Proteolytic maturation of vaccinia virus core proteins: identification of a conserved motif at the N termini of the 4b and 25K virion proteins

The vaccinia virus F10 protein is one of two virally encoded protein kinases. A phenotypic analysis of infections involving a tetracycline-inducible recombinant (v⌬iF10) indicated that F10 is involved in the early stages of virion morphogenesis, as previously reported for the mutants ts28 and ts15. The proteins encoded by ts28 and ts15 have primary defects in enzymatic activity and thermostability, respectively. Using a transient complementation assay, we demonstrated that the enzymatic activity of F10 is essential for its biological function and that both its enzymatic and biological functions depend upon N-terminal sequences that precede the catalytic domain. An execution point analysis indicated that in addition to its role at the onset of morphogenesis, F10 is also required at later stages, when membrane crescents surround virosomal contents and develop into immature virions. The F10 protein is phosphorylated in vivo, appears to be tightly associated with intracellular membranes, and can bind to specific phosphoinositides in vitro. When F10 is repressed or impaired, the phosphorylation of several cellular and viral proteins appears to increase in intensity, suggesting that F10 may normally intersect with cellular signaling cascades via the activation of a phosphatase or the inhibition of another kinase. These cascades may drive the F10-induced remodeling of membranes that accompanies virion biogenesis. Upon the release of ts28-infected cultures from a 40°C-induced block, a synchronous resumption of morphogenesis that culminates in the production of infectious virus can be observed. The pharmacological agents H89 and cerulenin, which are inhibitors of endoplasmic reticulum exit site formation and de novo lipid synthesis, respectively, block this recovery.Protein phosphorylation plays a vital role in the regulation of key cellular processes. From signal transduction to cell cycle control, the phosphorylation state of a protein has been known to influence its intracellular localization, enzymatic activity, and interactions with proteins, nucleic acids, ligands, or downstream effectors. Protein kinases and phosphatases can act as molecular switches, regulating the activation or inhibition of cellular pathways in response to intrinsic or extrinsic stimuli.The analogous regulation of the vaccinia virus life cycle by reversible protein phosphorylation is illustrated by the fact that the viral genome encodes one protein phosphatase (H1) and two protein kinases (B1 and F10), all of which are essential for virus viability. The 20-kDa phosphatase encoded by the H1 gene was the first member of the dual-specificity class of phosphatases to be identified (19). The protein is expressed at late times during infection, and ϳ200 to 300 copies of H1 are encapsidated in wild-type (wt) virions. An analysis of a recombinant virus in which the expression of H1 is regulated by the lac operator-repressor system (vindH1) (33) revealed that the repression of H1 does not compromise the number of viral particles produced but instead aff...

Section: Modification Of F10 Proteinmentioning

confidence: 99%

Cell Biological and Functional Characterization of the Vaccinia Virus F10 Kinase: Implications for the Mechanism of Virion Morphogenesis

Punjabi

Traktman

2005

“…Three of the major structural proteins found within the mature VV virion core, 4a, 4b, and 25K, were known to be produced from highermolecular-weight precursors at late times during infection (12). VanSlyke et al (13,14) demonstrated that a large number of VV core proteins, including 4a, 4b, and 25K, appear to be processed via a common morphogenic cleavage pathway that is intimately linked with virion assembly and maturation. Cleavage of the precursors occurs only within the context of the maturing virion.…”

mentioning

confidence: 99%

“…Figure 2 is a map of the three major core protein precursors, P4a, P4b, and P25K, which are products of the A10L, A3L, and L4R ORFs, respectively, with the previously determined cleavage sites indicated on them. These cleavage sites have all been mapped to an Ala-Gly-Xaa motif (14,16). P4a is the largest precursor protein, with a molecular size of 98 kDa, and contains both an Ala-Gly-Ser and an Ala-Gly-Thr cleavage site in the C-terminal region of the protein.…”

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confidence: 99%

Molecular Dissection of the Vaccinia Virus I7L Core Protein Proteinase

Byrd

Bolken

Hruby

2003

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.Vaccinia virus (VV) is a large double-stranded-DNA virus with a cytoplasmic site of replication. It has over 200 open reading frames (ORFs) and has been extensively used as a eukaryotic cloning and expression vector and for vaccine research. VV is closely related to variola virus, the causative agent of smallpox, and therefore is of interest as a surrogate target in the development of antiviral drugs and vaccines. It is therefore of interest that the gene product of the I7L ORF of VV, which is predicted to be the core protein protease (2), shares 99% identity with the homologous K7L gene of variola major virus.Most viruses, including poliovirus, human immunodeficiency virus, and adenovirus, use posttranslational proteolytic processing as an essential step in their replication cycles (8). Therefore, it was not surprising to discover that proteolytic maturation of orthopoxvirus core proteins appears to be required for infectious progeny to be produced (6). Three of the major structural proteins found within the mature VV virion core, 4a, 4b, and 25K, were known to be produced from highermolecular-weight precursors at late times during infection (12). VanSlyke et al. (13,14) demonstrated that a large number of VV core proteins, including 4a, 4b, and 25K, appear to be processed via a common morphogenic cleavage pathway that is intimately linked with virion assembly and maturation. Cleavage of the precursors occurs only within the context of the maturing virion. All of the precursor proteins appear to be cleaved at a novel Ala-Gly-Xaa motif. This motif is distinct from that utilized in any other viral system, although some of the cysteine proteinases identified in other systems cleave polyproteins at Gly-Gly-Xaa sites, as demonstrated by the yeast cysteine protease (9, 10), the adenovirus protease (15,3,5), and the African swine fever virus (ASFV) protease (1).The gene product of the I7L ORF of VV was originally identified as a putative proteinase due to its homology to an ubiquitin-like proteinase in yeast (9) and was recently shown to be one of the proteinases responsible for cleavage of the VV core proteins (2). While there is a relatively detailed understanding of the cis-signals (sequences and protein structure characteristics) that direct the cleavage of the core protein precursors, relatively little is known about the enzyme that carries out these reactions. It is not known whether the entire I7L protein is required for recognition and cleavage of the core precursor proteins or if just the predicted catalytic domain is required. Is I7L capable of cleaving each of the core protein precursors, ...

“…Morphogenesis begins with the formation of crescentshaped membranes that engulf dense viroplasm to form spherical immature virions (IV) (6). Maturation of the IV into infectious intracellular mature virions (IMV) involves a poorly understood process in which at least three core proteins, namely P4a, P4b, and P25, encoded by the A10L, A3L, and L4R open reading frames (ORFs), respectively, undergo proteolytic processing (12,16,26) at a consensus AG/X motif (21)(22)(23)28). Initial evidence that processing is coupled to virus maturation was obtained by using the drug rifampin, which reversibly blocks an early step in virus assembly and the cleavage of core proteins (12,17).…”

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confidence: 99%

Role of the I7 Protein in Proteolytic Processing of Vaccinia Virus Membrane and Core Components

Ansarah-Sobrinho

Moss

2004

Certain core and membrane proteins of vaccinia virus undergo proteolytic cleavage at consensus AG/X sites. The processing of core proteins is coupled to morphogenesis and is inhibited by the drug rifampin, whereas processing of the A17 membrane protein occurs at an earlier stage of assembly and is unaffected by the drug. A temperature-sensitive mutant with a lesion in the I7L gene exhibits blocks in morphogenesis and in cleavage of core proteins. We found that the mutant also failed to cleave the A17 membrane protein. To further investigate the role of the putative I7 protease, we constructed a conditional lethal mutant in which the I7L gene was regulated by the Escherichia coli lac repressor. In the absence of an inducer, the synthesis of I7 was repressed, proteolytic processing of the A17 membrane protein and the L4 core protein was inhibited, and virus morphogenesis was blocked. Under these conditions, expression of the wild-type I7 protein in trans restored protein processing. In contrast, rescue did not occur when the putative protease active site residue histidine 241 or cysteine 328 of I7 was converted to alanine. The mutation of an authentic AG/A and an alternative AG/S motif of L4 prevented substrate cleavage. Similarly, when AG/X sites of A17 were mutated, I7-induced cleavages at the N and C termini failed to occur. In conclusion, we provide evidence that I7 is a viral protease that is required for AG/X-specific cleavages of viral membrane and core proteins, which occur at early and late stages of virus assembly, respectively.Vaccinia virus (VV), the best-studied member of the Poxviridae, is a complex enveloped DNA virus that replicates in the cytoplasm of infected cells (15). Viral DNA replication, transcription, the accumulation of viral structural proteins, and the assembly of virions occur in discrete juxtanuclear viral factories. Morphogenesis begins with the formation of crescentshaped membranes that engulf dense viroplasm to form spherical immature virions (IV) (6). Maturation of the IV into infectious intracellular mature virions (IMV) involves a poorly understood process in which at least three core proteins, namely P4a, P4b, and P25, encoded by the A10L, A3L, and L4R open reading frames (ORFs), respectively, undergo proteolytic processing (12,16,26) at a consensus AG/X motif (21-23, 28). Initial evidence that processing is coupled to virus maturation was obtained by using the drug rifampin, which reversibly blocks an early step in virus assembly and the cleavage of core proteins (12, 17). Subsequently, numerous temperature-sensitive (ts) mutants were shown to have defects in virus assembly and the processing of core proteins under nonpermissive conditions. One IMV membrane protein, encoded by the A17L ORF, also undergoes proteolytic cleavages at AG/X sites (2,18,21,28). The processing of membrane and core proteins appears to be separable, as two studies described no effect of rifampin on the cleavage of A17 (2, 18) but a third study concluded that rifampin blocked cleavage of this protein (28...