Martin J. Vincent

Characterization of the Glycoproteins of Crimean-Congo Hemorrhagic Fever Virus

ABSTRACT Crimean-Congo hemorrhagic fever (CCHF) virus is the cause of an important tick-borne disease of humans throughout regions of Africa, Europe, and Asia. Like other members of the genus Nairovirus, family Bunyaviridae, the CCHF virus M genome RNA segment encodes the virus glycoproteins. Sequence analysis of the CCHF virus (Matin strain) M RNA segment revealed one major open reading frame that potentially encodes a precursor polyprotein 1,689 amino acids (aa) in length. Comparison of the deduced amino acid sequences of the M-encoded polyproteins of Nigerian, Pakistani, and Chinese CCHF virus strains revealed two distinct protein regions. The carboxyl-terminal 1,441 aa are relatively highly conserved (up to 8.4% identity difference), whereas the amino-terminal 243 to 248 aa are highly variable (up to 56.4% identity difference) and have mucin-like features, including a high serine, threonine, and proline content (up to 47.3%) and a potential for extensive O-glycosylation. Analysis of released virus revealed two major structural glycoproteins, G2 (37 kDa) and G1 (75 kDa). Virus protein analysis by various techniques, including pulse-chase analysis and/or reactivity with CCHF virus-specific polyclonal and antipeptide antibodies, demonstrated that the 140-kDa (which contains the mucin-like region) and 85-kDa nonstructural proteins are the precursors of the mature G2 and G1 proteins, respectively. The amino termini of the CCHF virus (Matin strain) G2 and G1 proteins were established by microsequencing to be equivalent to aa 525 and 1046, respectively, of the encoded polyprotein precursor. The tetrapeptides RRLL and RKPL are immediately upstream of the cleavage site for mature G2 and G1, respectively. These are completely conserved among the predicted polyprotein sequences of all the CCHF virus strains and closely resemble the tetrapeptides that represent the major cleavage recognition sites present in the glycoprotein precursors of arenaviruses, such as Lassa fever virus (RRLL) and Pichinde virus (RKLL). These results strongly suggest that CCHF viruses (and other members of the genus Nairovirus) likely utilize the subtilase SKI-1/S1P-like cellular proteases for the major glycoprotein precursor cleavage events, as has recently been demonstrated for the arenaviruses.

Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Proteolytic Processing by Subtilase SKI-1

ABSTRACT Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. The mature virus glycoproteins, Gn and Gc (previously referred to as G2 and G1), are generated by proteolytic cleavage from precursor proteins. The amino termini of Gn and Gc are immediately preceded by tetrapeptides RRLL and RKPL, respectively, leading to the hypothesis that SKI-1 or related proteases may be involved (A. J. Sanchez, M. J. Vincent, and S. T. Nichol, J. Virol. 76:7263-7275, 2002). In vitro peptide cleavage data show that an RRLL peptide representing the Gn processing site is efficiently cleaved by SKI-1 protease, whereas an RKPL peptide representing the Gc processing site is cleaved at negligible levels. The efficient cleavage of RRLL peptide is consistent with the known recognition sequences of SKI-1, including the sequence determinants involved in the cleavage of the Lassa virus (family Arenaviridae) glycoprotein precursor. These in vitro findings were confirmed by expression of wild-type or mutant CCHF virus glycoproteins in CHO cells engineered to express functional or nonfunctional SKI-1. Gn processing was found to be dependent on functional SKI-1, whereas Gc processing was not. Gn processing occurred in the endoplasmic reticulum-cis Golgi compartments and was dependent on an R at the −4 position within the RRLL recognition motif, consistent with the known cleavage properties of SKI-1. Comparison of SKI-1 cleavage efficiency between peptides representing Lassa virus GP2 and CCHF virus Gn cleavage sites suggests that amino acids flanking the RRLL may modulate the efficiency. The apparent lack of SKI-1 cleavage at the CCHF virus Gc RKPL site indicates that related proteases, other than SKI-1, are likely to be involved in the processing at this site and identical or similar sites utilized in several New World arenaviruses.

Conserved Receptor-binding Domains of Lake Victoria Marburgvirus and Zaire Ebolavirus Bind a Common Receptor

The GP1,2 envelope glycoproteins (GP) of filoviruses (marburg- and ebolaviruses) mediate cell-surface attachment, membrane fusion, and entry into permissive cells. Here we show that a 151-amino acid fragment of the Lake Victoria marburgvirus GP1 subunit bound filovirus-permissive cell lines more efficiently than full-length GP1. An homologous 148-amino acid fragment of the Zaire ebolavirus GP1 subunit similarly bound the same cell lines more efficiently than a series of longer GP1 truncation variants. Neither the marburgvirus GP1 fragment nor that of ebolavirus bound a nonpermissive lymphocyte cell line. Both fragments specifically inhibited replication of infectious Zaire ebolavirus, as well as entry of retroviruses pseudotyped with either Lake Victoria marburgvirus or Zaire ebolavirus GP1,2. These studies identify the receptor-binding domains of both viruses, indicate that these viruses utilize a common receptor, and suggest that a single small molecule or vaccine can be developed to inhibit infection of all filoviruses.

Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Precursor Is Cleaved by Furin-Like and SKI-1 Proteases To Generate a Novel 38-Kilodalton Glycoprotein

ABSTRACT Crimean-Congo hemorrhagic fever virus (genus Nairovirus, family Bunyaviridae) genome M segment encodes an unusually large (in comparison to members of other genera) polyprotein (1,684 amino acids in length) containing the two major structural glycoproteins, Gn and Gc, that are posttranslationally processed from precursors PreGn and PreGc by SKI-1 and SKI-1-like proteases, respectively. The characteristics of the N-terminal 519 amino acids located upstream of the mature Gn are unknown. A highly conserved furin/proprotein convertase (PC) cleavage site motif (RSKR247) is located between the variable N-terminal region that is predicted to have mucin-like properties and the rest of PreGn. Mutational analysis of the RSKR247 motif and use of a specific furin/PC inhibitor and brefeldin A demonstrate that furin/PC cleavage occurs at the RSKR247 motif of PreGn as the protein transits the trans Golgi network and generates a novel glycoprotein designated GP38. Immunoprecipitation analysis identified two additional proteins, GP85 and GP160, which contain both mucin and GP38 domain regions, and whose generation does not involve furin/PC cleavage. Consistent with glycosylation predictions, heavy O-linked glycosylation and moderate levels of N-glycans were detected in the GP85 and GP160 proteins, both of which contain the mucin domain. GP38, GP85, and GP160 are likely soluble proteins based on the lack of predicted transmembrane domains, their detection in virus-infected cell supernatants, and the apparent absence from virions. Analogy with soluble glycoproteins and mucin-like proteins encoded by other hemorrhagic fever-associated RNA viruses suggests these proteins could play an important role in viral pathogenesis.

Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Processing by the Endoprotease SKI-1/S1P Is Critical for Virus Infectivity

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV) causes severe human disease. The CCHFV medium RNA encodes a polyprotein which is proteolytically processed to yield the glycoprotein precursors PreGn and PreGc, followed by structural glycoproteins Gn and Gc. Subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) plays a central role in Gn processing. Here we show that CCHFV-infected cells deficient in SKI-1/S1P produce no infectious virus, although PreGn and PreGc accumulated normally in the Golgi apparatus, the site of virus assembly. Only nucleoprotein-containing particles which lacked virus glycoproteins (Gn/Gc or PreGn/PreGc) were secreted. Complementation of SKI-1/S1P-deficient cells with a SKI-1/S1P expression vector restored release of infectious virus (>106 PFU/ml), confirming that SKI-1/S1P processing is required for incorporation of viral glycoproteins. SKI-1/S1P may represent a promising antiviral target.

Function of the KKXX Motif in Endoplasmic Reticulum Retrieval of a Transmembrane Protein Depends on the Length and Structure of the Cytoplasmic Domain

Transmembrane glycoproteins with type 1 topology can be retrieved to the endoplasmic reticulum (ER) by a retrieval signal containing a di-lysine (KK) motif near the C terminus. To investigate the structural requirements for ER retrieval, we have constructed mutants of the simian immunodeficiency virus (SIV) envelope (Env) protein with cytoplasmic tails of different lengths and containing a KK motif at the −3 and −4 positions. Such proteins were found to be retained intracellularly when the signal was located 18 amino acids or more away from the membrane spanning domain. The retrieval signal was found to be functional even when placed at the distal end of the wild-type SIV Env protein with 164 amino acids in the cytoplasmic tail, as shown by the lack of proteolytic processing and lack of cell surface expression of the mutant proteins. However, proteins with a cytoplasmic tail length of 13 amino acids or less having the di-lysine motif at the −3 and −4 positions were not retrieved to the ER since they were found to be processed and transported to the cell surface. The surface-expressed proteins were found to be functional in inducing cell fusion, whereas the proteins retained intracellularly were defective in fusion activity. We also found that the KK motif introduced near an amphipathic helical region in the cytoplasmic tail was not functional. These results demonstrate that the ability of the KK motif to cause protein retrieval and retention in the endoplasmic reticulum depends on the length and structure of the cytoplasmic domain. The ER retrieval of the mutant proteins was found to correlate with increased intracellular binding to β COP proteins.

Enhancement of mucosal immune responses to the influenza virus HA protein by alternative approaches to DNA immunization

DNA immunization provides many advantages as an approach to prevent infectious diseases. However, although previous studies using this approach have demonstrated immune responses in serum, they were not successful in inducing significant levels of antibodies in secretions. In this study, plasmid DNAs expressing the influenza virus hemagglutinin glycoprotein have been evaluated for their ability to induce antibody responses in serum and saliva when used alone or along with either liposomes or bioadhesive polymers as mucosal delivery vehicles. Significant levels of virus-specific Ig in serum as well as secretory IgA in saliva were detected in mice following mucosal DNA immunization. These antibodies were found to block the infectivity of the virus using a plaque reduction assay. Our findings thus indicate that mucosal DNA immunization with specific delivery systems can elicit virus-specific antibody responses in serum as well as IgA responses at mucosal surfaces.