Influenza Virus-Induced Caspase-Dependent Enlargement of Nuclear Pores Promotes Nuclear Export of Viral Ribonucleoprotein Complexes

Abstract

Influenza A viruses (IAV) replicate their segmented RNA genome in the nucleus of infected cells and utilize caspase-dependent nucleocytoplasmic export mechanisms to transport newly formed ribonucleoprotein complexes (RNPs) to the site of infectious virion release at the plasma membrane. In this study, we obtained evidence that apoptotic caspase activation in IAV-infected cells is associated with the degradation of the nucleoporin Nup153, an integral subunit of the nuclear pore complex. Transmission electron microscopy studies revealed a distinct enlargement of nuclear pores in IAV-infected cells. Transient expression and subcellular accumulation studies of multimeric marker proteins in virus-infected cells provided additional evidence for increased nuclear pore diameters facilitating the translocation of large protein complexes across the nuclear membrane. Furthermore, caspase 3/7 inhibition data obtained in this study suggest that active, Crm1-dependent IAV RNP export mechanisms are increasingly complemented by passive, caspase-induced export mechanisms at later stages of infection. IMPORTANCEIn contrast to the process seen with most other RNA viruses, influenza virus genome replication occurs in the nucleus (rather than the cytoplasm) of infected cells. Therefore, completion of the viral replication cycle critically depends on intracellular transport mechanisms that ensure the translocation of viral ribonucleoprotein (RNP) complexes across the nuclear membrane. Here, we demonstrate that virus-induced cellular caspase activities cause a widening of nuclear pores, thereby facilitating nucleocytoplasmic translocation processes and, possibly, promoting nuclear export of newly synthesized RNPs. These passive transport mechanisms are suggested to complement Crm1-dependent RNP export mechanisms known to occur at early stages of the replication cycle and may contribute to highly efficient production of infectious virus progeny at late stages of the viral replication cycle. The report provides an intriguing example of how influenza virus exploits cellular structures and regulatory pathways, including intracellular transport mechanisms, to complete its replication cycle and maximize the production of infectious virus progeny.

Bik Mediates Caspase-Dependent Cleavage of Viral Proteins to Promote Influenza A Virus Infection

“…ER stress is involved in the pathogenesis of IAV (46), and BIK initiates ER Ca 21 release prior to the activation of effector caspases (11). In response to ER stress, GRP78 dissociates from the luminal domain of these sensors, leading to their activation (47).…”

“…More recently, studies have shown that Bik mediates activation of caspases in cells infected with Epstein-Barr virus (20). Recent studies have shown that IAVinduced caspase activation causes a widening of nuclear pores to facilitate passive transport of viral ribonucleic protein (RNP) to ensure efficient production of infectious virus progeny (21). Inhibitors of host cell proteases have been used effectively to reduce viral replication and treat infection, such as in the case of HIV-1 infection (22).…”

Caspases rule the intracellular trafficking cartel

“…Indeed, nucleuslocalized GFP via an NLS leaks into the cytosol and proteins up to 140 kDa enter the nucleus of apoptotic cells, while a 580-kDa GFP-tagged b-galactosidase fusion protein remains in the cytoplasm and 4',6-diamidino-2-phenylindole-stained DNA stays nuclear [119]. Enlargement of the NPC facilitates diffusion of medium-sized proteins through the NPC [123], including that of executioner caspases 3, 6, and 7 (50-70 kDa). Redistribution of several transport factors in the nucleus and cytoplasm is observed along with an accumulation of nuclear mRNAs [124].…”

Verapamil has Antiviral Activities that Target Different Steps of the Influenza Virus Replication Cycle

“…In an attempt to close this gap of knowledge between the previous observation and the current understanding of IAV replication we performed the present study and now provide evidence that Verapamil profoundly affects the replication and transcription activity of the viral RNA-dependent RNA polymerase (RdRp), resulting in reduced expression of viral proteins and, consequently, reduced production of virus progeny. Furthermore, we demonstrate that Verapamil interferes with the virus-induced activation of the transcription factor NF-κB, known to be involved in the expression of pro-apoptotic factors and subsequent apoptotic caspase-3 activation, the latter shown previously to promote viral RNP export [15,16]. Taken together, the data suggest that Verapamil has diverse effects on both virus and host cell functions that collectively result in strongly reduced IAV replication in vitro.…”

“…Additionally, it was demonstrated that activation of the apoptotic Caspase 3 is also important for efficient IV propagation [39] as IV-induced Caspase-dependent enlargement of nuclear pores promotes nuclear export of the viral RNPs [16]. Current understanding therefore proposes a model that connects the observed events, e.g.…”