The Role of Phlebovirus Glycoproteins in Viral Entry, Assembly and Release

Bunyaviruses are enveloped viruses with a tripartite RNA genome that can pose a serious threat to animal and human health. Members of the Phlebovirus genus of the family Bunyaviridae are transmitted by mosquitos and ticks to humans and include highly pathogenic agents like Rift Valley fever virus (RVFV) and severe fever with thrombocytopenia syndrome virus (SFTSV) as well as viruses that do not cause disease in humans, like Uukuniemi virus (UUKV). Phleboviruses and other bunyaviruses use their envelope proteins, Gn and Gc, for entry into target cells and for assembly of progeny particles in infected cells. Thus, binding of Gn and Gc to cell surface factors promotes viral attachment and uptake into cells and exposure to endosomal low pH induces Gc-driven fusion of the viral and the vesicle membranes. Moreover, Gn and Gc facilitate virion incorporation of the viral genome via their intracellular domains and Gn and Gc interactions allow the formation of a highly ordered glycoprotein lattice on the virion surface. Studies conducted in the last decade provided important insights into the configuration of phlebovirus Gn and Gc proteins in the viral membrane, the cellular factors used by phleboviruses for entry and the mechanisms employed by phlebovirus Gc proteins for membrane fusion. Here, we will review our knowledge on the glycoprotein biogenesis and the role of Gn and Gc proteins in the phlebovirus replication cycle

Evidence that Processing of the Severe Fever with Thrombocytopenia Syndrome Virus Gn/Gc Polyprotein Is Critical for Viral Infectivity and Requires an Internal Gc Signal Peptide.

The severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, highly pathogenic bunyavirus against which neither antivirals nor vaccines are available. The SFTSV glycoproteins, Gn and Gc, facilitate viral entry into host cells. Gn and Gc are generated from a precursor protein, Gn/Gc, but it is currently unknown how the precursor is converted into the single proteins and whether this process is required for viral infectivity. Employing a rhabdoviral pseudotyping system, we demonstrate that a predicted signal sequence at the N-terminus of Gc is required for Gn/Gc processing and viral infectivity while potential proprotein convertase cleavage sites in Gc are dispensable. Moreover, we show that expression of Gn or Gc alone is not sufficient for host cell entry while particles bearing both proteins are infectious, and we provide evidence that Gn facilitates Golgi transport and virion incorporation of Gc. Collectively, these results suggest that signal peptidase liberates mature Gc from the Gn/Gc precursor and that this process is essential for viral infectivity and thus constitutes a potential target for antiviral intervention

Inhibitors of signal peptide peptidase and subtilisin/kexin-isozyme 1 inhibit Ebola virus glycoprotein-driven cell entry by interfering with activity and cellular localization of endosomal cathepsins.

Emerging viruses such as severe fever and thrombocytopenia syndrome virus (SFTSV) and Ebola virus (EBOV) are responsible for significant morbidity and mortality. Host cell proteases that process the glycoproteins of these viruses are potential targets for antiviral intervention. The aspartyl protease signal peptide peptidase (SPP) has recently been shown to be required for processing of the glycoprotein precursor, Gn/Gc, of Bunyamwera virus and for viral infectivity. Here, we investigated whether SPP is also required for infectivity of particles bearing SFTSV-Gn/Gc. Entry driven by the EBOV glycoprotein (GP) and the Lassa virus glycoprotein (LASV-GPC) depends on the cysteine proteases cathepsin B and L (CatB/CatL) and the serine protease subtilisin/kexin-isozyme 1 (SKI-1), respectively, and was examined in parallel for control purposes. We found that inhibition of SPP and SKI-1 did not interfere with SFTSV Gn + Gc-driven entry but, unexpectedly, blocked entry mediated by EBOV-GP. The inhibition occurred at the stage of proteolytic activation and the SPP inhibitor was found to block CatL/CatB activity. In contrast, the SKI-1 inhibitor did not interfere with CatB/CatL activity but disrupted CatB localization in endo/lysosomes, the site of EBOV-GP processing. These results underline the potential of protease inhibitors for antiviral therapy but also show that previously characterized compounds might exert broader specificity than initially appreciated and might block viral entry via diverse mechanisms

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2

<div><p>The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.</p></div

Mutations K543A/R544A and R563A/K566A interfere with SARS S trafficking through the constitutive secretory pathway.

<p>(A) COS-7 cells were cotransfected with expression plasmids for SARS S wt or the indicated mutants and with a plasmid encoding YFP fused to a plasma membrane anchor (red color). After staining for SARS S with V5 antibody and anti-mouse Alexa Fluor 647 secondary antibody (green color) the cells were analyzed by confocal microscopy. Similar results were obtained in a second independent experiment. (B) The experiment was carried out as described for panel A, but GFP fused to a Golgi marker (red color) was used instead of YFP with plasma membrane anchor. The results were confirmed in a second separate experiment. For optimal visualization, colors were manually assigned with LSM Pascal 5 software version 3 and do not correspond to the actual emission spectra of YFP, GFP and Alexa Fluor 647.</p

Mutations R667A, T678S and R797N are compatible with robust S protein-driven cell entry.

<p>(A) 293T cells transfected to express MLV particles pseudotyped with SARS S wt or the indicated SARS S mutants and the corresponding supernatants were analyzed for expression of S protein (employing anti-V5 antibody) and MLV capsid protein (employing anti-p30 antibody) by Western blot. Cells transfected to express S protein alone or transfected with empty plasmid were used as controls. Similar results were obtained in two independent experiments. (B) 293T target cells transfected with empty plasmid (control) or ACE2 plasmid were transduced with equal volumes of pseudotypes bearing the indicated viral glycoproteins. Transduction efficiency was quantified by measuring luciferase activities in cell lysates. Results were normalized for SARS S-driven transduction of ACE2 expressing cells, which was set as 1-fold. The average of three to eight independent experiments is shown. Error bars indicate standard error of the mean (SEM).</p

R797 but not R667 is required for SARS S activation by TMPRSS2.

<p>293T target cells transfected with ACE2 plasmid or cotransfected with ACE2 and TMPRSS2 plasmid were pretreated with DMSO or the cathepsin B/L inhibitor MDL 28170 for 1 h before addition of equal volumes of pseudotypes bearing the indicated glycoproteins. Cellular entry was quantified by measurement of luciferase activity in cell lysates. Results were normalized for transduction of ACE2⁺, TMPRSS2^- cells in the absence of inhibitor, which was set as 1-fold. The average of three to six independent experiments is shown (R667A, n = 3; SARS S wt and VSV-G, n = 4; R797N, n = 6). Error bars indicate SEM. Statistical significance was assessed using one-tailed student’s t-est.</p

R667 but not R797 is required for SARS S cleavage by trypsin and TMPRSS2.

<p>Plasmids encoding SARS S wt or the indicated SARS S mutants were cotransfected with TMPRSS2 plasmid or empty plasmid into 293T cells. Subsequently, the cells were treated with trypsin or PBS and SARS S cleavage was analyzed by Western blot employing anti-V5 antibody. Detection of β-actin served as loading control. The results were confirmed in two to four separate experiments. The SARS S cleavage products generated by trypsin and TMPRSS2 are indicated by black and white filled triangles, respectively.</p