Vaccinia virus G1 protein, a predicted metalloprotease, is essential for morphogenesis of infectious virions but not for cleavage of major core proteins

Genes encoding orthologs of the vaccinia virus G1 protein are present in all poxviruses for which sequence information is available, yet neither the role of the protein nor its requirement for virus replication is known. G1 was predicted to be involved in the cleavage of core proteins, based on a transfection study and the presence of an HXXEH motif found in a subset of metallopeptidases. In the present study, we engineered a recombinant vaccinia virus containing a single copy of the G1L gene with a C-terminal epitope tag that is stringently regulated by the Escherichia coli lac repressor. In the absence of inducer, expression of G1 was repressed and virus replication was inhibited. Rescue of infectious virus was achieved by expression of wild-type G1 in trans, but not when the putative protease active site residues histidine-41, glutamate-44, or histidine-45 were mutated. Nevertheless, the synthesis and proteolytic processing of major core and membrane proteins appeared unaffected under nonpermissive conditions, distinguishing the phenotype of the G1L mutant from one in which the gene encoding the I7 protease was repressed. Noninfectious virus particles, assembled in the absence of inducer, did not attain the oval shape or characteristic core structure of mature virions. The polypeptide composition of these particles, however, closely resembled that of wild-type virus. Full-length and shorter forms of the G1 protein were found in the core fraction of virus particles assembled in the presence of inducer, suggesting that G1 is processed by self-cleavage or by another protease.     

The NS2 protein of hepatitis C virus is a transmembrane polypeptide.

The NS2 protein of hepatitis C virus (HCV) is released from its polyprotein precursor by two proteolytic cleavages. The N terminus of this protein is separated from the E2/p7 polypeptide by a cleavage thought to be mediated by signal peptidase, whereas the NS2-3 junction located at the C terminus is processed by a viral protease. To characterize the biogenesis of NS2 encoded by the BK strain of HCV, we have defined the minimal region of the polyprotein required for efficient cleavage at the NS2-3 site and analyzed the interaction of the mature polypeptide with the membrane of the endoplasmic reticulum (ER). We have observed that although cleavage can occur in vitro in the absence of microsomal membranes, synthesis of the polyprotein precursor in the presence of membranes greatly increases processing at this site. Furthermore, we show that the membrane dependency for efficient in vitro processing varies among different HCV strains and that host proteins located on the ER membrane, and in particular the signal recognition particle receptor, are required to sustain efficient proteolysis. By means of sedimentation analysis, protease protection assay, and site-directed mutagenesis, we also demonstrate that the NS2 protein derived from processing at the NS2-3 site is a transmembrane polypeptide, with the C terminus translocated in the lumen of the ER and the N terminus located in the cytosol.     

Regulation of vaccinia virus morphogenesis: phosphorylation of the A14L and A17L membrane proteins and C-terminal truncation of the A17L protein are dependent on the F10L kinase

This study focused on three vaccinia virus-encoded proteins that participate in early steps of virion morphogenesis: the A17L and A14L membrane proteins and the F10L protein kinase. We found that (i) the A17L protein was cleaved at or near an AGX consensus motif at amino acid 185, thereby removing its acidic C terminus; (ii) the nontruncated form was associated with immature virions, but only the C-terminal truncated protein was present in mature virions; (iii) the nontruncated form of the A17L protein was phosphorylated on serine, threonine, and tyrosine residues, whereas the truncated form was unphosphorylated; (iv) nontruncated and truncated forms of the A17L protein existed in a complex with the A14L membrane protein; (v) C-terminal cleavage of the A17L protein and phosphorylation of the A17L and A14L proteins failed to occur in cells infected with a F10L kinase mutant at the nonpermissive temperature; and (vi) the F10L kinase was the only viral late protein that was necessary for phosphorylation of the A17L protein, whereas additional proteins were needed for C-terminal cleavage. We suggest that phosphorylation of the A17L and A14L proteins is mediated by the F10L kinase and is required to form the membranes associated with immature virions. Removal of phosphates and the A17L acidic C-terminal peptide occur during the transition to mature virions.     

Proteolytic processing of the replicase ORF1a protein of equine arteritis virus.

To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire ORF1a protein, results in cleavage products of approximately 29, 61, 22, 31, 41, and 3 kDa, which were named nonstructural proteins (nsps) 1 through 6, respectively. Pulse-chase experiments revealed that the cleavages at the nsp1/2 and nsp2/3 junctions are the most rapid processing steps. The remaining nsp3456 precursor is first cleaved at the nsp4/5 site. Final processing of the nsp34 and nsp56 intermediates is extremely slow. As predicted from previous in vitro translation experiments (E. J. Snijder, A. L. M. Wassenaar, and W. J. M. Spaan, J. Virol. 66:7040-7048, 1992), a cysteine protease domain in nsp1 was shown to be responsible for the nsp1/2 cleavage. The other processing steps are carried out by the putative EAV serine protease in nsp4 and by a third protease, which remains to be identified.     

The PBN1 gene of Saccharomyces cerevisiae: an essential gene that is required for the post-translational processing of the protease B precursor.

The vacuolar hydrolase protease B in Saccharomyces cerevisiae is synthesized as an inactive precursor (Prb1p). The precursor undergoes post-translational modifications while transiting the secretory pathway. In addition to N- and O-linked glycosylations, four proteolytic cleavages occur during the maturation of Prb1p. Removal of the signal peptide by signal peptidase and the autocatalytic cleavage of the large amino-terminal propeptide occur in the endoplasmic reticulum (ER). Two carboxy-terminal cleavages of the post regions occur in the vacuole: the first cleavage is catalyzed by protease A and the second results from autocatalysis. We have isolated a mutant, pbn1-1, that exhibits a defect in the ER processing of Prb1p. The autocatalytic cleavage of the propeptide from Prb1p does not occur and Prb1p is rapidly degraded in the cytosol. PBN1 was cloned and is identical to YCL052c on chromosome III. PBN1 is an essential gene that encodes a novel protein. Pbn1p is predicted to contain a sub-C-terminal transmembrane domain but no signal sequence. A functional HA epitope-tagged Pbn1p fusion localizes to the ER. Pbn1p is N-glycosylated in its amino-terminal domain, indicating a lumenal orientation despite the lack of a signal sequence. Based on these results, we propose that one of the functions of Pbn1p is to aid in the autocatalytic processing of Prb1p.     

Participation of vaccinia virus l2 protein in the formation of crescent membranes and immature virions

Morphogenesis of vaccinia virus begins with the appearance of crescent-shaped membrane precursors of immature virions in cytoplasmic factories. During the initial characterization of the product of the L2R reading frame, we discovered that it plays an important role in crescent formation. The L2 protein was expressed early in infection and was associated with the detergent-soluble membrane fraction of mature virions, consistent with two potential membrane-spanning domains. All chordopoxviruses have L2 homologs, suggesting an important function. Indeed, we were unable to isolate an infectious L2R deletion mutant. Consequently, we constructed an inducible mutant with a conditional lethal phenotype. When L2 expression was repressed, proteolytic processing of the major core proteins and the A17 protein, which is an essential component of the immature virion membrane, failed to occur, suggesting an early block in viral morphogenesis. At 8 h after infection in the presence of inducer, immature and mature virions were abundantly seen by electron microscopy. In contrast, those structures were rare in the absence of inducer and were replaced by large, dense aggregates of viroplasm. A minority of these aggregates had short spicule-coated membranes, which resembled the beginnings of crescent formation, at their periphery. These short membrane segments at the edge of the dense viroplasm increased in number at later times, and some immature virions were seen. Although the L2 protein was not detected under nonpermissive conditions, minute amounts could account for stunted and delayed viral membrane formation. These findings suggested that L2 is required for the formation or elongation of crescent membranes.     

Differential requirements of NS4A for internal NS3 cleavage and polyprotein processing of hepatitis C virus

The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT(402)|S being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.     

A Bipartite Membrane-Binding Signal in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Required for the Proteolytic Processing of Gag Precursors in a Cell Type-Dependent Manner

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4⁺ T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4⁺ T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.     

Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis.

We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin. To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome. Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis.     

Sequential cleavage by metallopeptidases and proteasomes is involved in processing HIV-1 ENV epitope for endogenous MHC class I antigen presentation

Antigenic peptides derived from viral proteins by multiple proteolytic cleavages are bound by MHC class I molecules and recognized by CTL. Processing predominantly takes place in the cytosol of infected cells by the action of proteasomes. To identify other proteases involved in the endogenous generation of viral epitopes, specifically those derived from proteins routed to the secretory pathway, we investigated presentation of the HIV-1 ENV 10-mer epitope 318RGPGRAFVTI327 (p18) to specific CTL in the presence of diverse protease inhibitors. Both metalloproteinase and proteasome inhibitors decreased CTL recognition of the p18 epitope expressed from either native gp160 or from a chimera based on the hepatitis B virus secretory core protein as carrier protein. Processing of this epitope from both native ENV and the hepatitis B virus secretory core chimeric protein appeared to proceed by a TAP-dependent pathway that involved sequential cleavage by proteasomes and metallo-endopeptidases; however, other protease activities could replace the function of the lactacystin-sensitive proteasomes. By contrast, in a second TAP-independent pathway we detected no contribution of metallopeptidases for processing the ENV epitope from the chimeric protein. These results show that, in the classical TAP-dependent MHC class I pathway, endogenous Ag processing of viral proteins to yield the p18 10-mer epitope requires metallo-endopeptidases in addition to proteasomes.     

Molecular dissection of the vaccinia virus I7L core protein proteinase

The vaccinia virus I7L gene product is predicted to be a cysteine proteinase and is demonstrated in this study to be responsible for cleavage of each of the three major core protein precursors (P4a, P4b, and P25K) in vivo. Mutagenesis of the putative catalytic triad of I7L or of the cleavage sites in the core protein precursors inhibits processing. A truncated protein lost the ability to cleave the core protein precursors.     

Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes

The mechanism for efficient nucleocapsid (NC) uptake into flavivirus particles which form by budding through the membranes of the endoplasmic reticulum (ER) was investigated by using Murray Valley encephalitis virus as a model. Budding of flavivirus membranes is driven by the viral transmembrane proteins prM and E independently of NC interaction. We show that control of signalase cleavage of the multimembrane-spanning flavivirus polyprotein by the catalytic function of the viral protease is critical for efficient virus morphogenesis. In wild-type virus, signalase cleavage of prM remains inefficient until cleavage of capsid at the cytosolic side of the signal sequence separating the two proteins has occurred. This obligatory sequence of cleavages was uncoupled in a mutant virus with the consequence of greatly reduced incorporation of NC into budding membranes and augmented release of NC-free virus-like particles. Efficient signalase cleavage of prM in the mutant virus resulted in partial inhibition of cleavage of capsid by the viral NS2B-3 protease. Our results support a model for flavivirus morphogenesis involving temporal and spatial coordination of NC assembly and envelopment by regulated cleavages of an ER membrane-spanning capsid-prM intermediate.     

Importation of nuclease colicins into E coli cells: endoproteolytic cleavage and its prevention by the immunity protein

A major group of colicins comprises molecules that possess nuclease activity and kill sensitive cells by cleaving RNA or DNA. Recent data open the possibility that the tRNase colicin D, the rRNase colicin E3 and the DNase colicin E7 undergo proteolytic processing, such that only the C-terminal domain of the molecule, carrying the nuclease activity, enters the cytoplasm. The proteases responsible for the proteolytic processing remain unidentified. In the case of colicin D, the characterization of a colicin D-resistant mutant shows that the inner membrane protease LepB is involved in colicin D toxicity, but is not solely responsible for the cleavage of colicin D. The lepB mutant resistant to colicin D remains sensitive to other colicins tested (B, E1, E3 and E2), and the mutant protease retains activity towards its normal substrates. The cleavage of colicin D observed in vitro releases a C-terminal fragment retaining tRNase activity, and occurs in a region of the amino acid sequence that is conserved in other nuclease colicins, suggesting that they may also require a processing step for their cytotoxicity. The immunity proteins of both colicins D and E3 appear to have a dual role, protecting the colicin molecule against proteolytic cleavage and inhibiting the nuclease activity of the colicin. The possibility that processing is an essential step common to cell killing by all nuclease colicins, and that the immunity protein must be removed from the colicin prior to processing, is discussed.     

Cathepsin L is involved in proteolytic processing of the Hendra virus fusion protein

Proteolytic processing of paramyxovirus fusion (F) proteins is essential for the generation of a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of the newly emerged Hendra virus F protein occurs by a previously unidentified cellular protease following a single lysine at residue 109. We demonstrate here that the cellular protease cathepsin L is involved in converting the Hendra virus precursor F protein (F(0)) to the active F(1) + F(2) disulfide-linked heterodimer. To initially identify the class of protease involved in Hendra virus F protein cleavage, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be proteolytically processed by furin) were metabolically labeled and chased in the absence or presence of serine, cysteine, aspartyl, and metalloprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease cathepsin activity inhibited proteolytic processing of Hendra virus F but had no effect on simian virus 5 F processing. We next designed shRNA oligonucleotides to cathepsin L which dramatically reduced cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero cells transfected with pCAGGS-Hendra F demonstrated a nondetectable amount of cleavage of the Hendra virus F protein and significantly decreased membrane fusion activity. Additionally, we found that purified human cathepsin L processed immunopurified Hendra virus F(0) into F(1) and F(2) fragments. These studies introduce a novel mechanism for primary proteolytic processing of viral glycoproteins and also suggest a previously unreported biological role for cathepsin L.     

Site-specific mutations at a picornavirus VP3/VP1 cleavage site disrupt in vitro processing and assembly of capsid precursors.

Most proteolytic cleavages within the picornavirus polyproteins are carried out by viral protease 3C. For encephalomyocarditis virus, the protease 3C-catalyzed processing occurs between Gln-Gly or Gln-Ser amino acid pairs which are flanked by proline residues, but the sequence-specific constraints on recognition and cleavage by the enzyme are not completely understood. To examine alternative cleavage site sequences, we constructed a cDNA plasmid which expresses the viral L-P1-2A capsid precursor in vitro and introduced site-specific mutations into the Gln-Gly pair at the VP3/VP1 junction. The altered protein substrates were tested for cleavage activity in assays with protease 3C. The encephalomyocarditis virus 3C processed Gln-Ala as efficiently as its natural sites but did not cleave Gln-Val, Gln-Glu, Lys-Gly, Lys-Ala, Lys-Val, Lys-Glu, or Pro-Gly combinations. Displacement of the flanking proline residue by an engineered insertion slowed but did not prevent cleavage at this site. Also, a mutant defective in processing at the VP3/VP1 junction was unable to form 14S pentameric assembly intermediates in vitro.     

A trans-envelope protein complex needed for filamentous phage assembly and export

Assembly and export of filamentous phage requires four non-capsid proteins: the outer membrane protein, pIV; the inner membrane proteins, pI and pXI; and a cytoplasmic host factor, thioredoxin. Chemical cross-linking of intact cells demonstrates a trans-membrane complex containing pI and pIV. Formation of the complex protects pI from proteolytic cleavage by an endogenous protease. This protection also requires pXI, which is identical to the C-terminal portion of pI. This indicates that pXI, which is required for phage assembly in its own right, is also part of the complex. This complex forms in the absence of any other phage proteins or the DNA substrate; hence, it represents the first preinitiation step of phage morphogenesis. On the basis of protease protection data, we propose that the preinitiation complex is converted to an initiation complex by binding phage DNA, thioredoxin and the initiating minor coat protein(s).     

Foot-and-mouth disease virus 3C protease induces cleavage of translation initiation factors eIF4A and eIF4G within infected cells

Infection of cells by foot-and-mouth disease virus (FMDV) results in the rapid inhibition of host cell protein synthesis. This process is accompanied by the early cleavage of the translation initiation factor eIF4G, a component of the cap-binding complex eIF4F. This cleavage is mediated by the leader (L) protease. Subsequently, as the virus proteins accumulate, secondary cleavages of eIF4G occur. Furthermore, eIF4A (46 kDa), a second component of eIF4F, is also cleaved in these later stages of the infection cycle. The 33-kDa cleavage product of eIF4A has lost a fragment from its N terminus. Transient-expression assays demonstrated that eIF4A was not cleaved in the presence of FMDV L or with the poliovirus 2A protease (which also mediates eIF4G cleavage) but was cleaved when the FMDV 3C protease was expressed. The FMDV 3C protease was also shown in such assays to induce cleavage of eIF4G, resulting in the production of cleavage products different from those generated by the L protease. Consistent with these results, within cells infected with a mutant FMDV lacking the L protease or within cells containing an FMDV replicon lacking L-P1 coding sequences it was again shown that eIF4A and eIF4G were cleaved.     

Assembly of outer membrane lipoprotein in an Escherichia coli mutant with a single amino acid replacement within the signal sequence of prolipoprotein.

We have compared the rate of assembly of outer membrane proteins including the lipoprotein in a pair of isogenic mlpA+ (lpp+) and mlpA (lpp) strains by pulse-chase experiments. The rate of assembly of the mutant prolipoprotein into the outer membrane was slightly slower than that of the wild-type lipoprotein. The rate of assembly of protein I and protein H-2 was similar in the wild type and the mutant, whereas the rate of assembly of protein II into the outer membrane was slightly reduced in the mutant strain. The organization of outer membrane was slightly reduced in the mutant strain. The organization of outer membrane proteins in the mutant cells appeared not to be grossly altered, based on the apparent resistance (or susceptibility) of these proteins toward trypsin treatment and their resistance to solubilization by Sarkosyl. Like the wild-type lipoprotein, the mutant prolipoprotein in the outer membrane was resistant to trypsin. On the other hand, the prolipoprotein in the cytoplasmic membrane fraction of the mutant cell envelope was susceptible to trypsin digestion. We conclude from these data that proteolytic cleavage of prolipoprotein is not essential for the translocation and proper assembly of lipoprotein into outer membrane.     

Deletion of the carboxyl-terminal portion of the transit peptide affects processing but not import or assembly of the small subunit of ribulose-1,5-bisphosphate carboxylase

Import of the small subunit of ribulose-1,5-biphosphate carboxylase/oxygenase into the chloroplast has been proposed to involve two proteolytic cleavages which convert the 20-kDa precursor (pSSU) into the mature 14-kDa subunit (SSU) via an 18-kDa intermediate. A deletion mutant (PSd48/57) of pSSU which lacks 10 amino acids in a conserved region in the carboxyl-terminal portion of the transit peptide is converted into a series of 16-18-kDa polypeptides in addition to the mature 14-kDa SSU when imported into isolated pea chloroplasts. We examined import and processing of this mutant pSSU to determine whether the 16-18-kDa SSUs undergo further maturation in the chloroplast stroma to yield 14-kDa SSU. The ratio of incorrectly processed to 14-kDa SSU is stable up to 60 min following import. This indicates that processing of PSd48/57 involves a single proteolytic cleavage which occurs during or immediately following transit across the chloroplast envelope. The carboxyl-terminal portion of the transit peptide confers either sequence specificity for the processing protease or provides a three-dimensional structure necessary for consistent cleavage at the mature amino terminus of SSU. Incorrectly processed SSUs were incorporated into the holoenzyme demonstrating that removal of the entire transit sequence is not necessary for assembly of the holoenzyme.