The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2′ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.

Vaccines and therapies are urgently needed to address public health needs stemming from emerging pathogens and biological threat agents such as the filoviruses Ebola virus (EBOV) and Marburg virus (MARV). Ebola virus (EBOV) and Marburg virus (MARV) of the virus familyFiloviridae are emerging and reemerging pathogens that cause hemorrhagic fever with high mortality rates in humans and nonhuman primates 1-3 . The public health concern about filoviruses has increased in recent years as a result of increased awareness and frequency of cases in central Africa as evidenced by the current outbreak of MARV in Angola 4 and also because filoviruses are considered to be potential agents of bioterrorism 5 . Currently, there are no EBOV or MARV vaccines or therapies approved for human use. Recently, we generated live attenuated recombinant vesicular stomatitis viruses (rVSV) expressing the transmembrane glycoprotein of Zaire ebolavirus (ZEBOV; VSV ∴∆G/ZEBOVGP) and MARV (VSV∆G/MARVGP) 6 . Our study evaluated the utility of these rVSV vectors as candidate vaccines for EBOV and MARV using the cynomolgus macaque model.Filovirus vaccine research has been extensively reviewed in the past and has primarily focused on EBOV 7,8 . The first EBOV vaccine to protect nonhuman primates was a DNA prime-adenovirus boost approach using both the glycoprotein and nucleoprotein as target antigens 9 . This approach required several months for immunity to develop, which limited the utility of this strategy. More recently, an accelerated vaccine was described. A single immunization of nonhuman primates with 2 × 10 12 particles of an equal mixture of human adenovirus 5 vectors carrying either the gene encoding ZEBOV glycoprotein or the gene encoding ZEBOV nucleoprotein resulted in complete protection against ZEBOV 10 . Despite the intriguing success of the adenovirus vaccine, preexisting immunity rates of between 40 and 60% have been reported to adenovirus in the human population and this may eventually limit the utility of this approach [11][12][13] .A smaller number of efforts have focused on developing vaccines against MARV. Alphavirus replicons expressing MARV proteins protected cynomolgus monkeys from homologous MARV challenge 14 . Subsequent studies evaluating this platform as a vaccine for EBOV were less encouraging, as the EBOV counterpart of this alphavirus replicon platform was unable to protect any animal against lethal EBOV challenge under similar test conditions 7 . The ideal vaccine would protect humans from infection from all four EBOV species (ZEBOV, Sudan ebolavirus (SEBOV), Reston ebolavirus, Ivory Coast ebolavirus) and MARV. Although the adenovirusbased vaccine platform has completely protected nonhuman primates against ZEBOV 9,10 , and the platform based on alphavirus replicons protected monkeys against MARV 14 , no platform has demonstrably protected nonhuman primates against both of these viruses.Vaccines based on live attenuated rVSV have been highly effective in animal models and are particularly attractive because they can ...

We have previously reported that mutations in the polymerase proteins PB1, PB2, PA, and the nucleocapsid protein NP resulting in enhanced transcription and replication activities in mammalian cells are responsible for the conversion of the avian influenza virus SC35 (H7N7) into the mouse-adapted variant SC35M. We show now that adaptive mutations D701N in PB2 and N319K in NP enhance binding of these proteins to importin α1 in mammalian cells. Enhanced binding was paralleled by transient nuclear accumulation and cytoplasmic depletion of importin α1 as well as increased transport of PB2 and NP into the nucleus of mammalian cells. In avian cells, enhancement of importin α1 binding and increased nuclear transport were not observed. These findings demonstrate that adaptation of the viral polymerase to the nuclear import machinery plays an important role in interspecies transmission of influenza virus.

Plaque assay under Avicel-containing overlay media is easier, faster and more sensitive than assays under agar- and methylcellulose overlays. The assay can be readily performed in a 96-well plate format and seems particularly suitable for high-throughput virus titrations, serological studies and experiments on viral drug sensitivity. It may also facilitate work with highly pathogenic agents performed under hampered conditions of bio-safety labs.

Sialic acid linked to glycoproteins and gangliosides is used by many viruses as a receptor for cell entry. These viruses include important human and animal pathogens, such as influenza, parainfluenza, mumps, corona, noro, rota, and DNA tumor viruses. Attachment to sialic acid is mediated by receptor binding proteins that are constituents of viral envelopes or exposed at the surface of non-enveloped viruses. Some of these viruses are also equipped with a neuraminidase or a sialyl-O-acetyl-esterase. These receptor-destroying enzymes promote virus release from infected cells and neutralize sialic acid-containing soluble proteins interfering with cell surface binding of the virus. Variations in the receptor specificity are important determinants for host range, tissue tropism, pathogenicity, and transmissibility of these viruses.

Lassa virus is an enveloped virus with glycoprotein spikes on its surface. It contains an RNA ambisense genome that encodes the glycoprotein precursor GP-C, the nucleoprotein NP, the polymerase L, and the Z protein. Here we demonstrate that the Lassa virus Z protein (i) is abundant in viral particles, (ii) is strongly membrane associated, (iii) is sufficient in the absence of all other viral proteins to release enveloped particles, and (iv) contains two late domains, PTAP and PPXY, necessary for the release of virus-like particles. Our data provide evidence that Z is the Lassa virus matrix protein that is the driving force for virus particle release.Lassa virus belongs to the large family of Arenaviridae, including the closely related Lymphocytic choriomeningitis virus (LCMV) and other important human pathogens like Guanarito virus, Junin virus, and Machupo virus. Lassa virus is the etiologic agent of a hemorrhagic fever endemic in West Africa, where annually up to 100,000 cases of clinically apparent Lassa fever occur. Up to 20% of these patients develop hemorrhagic manifestations with a total mortality of 10 to 15% (28,29). In recent years this disease has been increasingly exported from regions where it is endemic to other parts of the world (36).Lassa virus consists of a helical nucleocapsid containing a bisegmented RNA genome surrounded by a lipid bilayer with integrated glycoprotein spikes. Each single-stranded RNA encodes two viral genes in an ambisense coding strategy separated by an intergenic region. The small RNA encodes the nucleoprotein NP (60 kDa) and the immature glycoprotein precursor pre-GP-C (80 kDa), which is cotranslationally cleaved by signal peptidase into GP-C (75 kDa) and a stable signal peptide of 58 amino acids (aa) (10). GP-C is cleaved posttranslationally by subtilase SKI-1/S1P into the N-terminal subunit GP-1 (40 kDa) and the membrane-bound subunit GP-2 (35 kDa). Both subunits are incorporated in virus particles (24,25). The large RNA segment encodes the RNAdependent RNA polymerase L (ϳ200 kDa) and the Z protein with a length of 99 amino acids and a molecular mass of approximately 11 kDa (33,35).During the last few years, the role of the arenavirus Z protein has been elucidated in respect to virus replication. The Z protein of Lassa virus and LCMV contains a RING motif and was shown to have zinc-binding activity (34). Most of the information so far indicates a regulatory role of Z, as the LCMV Z protein was shown to bind to the promyelotic leukemia protein and to relocate nuclear structures formed by the promyelotic leukemia protein (2, 4). The LCMV Z protein has also been reported to interact with the nuclear fraction of the ribosomal protein P0 and with the eukaryotic translation initiation factor eIF4E (3, 7). Furthermore, the Z protein of Tacaribe virus is implicated in RNA synthesis and genome replication in the early stage of infection (13). In contrast, the Z protein of LCMV was not required for RNA replication and transcription in a LCMV minigenome system. Moreover, the Z pro...

Wild-type (WT) influenza A/PR/8/34 virus and its variant lacking the NS1 gene (delNS1) have been compared for their ability to mediate apoptosis in cultured cells and chicken embryos. Cell morphology, fragmentation of chromatin DNA, and caspase-dependent cleavage of the viral NP protein have been used as markers for apoptosis. Another marker was caspase cleavage of the viral M2 protein, which was also found to occur in an apoptosis-specific manner. In interferon (IFN)-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses. Taken together, these data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.The influenza virus genome contains eight single-stranded RNA segments of negative polarity, coding for nine structural proteins and a nonstructural polypeptide designated NS1. NS1 is expressed together with the nucleocapsid protein NP soon after virus entry into the cell and acts at an early stage of infection (for a review, see reference 30). It has many regulatory functions, such as inhibition of host mRNA polyadenylation (40), inhibition of nuclear export of polyadenylated host mRNA (8), inhibition of mRNA splicing (14, 37, 56), stimulation of translation of viral RNA (2, 9, 13), and modulation of viral RNA transcription and replication (45). In addition, NS1 has the ability to bind double-stranded RNA (dsRNA) and thus to prevent intracellular dsRNA-activated protein kinase R (PKR) activation (20, 37). It was also shown that NS1 directly interacts with PKR, suppressing its function (52). Recently the important observation has been made that the NS1 gene is not absolutely necessary for virus replication. Influenza virus lacking this gene partly or completely was able to replicate efficiently in hosts defective in interferon (IFN) production, whereas replication of delNS1 virus (influenza A/PR/8/34 virus variant lacking the NS1 gene) in IFN-competent cells was significantly reduced (12,17). In a similar manner delNS1 virus replicated effectively in host organisms lacking PKR (4, 7). This replication phenotype of delNS1 virus has been explained by its inability to shut off activation of the IFN-PKR system (7,17,51). On the basis of these data it has been concluded that the NS1 protein is an antagonist of the IFN system.Influenza virus induces apoptosis in infected cells (24,48). It has been observed recently that the caspase 8 pathway is involved in influenza virus-mediated apoptosis (3) and that the viral nucleocapsid protein NP is specifically cleaved by host cell caspases into the truncated form aNP (62). Another interesting observation was that PKR sensitizes cells to apoptosis induced by influenza virus (3). The observ...

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