Viral Oncolysis That Targets Raf-1 Signaling Control of Nuclear Transport

Abstract

The central role of Raf protein kinase isoforms in human cancer demands specific anti-Raf therapeutic inhibitors. Parvoviruses are currently used in experimental cancer therapy due to their natural oncotropism and lytic life cycle. In searching for mechanisms underlying parvovirus oncolysis, we found that trimers of the major structural protein (VP) of the parvovirus minute virus of mice (MVM), which have to be imported into the nucleus for capsid assembly, undergo phosphorylation by the Raf-1 kinase. Purified Raf-1 phosphorylated the capsid subunits in vitro to the two-dimensional pattern found in natural MVM infections. VP trimers isolated from mammalian cells translocated into the nucleus of digitonin-permeabilized human cells. In contrast, VP trimers isolated from insect cells, which are devoid of Raf-1, were neither phosphorylated nor imported into the mammalian nucleus. However, the coexpression of a constitutively active Raf-1 kinase in insect cells restored VP trimer phosphorylation and nuclear transport competence. In MVM-infected normal and transformed cells, Raf-1 inhibition resulted in cytoplasmic retention of capsid proteins, preventing their nuclear assembly and progeny virus maturation. The level of Raf-1 activity in cancer cells was consistent with the extent of VP specific phosphorylation and with the permissiveness to MVM infection. Thus, Raf-1 control of nuclear translocation of MVM capsid assembly intermediates provides a novel target for viral oncolysis. MVM may reinforce specific therapies against frequent human cancers with deregulated Raf signaling.The Raf protein isoforms (A, B, C, or Raf-1) belong to the conserved Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling module. This signal cascade transduces mitogenic and other stimuli from the cell surface to the nucleus (reviewed in reference 41). Activation of the Raf kinases is primarily triggered by increased levels of the upstream regulator Ras-GTP protein, which directly binds and recruits cytosolic dimers of the Raf kinases to the plasma membrane (58). The membrane-associated Raf kinases are activated by phosphorylation and other events (48), assembling a MAPK signaling complex formed by the MEK and ERK kinases and scaffold proteins (8). Upon phosphorylation and dimerization (25), an active ERK kinase dissociates from the complex and translocates into the nucleus. Intranuclear ERK kinase promotes multiple protein phosphorylations and changes of gene expression that may be altered in oncogenic and deregulated signaling (50, 55). (40,67), which led to the use of lytic viruses dependent on this pathway (7) in ongoing preclinical and clinical trials (51). Molecular insights into the interactions between viral substrates and components of the MAPK pathway could thus increase specificity and therapeutic efficacy of oncolytic viruses.The replication of virus members of the Parvoviridae relies on functions provided by proliferative cells (24). Together with diverse factors that are linked to the neoplastic growth, the underlyi...

Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle

“…Like for other viruses, it has been shown that parvoviruses have functional NLSs in their capsid proteins (32,57). It has been demonstrated that these NLSs are necessary for nuclear import of capsid protein trimers prior to virion assembly in the nucleus (44) and that nuclear import of capsid protein trimers is dependent on phosphorylation by Raf-1 (45). One of the parvovirus NLSs, within VP2, is buried inside the capsid of assembled virions and could not be involved in nuclear entry of the incoming capsid during initial infection (1,31).…”

Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential

“…Indeed, in rat C6 and human U373MG glioblastoma cells infected with MVM, the characteristic pattern of VP phosphorylation by Raf-1 was conserved, correlating with a high activity of the MAPK signaling pathway (Riolobos et al 2010). Moreover these glioblastomas were efficiently killed by MVM and the virus underwent productive maturation to high yields (Rubio et al 2001).…”

“…Inside the nucleus the fully assembled icosahedral capsid harbors the NLS and NLM facing to the particle inward. Abbreviations: NPC, nuclear pore complex; NLM, nuclear localization motif; NLS, nuclear localization sequence (based on Lombardo et al 2000;Maroto et al 2000;Riolobos et al 2006;Riolobos et al 2010). …”

“…The important role of Raf-1 phosphorylation in VP nuclear transport is best illustrated in heterologous cell systems devoid of Raf-1. For example, the expression of parvovirus VP proteins in insect cells resulted in non-phosphorylated VP trimers and aggregates (Riolobos et al 2010) that, although at low efficiency, self-assembly into viruslike particles (VLPs) accumulating in the perinuclear region of the cytoplasm (Hernando et al 2000;Yuan and Parrish 2001). However the co-expression of a constitutively active Raf-1 kinase in the insect cells, as the truncated mutant Raf-22W with transforming activity (Stanton et al 1989), restored the phosphorylation and nuclear transport competence of the VP trimer (Riolobos et al 2010).…”

“…The VP proteins of MVM undergo cytoplasmic phosphorylation by the Raf-1 kinase (Riolobos et al 2010). Raf-1 (or C-RAF) (72-74 kDa) is a cytoplasmic major protein isoform of the conserved MAPK signaling module with intrinsic serine-threonine kinase activity.…”