingly, JNK1 was reported to be localized in focal adhesions, where it promotes cell migration by phosphorylating paxillin (16
), and more recently, JNK1 was shown to be part of the adherens junction complex, phosphorylate β-catenin (23
), control α-catenin-β-catenin binding (24
), and regulate cell-cell adhesion.
Our experiments showed that JNK activation was not specific to the viral envelope and that JNK inhibition decreased gene transfer with a lentivirus that was pseudotyped with either VSV-G or the amphotropic gp70 envelope. On the other hand, bafilomycin A1 treatment reduced JNK and c-Jun phosphorylation by VSV-G-LV, which enters cells by endocytosis (1
), but not that by ampho-LV, which enters via pH-independent membrane fusion (5
), indicating that JNK might be activated by internalized virions, regardless of the entry route. Although several HIV proteins, such as Tat, Nef, and Vpr, have been shown to induce activation of JNK (12
), none of these proteins are present in the 3rd-generation lentiviral vector, suggesting that another viral protein—perhaps the capsid protein p24 and/or the viral RNA—may be responsible for JNK activation.
An interesting question then arises: if JNK activation is triggered by entry of the viral nucleocapsid into the cytosol, how do the cell-bound or endosome-resident viruses initially promote JNK phosphorylation? An intriguing possibility may be that a “positive cooperativity” mechanism is at work, whereupon the first viral particles that enter the cytosol accelerate uptake of additional viruses through JNK activation. Such positive feedback generated by a small initial number of invading viruses may also explain why SP600125 or JNK siRNA significantly reduced but did not eliminate lentivirus entry and gene transfer. Regardless, the mechanism through which JNK activation may affect virus entry remains elusive. Previous studies have also shown that filopodium and lamellipodium extensions in the cell leading edge promote virus surfing to entry sites, where branched actin facilitates entry (19
). Coincidently, JNK was shown to phosphorylate the actin-binding protein Spir, which interacts with Wiskott-Aldrich syndrome protein (WASP) and actin-related protein 2/3 (Arp2/3) to promote actin polymerization and branching (44
). Indeed, actin stress fiber formation was shown to require activation of JNK through the RhoA-Rock-MEKK1 pathway (49
). Taking these data together with our findings, we propose that JNK1 phosphorylation by lentivirus may facilitate virus entry by promoting branched actin organization. This intriguing hypothesis warrants further investigation.