Inhibition of Infectious HIV-1 Production by Rerouting the Cellular Furin Inhibitor Serpin B8.

Serpins are a superfamily of proteins that regulate a variety of physiological processes by irreversibly inhibiting the enzymatic activity of different serine proteases. For example, Serpin Family B Member 8 (Serpin B8, also known as PI8 and CAP2) binds to and inhibits the proprotein convertase furin. Like many other viral pathogens, human immunodeficiency virus type 1 (HIV-1) exploits furin for the proteolytic activation of its envelope glycoprotein (Env). Since the furin inhibitor Serpin B8 is expressed in primary target cells of HIV-1 and induced under inflammatory conditions, we hypothesized that it might interfere with HIV-1 Env maturation and decrease infectivity of newly produced virions. Indeed, recombinant Serpin B8 reduced furin-mediated cleavage of an HIV-1 Env reporter substrate in vitro. However, Serpin B8 did not affect Env maturation or reduce HIV-1 particle infectivity when expressed in HIV-1-producing cells. Immunofluorescence imaging, dimerization assays and in silico sequence analyses revealed that Serpin B8 failed to inhibit intracellular furin since both proteins localized to different subcellular compartments. We therefore aimed at rendering Serpin B8 active against HIV-1 by relocalizing it to furin-containing secretory compartments. Indeed, the addition of a heterologous signal peptide conferred potent anti-HIV-1 activity to Serpin B8 and significantly decreased infectivity of newly produced viral particles. Thus, our findings demonstrate that subcellular relocalization of a cellular protease inhibitor can result in efficient inhibition of infectious HIV-1 production. IMPORTANCE Many cellular proteases serve as dependency factors during viral infection and are hijacked by viruses for the maturation of their own (glyco)proteins. Consequently, inhibition of these cellular proteases may represent a means to inhibit the spread of viral infection. For example, several studies have investigated the serine protease furin as a potential therapeutic target since this protease cleaves and activates several viral envelope proteins, including HIV-1 Env. Besides the development of small molecule inhibitors, cell-intrinsic protease inhibitors may also be exploited to advance current antiviral treatment approaches. Here, we show that Serpin B8, an endogenous furin inhibitor, can inhibit HIV-1 Env maturation and efficiently reduce infectious HIV-1 production when rerouted to the secretory pathway. The results of our study not only provide important insights into the biology of Serpins, but also show how protein engineering of an endogenous furin inhibitor can render it active against HIV-1.

Independent loss events of a functional tetherin gene in galliform birds.

IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

Attenuated replication and damaging effects of SARS-CoV-2 Omicron variants in an intestinal epithelial barrier model.

Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.