Wolfgang Garten

Influenza viruses cause an acute infection of the respiratory tract that affects millions of people during seasonal outbreaks and occasional pandemics. Furthermore, the recent outbreak of an avian H7N9 influenza virus in China, which caused severe respiratory disease and 43 deaths to date (WHO, July 4, 2013), demonstrated once again that the emergence of a novel influenza virus poses a continuous and unpredictable threat to public health. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin (HA) through binding to cell surface receptors and fusion of the viral envelope with the endosomal membrane in order to release the virus genome into the cell. HA is synthesized as a precursor protein HA0 and has to be cleaved by a host cell protease into the subunits HA1 and HA2 to gain its fusion capacity. Therefore, cleavage of HA is essential for virus infectivity and spread. We identified TMPRSS2 (transmembrane serine protease, S1 member 2) and HAT (human airway trypsin-like protease) as appropriate proteases present in the human airways. In the present study we investigated proteolytic activation and pathogenicity of the recent H7N9 isolate A/Anhui/1/13 (H7N9) and a 2009 pandemic H1N1 influenza virus in TMPRSS2-knockout (TMPRSS2-/-) mice. Knockout of TMPRSS2 inhibited virus replication of H7N9 in murine ex vivo trachea, bronchi and lung explants. Remarkably, TMPRSS2-/- mice infected with H7N9 did not display any symptoms of disease, whereas wildtype mice (WT) developed severe infection with a mortality of 100%. Resistance to H7N9 pathogenesis correlated with inhibition of virus replication in the lung of TMPRSS2-/- mice. Similarly, knockout of TMPRSS2 inhibited H1N1 replication in lung explants and prevented development of disease upon H1N1 infection of mice. In conclusion, our data demonstrate that TMPRSS2 is essential for pneumotropism and pathogenicity of H7N9 and H1N1 influenza virus in mice. The development of TMPRSS2-specific inhibitors therefore provides a promising approach for influenza treatment.

Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication. We demonstrate that stable cell lines inducibly expressing S1P-adapted α₁-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of α₁-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific α₁-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different α₁-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor. Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies. The virus family Arenaviridae includes several hemorrhagic fever causing agents such as Lassa, Guanarito, Junin, Machupo, and Sabia virus that pose a major public health concern to the human population in West African and South American countries. Current treatment options to control fatal outcome of disease are limited to the ribonucleoside analogue ribavirin, although its use has some significant limitations. The lack of effective treatment alternatives emphasizes the need for novel antiviral therapeutics to counteract these life-threatening infections. Maturation cleavage of the viral envelope glycoprotein by the host cell proprotein convertase site 1 protease (S1P) is critical for infectious virion production of several pathogenic arenaviruses. This finding makes this protease an attractive target for the development of novel anti-arenaviral therapeutics. We demonstrate here that highly selective S1P-adapted α₁-antitrypsins have the potential to efficiently inhibit glycoprotein processing, which resulted in reduced Lassa virus replication. Our findings suggest that S1P should be considered as an antiviral target and that further optimization of modified α₁-antitrypsins could lead to potent and specific S1P inhibitors with the potential for treatment of certain viral hemorrhagic fevers.

Plaque assays in cell culture monolayers under solid or semisolid overlay media are commonly used for quantification of viruses and antiviral substances. To overcome the pitfalls of known overlays, we tested suspensions of microcrystalline cellulose Avicel RC/CL™ as overlay media in the plaque and plaque-inhibition assay of influenza viruses. Significantly larger plaques were formed under Avicel-containing media, as compared to agar and methylcellulose (MC) overlay media. The plaque size increased with decreasing Avicel concentration, but even very diluted Avicel overlays (0.3%) ensured formation of localized plaques. Due to their low viscosity, Avicel overlays were easier to use than methylcellulose overlays, especially in the 96-well culture plates. Furthermore, Avicel overlay could be applied without prior removal of the virus inoculum thus facilitating the assay and reducing chances of cross-contamination. Using neuraminidase inhibitor oseltamivir carboxylate, we demonstrated applicability of the Avicel-based plaque reduction assay for testing of antiviral substances. 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.

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.

The matrix protein M of Borna disease virus (BDV) is associated with the inner viral membrane and is thought to be a mediator between the nucleocapsid and the lipid-containing envelope in stabilizing the virus shape. The full-length BDV-M gene encoding a 16 kDa protein was expressed in Escherichia coli. M was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. The crystals of M belong to the space group I432, with unit-cell parameters a = b = c = 144.6 A, and diffract to 3.1 A.

The surface glycoprotein of the Lassa virus, a member of the arenaviridae family, is synthesized as a 76-kDa precursor (GP-C) that is posttranslationally cleaved into an N-terminal 44-kDa subunit and a C-terminal membrane-anchored 36-kDa subunit. Cleavage occurs at the C-terminal end of the unusual recognition motif R-R-L-L. We show here that GP-C is cleaved in the endoplasmic reticulum by the cellular subtilase SKI-1/S1P, an enzyme that has so far been observed to be involved in cholesterol metabolism. Furthermore, we present evidence that only cleaved glycoprotein is incorporated into virions and that this is necessary for the formation of infectious virus. To our knowledge, there have been no previous reports of this type of viral glycoprotein processing, one that may be an interesting target for antiviral therapy.

The Lassa virus glycoprotein consists of an amino-terminal and a carboxy-terminal cleavage fragment designated GP-1 and GP-2, respectively, that are derived by proteolysis from the precursor GP-C. The membrane-anchored GP-2 obtained from purified virions of the Josiah strain revealed the N-terminal tripeptide GTF ₂₆₂ when analyzed by Edman degradation. Upstream of this site, GP-C contains the tetrapeptide sequence RRLL ₂₅₉ , which is conserved in all Lassa virus isolates published to date. Systematic mutational analysis of vector-expressed GP-C revealed that the motif R-X (L/I/V)-L ₂₅₉ (where X stands for L, I, or V) is essential for cleavage of the peptide bond between leucine ₂₅₉ and glycine ₂₆₀ . This cleavage motif is homologous to the consensus sequence recognized by a novel class of cellular endoproteases which have so far not been implicated in the processing of viral glycoproteins.

Antibodies specific to the N-terminal 34-aminoacid peptide of the major nucleocapsid protein NP of human influenza A viruses were obtained. The NP proteolytic cleavage occurring in infected cells late in infection has been demonstrated using this antibody. The antibody specifically reacted with noncleaved NP of 56 kD m.w. but not with cleaved NP of 53 kD m.w. This indicates that NP56-->NP53 intracellular cleavage released the N-terminal 3 kD peptide of Np molecule. The released 3 kD peptide did not accumulate in infected cells. Since the RNA-binding and nucleus migrating signals are located in the N-terminus of NP molecule, presumably, the terminal cleavage is important for intracellular NP functions.