Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus

Towner, Jonathan S.; Brown, David; Nguyen, Joseph H. C.; Semler, Bert L.

doi:10.1016/s0042-6822(03)00448-3

Cited by 13 publications

(22 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Similar results were obtained by Towner et al, who introduced mutations within the hydrophobic region of 3A and found a compensatory mutation within the first hydrophobic domain of the 2B protein (48).…”

Section: Construction Of 3a/3ab Mutants Suitable For Membrane-associasupporting

confidence: 88%

Membrane Topography of the Hydrophobic Anchor Sequence of Poliovirus 3A and 3AB Proteins and the Functional Effect of 3A/3AB Membrane Association upon RNA Replication

et al. 2007

View full text Add to dashboard Cite

Replication of poliovirus RNA takes place on the cytoplasmic surface of membranous vesicles that form after infection of the host cell. It is generally accepted that RNA polymerase 3D pol interacts with membranes in a complex with viral protein 3AB, which binds to membranes by means of a hydrophobic anchor sequence that is located near the C-terminus of the 3A domain. In this study, we used fluorescence and fluorescence quenching methods to define the topography of the anchor sequence in context of 3A and 3AB proteins inserted in model membranes. Mutants with a single tryptophan near the center of the anchor sequence but lacking Trp elsewhere in 3A/3AB were constructed which, after the emergence of suppressor mutations, replicated well in HeLa cells. When a peptide containing the mutant anchor sequence was incorporated in model membrane vesicles, measurements of Trp depth within the lipid bilayer indicated formation of a transmembrane topography. However, rather than the 22 residue length predicted from hydrophobicity considerations, the transmembrane segment had an effective length of 16 residues, such that Gln64 likely formed the N-terminal boundary. Analogous experiments using full length proteins bound to pre-formed model membrane vesicles showed that the anchor sequence formed a mixture of transmembrane and non-transmembrane topographies in the 3A protein but adopted only the nontransmembrane configuration in the context of 3AB protein. Studies of the function of 3A/3AB inserted into model membrane vesicles showed that membrane-bound 3AB is highly efficient in stimulating the activity of 3D pol in vitro while membrane-bound 3A totally lacks this activity. Moreover, in vitro uridylylation reactions showed that membrane-bound 3AB is not a substrate for 3D pol but free VPg released by cleavage of 3AB with proteinase 3CD pro could be uridylylated.After poliovirus (PV) enters the host cell its plus strand RNA genome is translated into a polyprotein that contains one structural (P1) and two nonstructural domains (P2, P3; Fig. 1A). There is an efficient and regulated cascade of protein processing that produces cleavage products with function distinct from those of the precursor proteins (Fig. 1A). A variety of precursor and mature proteins are released from the PV polyprotein by cleavage with *Corresponding author: Erwin London, Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215, Tel: (631) FAX: (631) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript proteinases 2A pro , 3C pro /3CD pro . The proteins of the P1 domain (VP1-VP4) assemble to form the capsid while those derived from P2 (2A pro , 2B, 2BC, 2C ATPase ) are primarily responsible for the biochemical and structural changes that occur in the infected cell. The proteins of the P3 domain are those most directly involved in RNA synthesis. These include two important and relatively stable precursors, proteinase 3CD pro and 3AB, which were shown to specifically interact with a cl...

show abstract

Section: Construction Of 3a/3ab Mutants Suitable For Membrane-associasupporting

confidence: 88%

Membrane Topography of the Hydrophobic Anchor Sequence of Poliovirus 3A and 3AB Proteins and the Functional Effect of 3A/3AB Membrane Association upon RNA Replication

et al. 2007

View full text Add to dashboard Cite

show abstract

“…Interestingly, a similar conclusion was reached based on the analysis of chimeric PV genomes in which the hydrophobic region in protein 3A was exchanged for the orthologous element from HRV14. Those chimeric viruses also were viable but demonstrated defects in replication that were improved by secondary mutations in 2B-encoding sequences (34). The postulated interaction between 2C and/or 2BC protein and 3A protein seems quite plausible, since all these proteins are localized together in membranous replication complexes.…”

Section: Discussionmentioning

confidence: 97%

Evidence for Functional Protein Interactions Required for Poliovirus RNA Replication

Teterina

Levenson

Rinaudo

et al. 2006

J Virol

View full text Add to dashboard Cite

Poliovirus protein 2C contains a predicted N-terminal amphipathic helix that mediates association of the protein with the membranes of the viral RNA replication complex. A chimeric virus that contains sequences encoding the 18-residue core from the orthologous amphipathic helix from human rhinovirus type 14 (HRV14) was constructed. The chimeric virus exhibited defects in viral RNA replication and produced minute plaques on HeLa cell monolayers. Large plaque variants that contained mutations within the 2C-encoding region were generated upon subsequent passage. However, the majority of viruses that emerged with improved growth properties contained no changes in the region encoding 2C. Sequence analysis and reconstruction of genomes with individual mutations revealed changes in 3A or 2B sequences that compensated for the HRV14 amphipathic helix in the polio 2C-containing proteins, implying functional interactions among these proteins during the replication process. Direct binding between these viral proteins was confirmed by mammalian cell twohybrid analysis.Poliovirus (PV) RNA replication takes place in replication complexes that form de novo in cultured cells after virus infection. Synthesis of viral proteins induces extensive rearrangement of intracellular membrane structures that produce perinuclear foci of vesicle-associated viral proteins and RNA (reference 11 and references therein). These coalesce into large clusters of vesicles engaged in viral RNA synthesis, accompanied by the loss of preexisting Golgi stacks and endoplasmic reticulum (ER). All viral nonstructural proteins, derived from the P2 and P3 polyprotein regions of the single open reading frame in the viral genome, are found associated with the membranous replication complexes and have been implicated by genetic analysis as playing essential roles in the process of viral RNA replication. Proteins containing 2B, 2C, or 3A sequences manifest inherent membrane-binding properties. It is not known how the other viral proteins are recruited to and/or retained in the replication complexes. They may enter or induce formation of the complexes as larger precursor proteins prior to cleavage and maintain their associations via protein-protein or protein-RNA interactions, a hypothesis supported by the observation that complementation of defective proteins by expression of individual functional gene products does not occur readily in infected cells (32) and requires expression of whole P2 or P3 precursor proteins in vitro (16,35).The precise biochemical roles in viral RNA synthesis played by each of the nonstructural proteins are poorly defined (summarized in reference 21). From the P3 region, protein 3D catalyzes polynucleotide chain elongation as well as uridylylation of VPg (protein 3B) to form a primer for RNA chain initiation. Protein 3C is the protease responsible for the majority of polyprotein cleavages, both in cis and in trans. Protein 3CD, in addition to serving a proteinase function for generation of capsid proteins from P1 precursors, binds and sti...

show abstract

“…These chimeric viruses are viable but do not replicate as well as wild-type virus. Viruses with improved replication were isolated that contain compensatory changes in the 2B protein, suggesting that 2B and 3A may interact during infection (71). Recent data also suggest interactions between 2B, 2C, and 3A (65).…”

Section: Discussionmentioning

confidence: 99%

“…The arrangement of viral proteins in the replication complex is unknown, but it is suspected that many of the nonstructural proteins interact with each other. For example, it has been suggested that proteins 2B and 3A interact (71) and that 2C binds 2B and 3A in the replication complex (65).…”

mentioning

confidence: 99%

Amino Acid Changes in Proteins 2B and 3A Mediate Rhinovirus Type 39 Growth in Mouse Cells

Harris

Racaniello

2005

J Virol

View full text Add to dashboard Cite

Many steps of viral replication are dependent on the interaction of viral proteins with host cell components. To identify rhinovirus proteins involved in such interactions, human rhinovirus 39 (HRV39), a virus unable to replicate in mouse cells, was adapted to efficient growth in mouse cells producing the viral receptor ICAM-1 (ICAM-L cells). Amino acid changes were identified in the 2B and 3A proteins of the adapted virus, RV39/L. Changes in 2B were sufficient to permit viral growth in mouse cells; however, changes in both 2B and 3A were required for maximal viral RNA synthesis in mouse cells. Examination of infected HeLa cells by electron microscopy demonstrated that human rhinoviruses induced the formation of cytoplasmic membranous vesicles, similar to those observed in cells infected with other picornaviruses. Vesicles were also observed in the cytoplasm of HRV39-infected mouse cells despite the absence of viral RNA replication. Synthesis of picornaviral nonstructural proteins 2C, 2BC, and 3A is known to be required for formation of membranous vesicles. We suggest that productive HRV39 infection is blocked in ICAM-L cells at a step posttranslation and prior to the formation of a functional replication complex. The observation that changes in HRV39 2B and 3A proteins lead to viral growth in mouse cells suggests that one or both of these proteins interact with host cell proteins to promote viral replication.Picornaviruses are small RNA viruses with a single-stranded, positive-sense genome of approximately 7 to 8 kb enclosed in a nonenveloped, icosahedral capsid. These viruses are responsible for a wide range of diseases including foot-and-mouth disease, hepatitis, poliomyelitis, and the common cold. Following cell entry, the viral genome is translated as a single polyprotein that is co-and posttranslationally cleaved by viral proteases, yielding both precursor proteins and end products. Many of the precursor proteins are essential for virus replication, with functions distinct from the end products (29,40,42). Four structural proteins produced from the P1 region of the genome form the viral capsid. Nonstructural proteins of the P2 and P3 region of the genome encode all of the viral proteins responsible for genomic RNA replication.Viral genome replication occurs on the surface of cytoplasmic vesicles induced by the synthesis of viral proteins 2C and 3A and the precursor 2BC (70). These vesicles are formed in conjunction with massive cell membrane proliferation (4, 7) and increased cellular synthesis of phospholipids (32). With the exception of the two viral proteases 2A pro and 3C pro , all of the nonstructural proteins have been found on the surface of these vesicles and are thought to interact in the replication complex (26,59,61,70). The vesicles assemble into a rosette, and the replication complex forms on the inner face of the rosette (13, 26). Salt-induced dissociation of the rosette demonstrated that the individual vesicles themselves can also support replication initiation and elongation (26). Neither t...

show abstract

Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus

Cited by 13 publications

References 51 publications

Membrane Topography of the Hydrophobic Anchor Sequence of Poliovirus 3A and 3AB Proteins and the Functional Effect of 3A/3AB Membrane Association upon RNA Replication

Membrane Topography of the Hydrophobic Anchor Sequence of Poliovirus 3A and 3AB Proteins and the Functional Effect of 3A/3AB Membrane Association upon RNA Replication

Evidence for Functional Protein Interactions Required for Poliovirus RNA Replication

Amino Acid Changes in Proteins 2B and 3A Mediate Rhinovirus Type 39 Growth in Mouse Cells

Contact Info

Product

Resources

About