Cathepsin B plays a key role in optimal production of the influenza A virus.

BACKGROUND: Influenza A virus (IAV) is the etiologic agent of the febrile respiratory illness, commonly referred to as 'flu'. The lysosomal protease cathepsin B (CTSB) has shown to be involved in the lifecycle of various viruses. Here, we examined the role of CTSB in the IAV lifecycle. METHODS: CTSB-deficient (CTSB-/-) macrophages and the human lung epithelial cell line A549 cells treated with CA-074Me were infected with the A/Puerto Rico/8/34 strain of IAV (IAV-PR8). Viral entry and propagation were measured through quantitative real-time RT-PCR; production and localization of hemagglutinin (HA) protein in the infected host cells were analysed by Western blots, flow cytometry and confocal microscopy; production of progeny viruses were measured by a hemagglutination assay. RESULTS: CTSB-/- macrophages and CA-074Me-treated A549 cells had no defects in incorporating IAV-PR8 virions and permitting viral RNA synthesis. However, these cells produced significantly lower amounts of HA protein and progeny virions than wild-type or untreated cells. CONCLUSION: These data indicate that CTSB is involved in the expression of IAV-PR8 HA protein and subsequent optimal production of IAV-PR8 progeny virions. Targeting CTSB can be a novel therapeutic strategy for treating IAV infection.

Interferon-induced HERC5 is evolving under positive selection and inhibits HIV-1 particle production by a novel mechanism targeting Rev/RRE-dependent RNA nuclear export.

BACKGROUND: Type I interferon (IFN) inhibits virus replication by activating multiple antiviral mechanisms and pathways. It has long been recognized that type I IFNs can potently block HIV-1 replication in vitro; as such, HIV-1 has been used as a system to identify and characterize IFN-induced antiviral proteins responsible for this block. IFN-induced HERC5 contains an amino-terminal Regulator of Chromosome Condensation 1 (RCC1)-like domain and a carboxyl-terminal Homologous to the E6-AP Carboxyl Terminus (HECT) domain. HERC5 is the main cellular E3 ligase that conjugates the IFN-induced protein ISG15 to proteins. This E3 ligase activity was previously shown to inhibit the replication of evolutionarily diverse viruses, including HIV-1. The contribution of the RCC1-like domain to the antiviral activity of HERC5 was previously unknown. RESULTS: In this study, we showed that HERC5 inhibits HIV-1 particle production by a second distinct mechanism that targets the nuclear export of Rev/RRE-dependent RNA. Unexpectedly, the E3 ligase activity of HERC5 was not required for this inhibition. Instead, this activity required the amino-terminal RCC1-like domain of HERC5. Inhibition correlated with a reduction in intracellular RanGTP protein levels and/or the ability of RanGTP to interact with RanBP1. Inhibition also correlated with altered subcellular localization of HIV-1 Rev. In addition, we demonstrated that positive evolutionary selection is operating on HERC5. We identified a region in the RCC1-like domain that exhibits an exceptionally high probability of having evolved under positive selection and showed that this region is required for HERC5-mediated inhibition of nuclear export. CONCLUSIONS: We have identified a second distinct mechanism by which HERC5 inhibits HIV-1 replication and demonstrate that HERC5 is evolving under strong positive selection. Together, our findings contribute to a growing body of evidence suggesting that HERC5 is a novel host restriction factor.

Inhibition or deficiency of cathepsin B leads defects in HIV-1 Gag pseudoparticle release in macrophages and HEK293T cells.

Human immunodeficiency virus type 1 (HIV-1) egresses from infected cells through utilizing the host membrane budding mechanisms. Assembly of HIV-1 Gag particles occurs on membranes where the Gag multimers subsequently bud off and form enveloped viral particles. In certain cell types such as macrophages, HIV-1 Gag particles have shown to be released into intracellular virus containing compartments (VCC) such as late endosomes, multivesicular bodies (MVBs) or invaginated plasma membrane pockets. Here, we showed that macrophages or HEK293T cells treated with the cathepsin B (CTSB)-specific inhibitor CA-074Me or cells deficient in CTSB failed to release HIV-1 Gag pseudoparticles into the extracellular environment. Based on immunofluorescence and electron microscopy, these cells retained the pseudoparticles in heterogeneous intracellular VCC. CA-074Me was also able to inhibit propagation of two enveloped viruses, herpes simplex virus and influenza A virus, but not non-enveloped enterovirus. These results suggest that CTSB is required for the efficient release of HIV-1 Gag pseudoparticles and targeting CTSB can be a new therapeutic strategy for inhibiting egress of HIV-1 and other enveloped viruses.