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    Topic review

    Proprotein Convertases and Enveloped Viruses

    Subjects: Virology
    View times: 8
    Submitted by: Nabil G. Seidah


    There are seven known human coronaviruses (CoV), which are enveloped positive-stranded RNA viruses belonging to the orderNidoviralesand are mostly responsible for upper respiratory tract infections. All these coronaviruses exhibit a “crown-like” structure composed of a trimeric spike (S) protein. The S-protein is a ~180–200 kDa type I transmembrane protein, with the N-terminus facing the extracellular space and anchored to the viral membrane via its transmembrane domain followed by a C-terminal short tail facing the cytosol.

    1. Introduction

    Infectious diseases that threatened the life of humans since at least the Neolithic times, are believed to have started around 12,000 years ago, when roaming human hunter-gatherers became sedentary and settled into small camps and villages to domesticate animals and cultivate crops [1][2]. The domestication of animals and their proximity to humans favored the transmission of animal-human (zoonotic) diseases. The latter implicated various infectious microorganisms, such as bacteria, fungi, parasites, and viruses. Some of these pathogens caused widespread disease worldwide with “pandemic proportions” resulting in the death of a significant fraction of the human population.

    2. Proprotein Convertases and Enveloped Viruses

    2.1. Furin in Viral Infections and Pathogenicity

    Furin is the third and best characterized member of the PCSK family of secretory convertases. It cleaves at basic amino acid motifs and recognizes a prototypical sequence R-X-(K/R)-R↓ [3]. The presence of this Furin(-like) motif in viral envelope glycoproteins constitutes a way to activate the fusion-dependant entry of several viruses such as HIV gp160, influenza hemagglutinin (Table 2), and some coronaviruses spike proteins [4][5].
    Table 2. Various enveloped viruses and the sequences of surrounding their surface glycoprotein cleavage sites (designated by an arrow) by Furin(-like) proprotein convertases. The bold and underlined residues at positions P8, P6, P4, P2, P1 and P2′ emphasize the importance of these amino acids for protease recognition.
    Virus Glycoprotein P8   P6   P4   P2 P2′
    HIV gp160   V Q R E K R A V
    H7N1 A/FPV/Rostock/34 HA   K K R E K R G L
    Avian H5N8 TKY/IRE HA   R K R K K R G L
    Avian H5N1 A/HK/97 HA R E R R R K K R G L
    Avian H5N1 TKY/ENG HA N T P Q R K K R G L
    Human CMV gB   H N R T K R S T
    Human MPV F Protein   N P R Q S R F V
    Human RSV F Protein   K K R K R R F L
    Dengue Virus (DENG2) PrM   H R R E K R S V
    Ebola Virus gp160   G R R T R R E A
    Chikungunya (CHIKV) E3E2   P R R Q R R S I
    Zika Virus PrM   A R R S R R A V
    SARS-CoV-2 S   S P R R A R S V
    In 1992, Furin was identified to be the cellular protease cleaving hemagglutinin (HA) of fowl plague/influenza virus (FPV; H7N1 A/FPV/Rostock/34), as well as the HIV glycoprotein gp160 into gp120 and gp41 [6][7][8]. The presence of a Furin cleavage sequence can shift the pathogenicity of certain viruses from non-virulent to virulent. For instance, Newcastle disease virus (NDV) fusion protein contains only a monobasic cleavage site, while the virulent NDV fusion protein possesses a polybasic sequence of amino acids that can be cleaved by Furin [9]. Similarly, the acquisition of a Furin cleavage site has been linked to the high pathogenicity of avian influenza [10], especially for the Hong Kong variant (H5N1 A/HK/97) [11][12] that has an insertion of RERR between the NTPQ and the Furin-site RKKR↓GL in HA (Table 2), resulting in a very efficient cleavage by more than one convertase, such as Furin, PC5 and PC7 [13]. Other pathogenic influenza virus do not possesses a Furin cleavage site and rely on trypsin-like proteases, e.g., HAT and TMPRSS2 [14]. The expression of these enzymes is restricted to the upper respiratory tract [15], while Furin is ubiquitously expressed, and the acquisition of a Furin(-like) site enhances the cellular tropism of influenza. However, the presence of a polybasic Furin(-like) cleavage site is not the only determining factor for cleavage by Furin, as the amino-acids adjacent to the Furin(-like) sequence are important too, as described in Table 2. Furthermore, the presence of oligosaccharides near the cleavage site can prevent processing [16], unless it is compensated by an increased number of basic residues as described in the non-pathogenic variant of influenza hemagglutinin A of H5N2 [17].
    Aside from glycoproteins from NDV and influenza hemagglutinin, HIV glycoprotein, as well as other retrovirus glycoproteins such as Rous sarcoma virus and murine leukemia virus are cleaved by Furin. Interestingly, these glycoproteins form trimers, and some viruses assemble with un-cleaved glycoproteins that remain either inactive or can be cleaved at the plasma membrane. The Furin-specific cleavage of gp160 in HIV-1 has been described as dispensable as other proprotein convertases [18] could substitute for Furin in some tissues, and plasmin could activate the gp160 at the plasma membrane. However, these hypotheses remain to be validated in vivo in humans.
    Human metapneumovirus (hMPV) is a paramyxovirus responsible for acute respiratory tract infections which can result in hospitalization of both children and adults. We showed that blocking the activity of the protease-activated receptor 1 (PAR1), a G-coupled receptor, induced by inflammation, protects against hMPV infections [19]. Furthermore, Furin activates the envelope F-protein of hMPV by cleavage at NPRQSR102↓FV (Table 2, where bold residues emphasise the critical P1 and P4 positions) [19]. Unexpectedly, PAR1 itself potently inhibits cellular Furin activity [20]. Indeed, PAR1 exhibits in its second luminal loop a Furin(-like) motif (PRSFLLR46-NP) with a P1 and P6 Arg and is cleaved by PC5A and PACE4 but not by Furin. The presence of an Asn47 at the P1′ site rather made it a potent Furin-inhibitor that binds its catalytic subunit and sequesters the [PAR1-Furin] complex in the trans-Golgi network (TGN), thereby inactivating Furin and preventing PAR1 from reaching the cell surface [20]. The overexpression of PAR1 has been described in the brain upon neuroinflammation and in patients with neurocognitive disorders associated with HIV infection (HAND) [20]. Interestingly, while PAR1 binds and inhibits Furin, PAR2 is cleaved by Furin at the motif RSSKGR36↓SL, which also exhibits a P1 and P6 Arg but a favorable Ser at P1′ and Leu at P2′ (Table 2) [16]. Recently, another strategy of viral host-cell defense has been identified. Guanylate-binding proteins (GBPs) are interferon stimulated genes. Upon HIV infection, the cytosolic GBP2 and 5 expressions are induced, and these proteins can bind the cytosolic tail of Furin and prevent its trafficking beyond the cis/medial Golgi, thus keeping it in an inactive state bound to its inhibitory prodomain. This effectively inhibits the Furin processing of gp160 into gp120 and gp41 [21][22]. Thus, while PAR1 is the first secretory endogenous natural inhibitor of Furin that blocks its enzymatic activity and prevents its exit from the TGN, the cytosolic GBP2,5 also inhibits Furin activity by blocking it in the cis/medial Golgi in an inactive state.
    Another family of viruses requiring Furin is the filoviridae. Marburg and Ebola are enveloped single negative strand RNA viruses of the filoviridae family. These viruses are highly pathogenic in humans causing hemorrhagic fever with a very high mortality rate. The glycoprotein of Marburg and Ebola (GPs) are cleaved by Furin into GP1 containing the receptor binding domain (RBD) and membrane-bound GP2 [23]. Pathogenic viruses of the family possess a Furin(-like) cleavage site, but experiments design to establish the role of Furin in pathogenicity indicated that Furin cleavage is not required for replication in cell culture [24] and disease severity in non-human primates [25]. Recent work identified a new Furin site in Ebola GP that requires N-glycosylation to be processed [26]. Interestingly, MARCH8 a member of the Membrane-associated RING-CH-type 8 (MARCH8) that has been described to have broad antiviral activity against several viruses, can inhibit Furin by forming a complex with the GP and Furin thereby sequestering it in the Golgi, and preventing the maturation of the viral particles [27]. This is the third example of endogenous Furin-inhibitors that sequester the enzyme in a subcellular Golgi compartment where it remains inactive.
    The Flaviviruses envelope also requires Furin to get activated. The first evidence was reported for the Tick Borne Encephalitis virus. Flavivirus are enveloped positive strand RNA viruses. Their surface protein is composed of a heterodimer formed by prM and E [28]. The immature prM is cleaved by Furin at GSRTRR205↓SV before the viral particles are released from the cell. The cleavage allows fusion and is required for infectivity. An acidic pH is required to promote a change of conformation of the heterodimer to facilitate Furin cleavage. While, the cleavage of prM into M is inefficient especially for Dengue virus (HRREKR205↓SV) (Table 2), likely due to the presence of a Glu at P3 [29], Furin has been confirmed to be required for infectivity of Dengue virus in vitro by using LoVo cells that are Furin deficient [30]. Additionally, the role of Furin in the antibody-dependent-enhancement (ADE) of Dengue virus infection is intriguing. ADE is the mechanism by which the immunisation against Dengue could exacerbate the pathogenicity of Dengue in the context of a second infection. This has been explained by the fact that the binding of antibodies to an immature particle could enhance its infectivity. Interestingly, while Furin inhibition has been described to block anti-Envelope dependant enhancement [31], antibodies against prM (uncleaved by Furin) allow tissue entry of immature particles [32]. Recently, broadly neutralizing monoclonal antibodies were reported to protect against multiple tick-borne flaviviruses (TBFVs) [33], opening the way to the design of vaccines and antibody therapeutics against clinically relevant TBFVs.
    Chikungunya virus (CHIKV) is a mosquito-transmitted α-virus that causes in humans an acute infection characterized by polyarthralgia, fever, myalgia, and headache. Since 2005 this virus has been responsible for an epidemic outbreak of unprecedented magnitude. By analogy with other α-viruses, it is thought that cellular proteases can process the viral precursor protein E3E2 to produce the receptor binding E2 protein that associates as a heterodimer with E1. Destabilization of the heterodimer by exposure to low pH allows viral fusion and infection. We demonstrated that membrane-bound Furin but also PC5B can process E3E2 from African CHIKV strains at the HRQRR642↓ST site, whereas a CHIKV strain of Asian origin is cleaved at RRQRR642↓SI (Table 2) by membranous and soluble Furin, PC5A, PC5B, and PACE4 but not by PC7 or SKI-1/S1P [34]. This cleavage was also observed in CHIKV-infected cells and could be blocked by Furin inhibitor decanoyl-RVKR-chloromethyl ketone. This inhibitor was compared with chloroquine for its ability to inhibit CHIKV spreading in myoblast cell cultures, a cell-type previously described as a natural target of this virus [34]. We observed an additive effect of dec-RVKR-cmk and chloroquine, supporting the concept that these two drugs act by essentially distinct mechanisms. The combinatory action of chloroquine and dec-RVKR-cmk led to almost total suppression of viral spread and yield [34].
    Furin cleavage of viral glycoproteins of the Herpesviridae family have been reported for Herpes simplex virus 1 and 2 and Varicella Zoster (Table 1), contributing to the pathogenesis of the later in vivo [35][36]. Finally, while Furin cleaves many glycoproteins, its activity is required for other viral proteins such as for Hepatitis B virus (HBV) core antigen. For reviews see [29][21].
    Table 1. Families of pathogenic viruses that depend on the basic aa-specific proprotein convertases for host cell entry. RT = reverse transcriptase. Modified from [29].
    Family Virus Capsid Genome
    Retroviridae HIV, Leukemia viruses Enveloped Linear ssRNA(−), RT
    Flaviridae HCV, Dengue, Zika, West Nile Enveloped Linear ssRNA(+)
    Togaviridae Chikungunya Enveloped Linear ssRNA(+)
    Coronaviridae SARS-CoV-1,2, MERS Enveloped Linear ssRNA(+)
    Filoviridae Ebola, Marburg Enveloped Linear ssRNA(−)
    Orthomyxoviridae Avian Influenza H5N1 Enveloped Linear ssRNA(−)
    Paramixoviridae Measle, RSV, Nipah, MPV Enveloped Linear ssRNA(−)
    Hepadnaviridae Hepatitis B Enveloped Linear ssDNA (−), RT
    Herpesviridae Herpes, CMV, Varicella-Zoster Enveloped Linear dsDNA
    Papillomaviridae HPV Naked Circular dsDNA
    HIV-1 encodes four accessory gene products—Vpr, Vif, Vpu, and Nef—which are thought to collectively manipulate host cell biology to promote viral replication, persistence, and immune escape [37]. Increasing evidence suggests that extracellular Vpr could contribute to HIV pathogenesis through its effect on bystander cells. Soluble forms of Vpr have been detected in the sera and cerebrospinal fluids of HIV-1-infected patients, and in vitro studies have implicated extracellular Vpr as an effector of cellular responses, including G2 arrest, apoptosis, and induction of cytokines and chemokines production, presumably through its ability to transduce into multiple cell types. Thus, Vpr is a true virulence factor and is a potential and promising target in different strategies aiming to fight infected cells including latently HIV-infected cells [38]. While Furin is clearly implicated in the processing activation of multiple viral surface glycoproteins, including gp160 of HIV-1, other cell-surface associated convertases such as PC5A and PACE4 [39][40] seem to negatively regulate the activity of the HIV-1 by cleavage-inactivation of secreted Vpr at RQRR88[41]. PC-mediated processing of extracellular Vpr results in a truncated Vpr product that was defective for the induction of cell cycle arrest and apoptosis when expressed in human cells [41]. Thus, inhibitors of PC5/PACE4 may enhance HIV-1 virulence, and Furin-specific inhibitors would restrict HIV-1 infectivity, without affecting Vpr inactivation by PC5A/PACE4.

    2.2. PCSK9 and Viral Infections

    The secreted protein PCSK9 is the 9th member of the PCSKs family that autocatalytically cleaves its prodomain in the ER allowing its exit from this compartment and its secretion from cells [42], as a protease-inactive [prodomain-PCSK9] complex [43][44]. Its high expression in the liver and the presence of its gene on chromosome 1p32 [42], led to the genetic association of gain-of-function (GOF) variants of PCSK9 with an autosomal dominant form of hypercholesterolemia [45]. This strongly suggested that PCSK9 may play a critical role in low density lipoprotein- cholesterol (LDLc) regulation. Indeed, while GOF variants were associated with high levels LDLc [45], the reverse was true for loss-of-function (LOF) variants [46]. Mechanistically, circulating PCSK9 was shown to reduce the levels of liver LDL-receptor (LDLR) protein by sorting the PCSK9-LDLR complex to lysosomes for degradation, implicating an interaction of the PCSK9 catalytic domain with the EGF-A domain of the LDLR [43][47][48].
    In a first study, we showed that PCSK9 can reduce HCV infection of HuH7 cells using the cell culture-derived HCV clone JFH1 (genotype 2a). We demonstrated that the effect was due to the PCSK9-induced decrease of the LDLR protein levels on the surface of HuH7 cells. Also, HCV upregulates the expression of LDLR in vitro and in the chronically infected liver [49], likely due to an upregulation of the LDLR promotor activity through SREBPs and a decreased expression of PCSK9 [50]. Befittingly, Alirocumab, a therapeutic human monoclonal antibody (mAb) to PCSK9 used to treat hypercholesterolemia through an increase of cell surface LDLR failed to reduce HCV entry and infectivity in hepatocyte in vitro [51].
    Additionally, the role of PCSK9 during natural HCV infection is not fully understood. We showed that HCV genotype 3 (G3) infected patients with high viral load have low levels of PCSK9 and LDLc compared to HCV genotype 1 (G1) infected patients, suggesting that the particular interplay between HCV and lipid metabolism and PCSK9 might be genotype specific [52]. In contrast, in a retrospective study, PCSK9 levels were found to be elevated especially in chronic genotype 2 (G2) HCV patients with or without hepatocellular carcinoma (HCC), but the increased PCSK9 levels were not significantly associated with changes in LDLc [53]. Overall, the data reported illustrate the complexity of the relation between HCV and lipid metabolism and more work would be required to better understand the role of PCSK9 in HCV infection.
    Dengue virus (DENV), a single positive-stranded RNA virus of the family Flaviviridae, is transmitted to humans by the urban-adapted Aedes mosquitoes. It is estimated that 400 M+ individuals are infected with 1 of the 4 types of DENV [54]. The DENV surface glycoprotein prM-E complex needs cleavage by Furin(-like) enzymes to remove the prodomain of the envelope glycoprotein prM and allow the formation of the activated M-E complex and acquisition of infectivity. However, unlike cleavage of the prM of other flaviviruses, cleavage of DENV prM is incomplete in many cell lines, likely due to the negative influence of the aspartic acid (D) at the P3 position of the processing site HRRDKR↓SV at the pr-M junction (Table 2) [55]. Therefore, it is not surprising that inhibitors of Furin(-like) enzymes, while significantly reducing viral entry, did not completely block viral infections [56] (unpublished results).
    Table 2. Various enveloped viruses and the sequences of surrounding their surface glycoprotein cleavage sites (designated by an arrow) by Furin(-like) proprotein convertases. The bold and underlined residues at positions P8, P6, P4, P2, P1 and P2′ emphasize the importance of these amino acids for protease recognition.
    Virus Glycoprotein P8   P6   P4   P2 P2′
    HIV gp160   V Q R E K R A V
    H7N1 A/FPV/Rostock/34 HA   K K R E K R G L
    Avian H5N8 TKY/IRE HA   R K R K K R G L
    Avian H5N1 A/HK/97 HA R E R R R K K R G L
    Avian H5N1 TKY/ENG HA N T P Q R K K R G L
    Human CMV gB   H N R T K R S T
    Human MPV F Protein   N P R Q S R F V
    Human RSV F Protein   K K R K R R F L
    Dengue Virus (DENG2) PrM   H R R E K R S V
    Ebola Virus gp160   G R R T R R E A
    Chikungunya (CHIKV) E3E2   P R R Q R R S I
    Zika Virus PrM   A R R S R R A V
    SARS-CoV-2 S   S P R R A R S V
    Very recently, we showed that DENV infection reduces the antiviral response of the host hepatocytes. Thus, DENV infection induces expression of PCSK9, thereby reducing cell surface levels of LDLR and LDLc uptake resulting in enhanced de novo cholesterol synthesis and its enrichment in the ER. In turn, high levels of ER cholesterol suppressed the phosphorylation and activation of the ER-resident stimulator of interferon (IFN) gene (STING), leading to reduction of type I interferon (IFN) signaling through antiviral IFN-stimulated genes (ISGs) [57]. This was supported by the detection of elevated plasma PCSK9 levels in patients infected with DENV resulting in high viremia and increased severity of plasma leakage. This unexpected role of PCSK9 in dengue pathogenesis, led us to test the effect of inhibition of PCSK9 function by the mAb Alirocumab. Befittingly, this treatment resulted in higher LDLR levels and lower viremia. Our data suggested that PCSK9 inhibitors could be a suitable host-directed treatment for patients with dengue [57], possibly in combination with Furin(-like) inhibitors.

    3. Discussion

    From the above considerations, it became clear that most enveloped viruses require the processing of their surface glycoproteins for productive infectivity. Enhanced pathogenicity has been associated with the apparition of a new cleavage site in certain viruses. While clearly favoring Furin cleavage [58][59], the exact role of the acquisition of a Furin site in SARS-CoV-2 [60] and its role in the spread of the virus and/or its pathogenicity have not been adequately established. Experimental evidence is not easy to obtain. One would need to generate a virus mutant to be tested in an animal model exhibiting the multiple pathogeneses that mimic the human clinical manifestation of COVID-19. A clear advantage of SARS-CoV-2 over SARS-CoV-1 has been its ability to spread asymptomatically and with great efficiency in a large portion of the population. One can speculate that the acquisition of a Furin cleavage site has contributed to confer to SARS-CoV-2 such an advantage and to enhance the tropism of this virus, as Furin is widely expressed in most tissues, whereas TMPRSS2 implicated in SARS-CoV-1 activation [61] is more limited in its tissue expression.

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    1. Dobson, A.P.; Carper, E.R. Infectious Diseases and Human Population History: Throughout history the establishment of disease has been a side effect of the growth of civilization. Bioscience 1996, 46, 115–126.
    2. Morens, D.M.; Folkers, G.K.; Fauci, A.S. Emerging infections: A perpetual challenge. Lancet Infect. Dis. 2008, 8, 710–719.
    3. Seidah, N.G.; Chretien, M. Proprotein and prohormone convertases: A family of subtilases generating diverse bioactive polypeptides. Brain Res. 1999, 848, 45–62.
    4. Le Coupanec, A.; Desforges, M.; Meessen-Pinard, M.; Dube, M.; Day, R.; Seidah, N.G.; Talbot, P.J. Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System. PLoS Pathog. 2015, 11, e1005261.
    5. Tang, T.; Bidon, M.; Jaimes, J.A.; Whittaker, G.R.; Daniel, S. Coronavirus membrane fusion mechanism offers as a potential target for antiviral development. Antivir. Res. 2020, 178, 104792.
    6. Seidah, N.G. The proprotein convertases, 20 years later. Methods Mol. Biol. 2011, 768, 23–57.
    7. Hallenberger, S.; Bosch, V.; Angliker, H.; Shaw, E.; Klenk, H.D.; Garten, W. Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160. Nature 1992, 360, 358–361.
    8. Stieneke-Grober, A.; Vey, M.; Angliker, H.; Shaw, E.; Thomas, G.; Roberts, C.; Klenk, H.D.; Garten, W. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J. 1992, 11, 2407–2414.
    9. Li, Z.; Sergel, T.; Razvi, E.; Morrison, T. Effect of cleavage mutants on syncytium formation directed by the wild-type fusion protein of Newcastle disease virus. J. Virol. 1998, 72, 3789–3795.
    10. Walker, J.A.; Molloy, S.S.; Thomas, G.; Sakaguchi, T.; Yoshida, T.; Chambers, T.M.; Kawaoka, Y. Sequence specificity of furin, a proprotein-processing endoprotease, for the hemagglutinin of a virulent avian influenza virus. J. Virol. 1994, 68, 1213–1218.
    11. Gao, P.; Watanabe, S.; Ito, T.; Goto, H.; Wells, K.; McGregor, M.; Cooley, A.J.; Kawaoka, Y. Biological heterogeneity, including systemic replication in mice, of H5N1 influenza A virus isolates from humans in Hong Kong. J. Virol. 1999, 73, 3184–3189.
    12. Claas, E.C.; Osterhaus, A.D.; van Beek, R.; de Jong, J.C.; Rimmelzwaan, G.F.; Senne, D.A.; Krauss, S.; Shortridge, K.F.; Webster, R.G. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 1998, 351, 472–477.
    13. Basak, A.; Zhong, M.; Munzer, J.S.; Chretien, M.; Seidah, N.G. Implication of the proprotein convertases furin, PC5 and PC7 in the cleavage of surface glycoproteins of Hong Kong, Ebola and respiratory syncytial viruses: A comparative analysis with fluorogenic peptides. Biochem. J. 2001, 353, 537–545.
    14. Böttcher, E.; Matrosovich, T.; Beyerle, M.; Klenk, H.D.; Garten, W.; Matrosovich, M. Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium. J. Virol. 2006, 80, 9896–9898.
    15. Murza, A.; Dion, S.P.; Boudreault, P.L.; Désilets, A.; Leduc, R.; Marsault, É. Inhibitors of type II transmembrane serine proteases in the treatment of diseases of the respiratory tract—A review of patent literature. Expert Opin. Ther. Pat. 2020, 30, 807–824.
    16. Sachan, V.; Lodge, R.; Mihara, K.; Hamelin, J.; Power, C.; Gelman, B.B.; Hollenberg, M.D.; Cohen, É.A.; Seidah, N.G. HIV-induced neuroinflammation: Impact of PAR1 and PAR2 processing by Furin. Cell Death Differ. 2019, 26, 1942–1954.
    17. Ohuchi, M.; Orlich, M.; Ohuchi, R.; Simpson, B.E.; Garten, W.; Klenk, H.D.; Rott, R. Mutations at the cleavage site of the hemagglutinin after the pathogenicity of influenza virus A/chick/Penn/83 (H5N2). Virology 1989, 168, 274–280.
    18. Gu, M.; Rappaport, J.; Leppla, S.H. Furin is important but not essential for the proteolytic maturation of gp160 of HIV-1. FEBS Lett. 1995, 365, 95–97.
    19. Aerts, L.; Hamelin, M.E.; Rhaume, C.; Lavigne, S.; Couture, C.; Kim, W.; Susan-Resiga, D.; Prat, A.; Seidah, N.G.; Vergnolle, N.; et al. Modulation of protease activated receptor 1 influences human metapneumovirus disease severity in a mouse model. PLoS ONE 2013, 8, e72529.
    20. Kim, W.; Zekas, E.; Lodge, R.; Susan-Resiga, D.; Marcinkiewicz, E.; Essalmani, R.; Mihara, K.; Ramachandran, R.; Asahchop, E.; Gelman, B.; et al. Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder. Mol. Cell Biol. 2015, 35, 3684–3700.
    21. Braun, E.; Sauter, D. Furin-mediated protein processing in infectious diseases and cancer. Clin. Transl. Immunol. 2019, 8, e1073.
    22. Braun, E.; Hotter, D.; Koepke, L.; Zech, F.; Groß, R.; Sparrer, K.M.J.; Müller, J.A.; Pfaller, C.K.; Heusinger, E.; Wombacher, R.; et al. Guanylate-Binding Proteins 2 and 5 Exert Broad Antiviral Activity by Inhibiting Furin-Mediated Processing of Viral Envelope Proteins. Cell Rep. 2019, 27, 2092–2104.e10.
    23. Volchkov, V.E.; Feldmann, H.; Volchkova, V.A.; Klenk, H.D. Processing of the Ebola virus glycoprotein by the proprotein convertase furin. Proc. Natl. Acad. Sci. USA 1998, 95, 5762–5767.
    24. Neumann, G.; Feldmann, H.; Watanabe, S.; Lukashevich, I.; Kawaoka, Y. Reverse genetics demonstrates that proteolytic processing of the Ebola virus glycoprotein is not essential for replication in cell culture. J. Virol. 2002, 76, 406–410.
    25. Neumann, G.; Geisbert, T.W.; Ebihara, H.; Geisbert, J.B.; Daddario-DiCaprio, K.M.; Feldmann, H.; Kawaoka, Y. Proteolytic processing of the Ebola virus glycoprotein is not critical for Ebola virus replication in nonhuman primates. J. Virol. 2007, 81, 2995–2998.
    26. Wang, B.; Wang, Y.; Frabutt, D.A.; Zhang, X.; Yao, X.; Hu, D.; Zhang, Z.; Liu, C.; Zheng, S.; Xiang, S.H.; et al. Mechanistic understanding of N-glycosylation in Ebola virus glycoprotein maturation and function. J. Biol. Chem. 2017, 292, 5860–5870.
    27. Yu, C.; Li, S.; Zhang, X.; Khan, I.; Ahmad, I.; Zhou, Y.; Li, S.; Shi, J.; Wang, Y.; Zheng, Y.H. MARCH8 Inhibits Ebola Virus Glycoprotein, Human Immunodeficiency Virus Type 1 Envelope Glycoprotein, and Avian Influenza Virus H5N1 Hemagglutinin Maturation. mBio 2020, 11, e01882-20.
    28. Stadler, K.; Allison, S.L.; Schalich, J.; Heinz, F.X. Proteolytic activation of tick-borne encephalitis virus by furin. J. Virol. 1997, 71, 8475–8481.
    29. Izaguirre, G. The Proteolytic Regulation of Virus Cell Entry by Furin and Other Proprotein Convertases. Viruses 2019, 11, 837.
    30. Zybert, I.A.; van der Ende-Metselaar, H.; Wilschut, J.; Smit, J.M. Functional importance of dengue virus maturation: Infectious properties of immature virions. J. Gen. Virol. 2008, 89, 3047–3051.
    31. Da Silva Voorham, J.M.; Rodenhuis-Zybert, I.A.; Ayala Nuñez, N.V.; Colpitts, T.M.; van der Ende-Metselaar, H.; Fikrig, E.; Diamond, M.S.; Wilschut, J.; Smit, J.M. Antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype 2. PLoS ONE 2012, 7, e29957.
    32. Rodenhuis-Zybert, I.A.; van der Schaar, H.M.; da Silva Voorham, J.M.; van der Ende-Metselaar, H.; Lei, H.Y.; Wilschut, J.; Smit, J.M. Immature dengue virus: A veiled pathogen? PLoS Pathog. 2010, 6, e1000718.
    33. VanBlargan, L.A.; Errico, J.M.; Kafai, N.M.; Burgomaster, K.E.; Jethva, P.N.; Broeckel, R.M.; Meade-White, K.; Nelson, C.A.; Himansu, S.; Wang, D.; et al. Broadly neutralizing monoclonal antibodies protect against multiple tick-borne flaviviruses. J. Exp. Med. 2021, 218, e20210174.
    34. Ozden, S.; Lucas-Hourani, M.; Ceccaldi, P.E.; Basak, A.; Valentine, M.; Benjannet, S.; Hamelin, J.; Jacob, Y.; Mamchaoui, K.; Mouly, V.; et al. Inhibition of Chikungunya Virus Infection in Cultured Human Muscle Cells by Furin Inhibitors: Impairment of the maturation of the E2 surface glycoprotein. J. Biol. Chem. 2008, 283, 21899–21908.
    35. Kropp, K.A.; Srivaratharajan, S.; Ritter, B.; Yu, P.; Krooss, S.; Polten, F.; Pich, A.; Alcami, A.; Viejo-Borbolla, A. Identification of the Cleavage Domain within Glycoprotein G of Herpes Simplex Virus Type 2. Viruses 2020, 12, 1428.
    36. Oliver, S.L.; Sommer, M.; Zerboni, L.; Rajamani, J.; Grose, C.; Arvin, A.M. Mutagenesis of varicella-zoster virus glycoprotein B: Putative fusion loop residues are essential for viral replication, and the furin cleavage motif contributes to pathogenesis in skin tissue in vivo. J. Virol. 2009, 83, 7495–7506.
    37. Emerman, M.; Malim, M.H. HIV-1 regulatory/accessory genes: Keys to unraveling viral and host cell biology. Science 1998, 280, 1880–1884.
    38. Wallet, C.; Rohr, O.; Schwartz, C. Evolution of a concept: From accessory protein to key virulence factor, the case of HIV-1 Vpr. Biochem. Pharmacol. 2020, 180, 114128.
    39. Seidah, N.G.; Prat, A. The biology and therapeutic targeting of the proprotein convertases. Nat. Rev. Drug Discov. 2012, 11, 367–383.
    40. Mayer, G.; Hamelin, J.; Asselin, M.C.; Pasquato, A.; Marcinkiewicz, E.; Tang, M.; Tabibzadeh, S.; Seidah, N.G. The regulated cell surface zymogen activation of the proprotein convertase PC5A directs the processing of its secretory substrates. J. Biol. Chem. 2008, 283, 2373–2384.
    41. Xiao, Y.; Chen, G.; Richard, J.; Rougeau, N.; Li, H.; Seidah, N.G.; Cohen, E.A. Cell-surface processing of extracellular human immunodeficiency virus type 1 Vpr by proprotein convertases. Virology 2008, 372, 384–397.
    42. Seidah, N.G.; Benjannet, S.; Wickham, L.; Marcinkiewicz, J.; Jasmin, S.B.; Stifani, S.; Basak, A.; Prat, A.; Chretien, M. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation. Proc. Natl. Acad. Sci. USA 2003, 100, 928–933.
    43. Benjannet, S.; Rhainds, D.; Essalmani, R.; Mayne, J.; Wickham, L.; Jin, W.; Asselin, M.C.; Hamelin, J.; Varret, M.; Allard, D.; et al. NARC-1/PCSK9 and its natural mutants: Zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol. J. Biol. Chem. 2004, 279, 48865–48875.
    44. Li, J.; Tumanut, C.; Gavigan, J.A.; Huang, W.J.; Hampton, E.N.; Tumanut, R.; Suen, K.F.; Trauger, J.W.; Spraggon, G.; Lesley, S.A.; et al. Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity. Biochem. J. 2007, 406, 203–207.
    45. Abifadel, M.; Varret, M.; Rabes, J.P.; Allard, D.; Ouguerram, K.; Devillers, M.; Cruaud, C.; Benjannet, S.; Wickham, L.; Erlich, D.; et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 2003, 34, 154–156.
    46. Cohen, J.; Pertsemlidis, A.; Kotowski, I.K.; Graham, R.; Garcia, C.K.; Hobbs, H.H. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9. Nat. Genet. 2005, 37, 161.
    47. Maxwell, K.N.; Breslow, J.L. Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype. Proc. Natl. Acad. Sci. USA 2004, 101, 7100–7105.
    48. Park, S.W.; Moon, Y.A.; Horton, J.D. Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver. J. Biol. Chem. 2004, 279, 50630–50638.
    49. Syed, G.H.; Tang, H.; Khan, M.; Hassanein, T.; Liu, J.; Siddiqui, A. Hepatitis C virus stimulates low-density lipoprotein receptor expression to facilitate viral propagation. J. Virol. 2014, 88, 2519–2529.
    50. Li, Z.; Liu, Q. Hepatitis C virus regulates proprotein convertase subtilisin/kexin type 9 promoter activity. Biochem. Biophys. Res. Commun. 2018, 496, 1229–1235.
    51. Ramanathan, A.; Gusarova, V.; Stahl, N.; Gurnett-Bander, A.; Kyratsous, C.A. Alirocumab, a Therapeutic Human Antibody to PCSK9, Does Not Affect CD81 Levels or Hepatitis C Virus Entry and Replication into Hepatocytes. PLoS ONE 2016, 11, e0154498.
    52. Bridge, S.H.; Sheridan, D.A.; Felmlee, D.J.; Crossey, M.M.; Fenwick, F.I.; Lanyon, C.V.; Dubuc, G.; Seidah, N.G.; Davignon, J.; Thomas, H.C.; et al. PCSK9, apolipoprotein E and lipoviral particles in chronic hepatitis C genotype 3: Evidence for genotype-specific regulation of lipoprotein metabolism. J. Hepatol. 2014, 62, 763–770.
    53. Fasolato, S.; Pigozzo, S.; Pontisso, P.; Angeli, P.; Ruscica, M.; Savarino, E.; De Martin, S.; Lupo, M.G.; Ferri, N. PCSK9 Levels Are Raised in Chronic HCV Patients with Hepatocellular Carcinoma. J. Clin. Med. 2020, 9, 3134.
    54. Bhatt, S.; Gething, P.W.; Brady, O.J.; Messina, J.P.; Farlow, A.W.; Moyes, C.L.; Drake, J.M.; Brownstein, J.S.; Hoen, A.G.; Sankoh, O.; et al. The global distribution and burden of dengue. Nature 2013, 496, 504–507.
    55. Junjhon, J.; Lausumpao, M.; Supasa, S.; Noisakran, S.; Songjaeng, A.; Saraithong, P.; Chaichoun, K.; Utaipat, U.; Keelapang, P.; Kanjanahaluethai, A.; et al. Differential modulation of prM cleavage, extracellular particle distribution, and virus infectivity by conserved residues at nonfurin consensus positions of the dengue virus pr-M junction. J. Virol. 2008, 82, 10776–10791.
    56. Rana, J.; Slon Campos, J.L.; Poggianella, M.; Burrone, O.R. Dengue virus capsid anchor modulates the efficiency of polyprotein processing and assembly of viral particles. J. Gen. Virol. 2019, 100, 1663–1673.
    57. Gan, E.S.; Tan, H.C.; Le, D.H.T.; Huynh, T.T.; Wills, B.; Seidah, N.G.; Ooi, E.E.; Yacoub, S. Dengue virus induces PCSK9 expression to alter antiviral responses and disease outcomes. J. Clin. Investig. 2020, 130, 5223–5234.
    58. Essalmani, R.; Jain, J.; Susan-Resiga, D.; Andréo, U.; Evagelidis, A.; Derbali, R.M.; Huynh, D.N.; Dallaire, F.; Laporte, M.; Delpal, A.; et al. Furin cleaves SARS-CoV-2 spike-glycoprotein at S1/S2 and S2′ for viral fusion/entry: Indirect role of TMPRSS2. bioRxiv 2020.
    59. Bestle, D.; Heindl, M.R.; Limburg, H.; Pilgram, O.; Moulton, H.; Stein, D.A.; Hardes, K.; Eickmann, M.; Dolnik, O.; Rohde, C.; et al. TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells. Life Sci. Alliance 2020, 3.
    60. Coutard, B.; Valle, C.; de Lamballerie, X.; Canard, B.; Seidah, N.G.; Decroly, E. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antivir. Res. 2020, 176, 104742.
    61. Millet, J.K.; Whittaker, G.R. Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis. Virus Res. 2015, 202, 120–134.