Characterization of a Newly Identified 35-Amino-Acid Component of the Vaccinia Virus Entry/Fusion Complex Conserved in All Chordopoxviruses

Here we examine the protein covalent structure of the vaccinia virus virion. Within two virion preparations, >88% of the theoretical vaccinia virus-encoded proteome was detected with high confidence, including the first detection of products from 27 open reading frames (ORFs) previously designated "predicted," "uncharacterized," "inferred," or "hypothetical" polypeptides containing as few as 39 amino acids (aa) and six proteins whose detection required nontryptic proteolysis. We also detected the expression of four short ORFs, each of which was located within an ORF ("ORF-within-ORF"), including one not previously recognized or known to be expressed. Using quantitative mass spectrometry (MS), between 58 and 74 proteins were determined to be packaged. A total of 63 host proteins were also identified as candidates for packaging. Evidence is provided that some portion of virion proteins are "nicked" via a combination of endoproteolysis and concerted exoproteolysis in a manner, and at sites, IMPORTANCEPoxviruses are among the most complex and irregular virions, about whose internal structure little is known. To better understand poxvirus virion structure, imaging should be supplemented with other tools. Here, we provide a deep study of the covalent structure of the vaccinia virus virion using the various tools of contemporary mass spectrometry. C ontemporary mass spectrometry (MS) instrumentation provides an avenue for proteome coverage with enormous breadth or depth, depending upon the complexity of the proteome. Typically, the power of the approach is exploited for breadth in the analysis of increasingly complex proteomes. Here, we have taken a relatively narrow proteome, that of a virus particle, and explored proteome depth, developing analytical approaches and tools as required.Sporadic information is available on the covalent (primary) molecular structure of the vaccinia virus virion. Around 75 proteins can be detected in two-dimensional (2D) gels of purified vaccinia virus virions (1). Three published MS studies provided early compendia of viral and host gene products present within vaccinia virus and human monkeypox virion preparations via MS (2-5). These studies built upon earlier, less comprehensive studies that employed a combination of MS and N-terminal sequencing (1, 6). Overlapping approaches in terms of instrumentation, preparation, and data analysis led to an overlapping set of detected gene products.In terms of covalent protein modifications, six vaccinia virus proteins can be labeled in vivo with myristate, five of which have been identified as L1R (7,8), G9R, A16L, and E7R (9), and A14L (10). All except A14L possess a glycine at position 2 in the protein.At least eight vaccinia virus-encoded proteins may be palmitoylated, as evidenced by the incorporation of 3 H-labeled palmitate in culture (11). These proteins include wrapped virus (WV)-specific protein p37 (F13L) (7,(12)(13)(14) and products of open reading frames (ORFs) A33R (15), B5R (16), F13L (17), the A22R Holliday resolvase (11), A...

Section: Resultsmentioning

confidence: 99%

Protein Primary Structure of the Vaccinia Virion at Increased Resolution

Ngo

Mirzakhanyan

Moussatché

et al. 2016

“…The small I2 protein is a good candidate, since the phenotype of a conditional lethal mutant is similar to that of EFC proteins with the exception that repression decreases the amounts of EFC components in MVs (26). Because of its small size and hydrophobicity, the I2 protein, like the O3 EFC protein (30), may have been missed by mass spectroscopy of the purified complex (33). The overall architecture of the EFC has not been determined, although a few protein-protein interactions that resist destabilization of the complex have been identified (25,42,43).…”

Section: Discussionmentioning

confidence: 99%

“…Four proteins have been reported to participate in attachment by binding to glycosaminoglycans and laminin (6,7,15,20). Analysis of conditional lethal mutants established roles in entry and membrane fusion for 11 viral proteins with homologs in all poxviruses: A16 (28), A21 (37), A28 (34), F9 (5), G3 (17), G9 (27), H2 (32), I2 (26), L1 (4), L5 (36), and O3 (30). Each of these proteins, except I2, has been shown to be associated in a stable complex that can be extracted from membranes and purified.…”

mentioning

confidence: 99%

Transcriptional Repression and RNA Silencing Act Synergistically To Demonstrate the Function of the Eleventh Component of the Vaccinia Virus Entry-Fusion Complex

Wolfe

Ojeda

Moss

2012

Self Cite

Poxviruses have an elaborate system for infecting cells comprising several proteins for attachment and a larger number dedicated to membrane fusion and entry. Thus far, 11 proteins have been identified as components of the vaccinia virus (VACV) entry-fusion complex (EFC), and 10 of these proteins have been shown to be required for entry. J5, the remaining functionally uncharacterized component of the complex, is conserved in all poxviruses, has a predicted C-terminal transmembrane domain, and is an N-terminally truncated paralog of two other EFC proteins. To determine the role of J5, we constructed a mutant that inducibly regulates J5 transcription. Although the virus yield was reduced only about 80% without inducer, the inability to isolate a J5 deletion mutant suggested an essential function. To enhance stringency, we employed RNA silencing alone and together with transcriptional repression of the inducible mutant. The yield of infectious virus was reduced 4-to 5-fold by repression, 2-fold by silencing, and 60-fold by the combination of the two. Virus particles made under the latter conditions appeared to contain a full complement of proteins excluding J5 but had very low infectivity. Further studies indicated that after binding to cells, J5-deficient virions had a defect in core entry and an inability to induce syncytium formation. In addition, we confirmed that J5 is associated with the EFC by affinity purification. These data indicate that J5 is a functional component of the EFC and highlights the advantage of combining transcriptional repression and RNA silencing for stringent reduction of gene expression. The members of the virus family Poxviridae are linear doublestranded DNA viruses that replicate exclusively in the cytoplasm of the host cell (23). Vaccinia virus (VACV), the prototype poxvirus, has a 190-kbp genome predicted to encode nearly 200 proteins of which approximately 80 have been identified as components of the mature virion (MV) (8,29,48). MVs enter cells by neutral-and low-pH fusion with plasma and endosomal membranes, respectively (3, 38) utilizing macropinocytosis or fluid phase uptake (16,22). Four proteins have been reported to participate in attachment by binding to glycosaminoglycans and laminin (6,7,15,20). Analysis of conditional lethal mutants established roles in entry and membrane fusion for 11 viral proteins with homologs in all poxviruses: A16 (28), A21 (37), A28 (34), F9 (5), G3 (17), G9 (27), H2 (32), I2 (26), L1 (4), L5 (36), and O3 (30). Each of these proteins, except I2, has been shown to be associated in a stable complex that can be extracted from membranes and purified. However, the complex could not be isolated if either A16, A21, A28, G3, G9, H2, L5, or O3 was repressed even though the remaining entry proteins were still incorporated into virions, suggesting that the complex is held together by multiple protein interactions. L1 and F9 are not required for isolation of the complex, which suggests that they are peripherally associated with the EFC. On this basis, A16, A21...

“…First, vaccinia MV binds to multiple cell surface components, such as glycosaminoglycans (11,29,42), extracellular matrix laminin (9), and sulfatides (49). Furthermore, vaccinia MV contains an entry fusion complex (EFC) of 12 viral envelope proteins that are evolutionarily conserved and essential for membrane fusion (6,7,33,47,52,56,68). Additionally, different strains of vaccinia MV harboring mutations in the viral envelope proteins A25 and A26 enter cells differently (8).…”

mentioning

confidence: 99%

Integrin β1 Mediates Vaccinia Virus Entry through Activation of PI3K/Akt Signaling

Izmailyan

Hsao

Chung

et al. 2012

107

Vaccinia virus has a broad range of infectivity in many cell lines and animals. Although it is known that the vaccinia mature virus binds to cell surface glycosaminoglycans and extracellular matrix proteins, whether additional cellular receptors are required for virus entry remains unclear. Our previous studies showed that the vaccinia mature virus enters through lipid rafts, suggesting the involvement of raft-associated cellular proteins. Here we demonstrate that one lipid raft-associated protein, integrin β1, is important for vaccinia mature virus entry into HeLa cells. Vaccinia virus associates with integrin β1 in lipid rafts on the cell surface, and the knockdown of integrin β1 in HeLa cells reduces vaccinia mature virus entry. Additionally, vaccinia mature virus infection is reduced in a mouse cell line, GD25, that is deficient in integrin β1 expression. Vaccinia mature virus infection triggers the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, and the treatment of cells with inhibitors to block P13K activation reduces virus entry in an integrin β1-dependent manner, suggesting that integrin β1-mediates PI3K/Akt activation induced by vaccinia virus and that this signaling pathway is essential for virus endocytosis. The inhibition of integrin β1-mediated cell adhesion results in a reduction of vaccinia virus entry and the disruption of focal adhesion and PI3K/Akt activation. In summary, our results show that the binding of vaccinia mature virus to cells mimics the outside-in activation process of integrin functions to facilitate vaccinia virus entry into HeLa cells.