Identification of tolerated insertion sites in poliovirus non-structural proteins

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Poliovirus proteins 3A and 3AB are small, membrane-binding proteins that play multiple roles in viral RNA replication complex formation and function. In the infected cell, these proteins associate with other viral and cellular proteins as part of a supramolecular complex whose structure and composition are unknown. We isolated viable viruses with three different epitope tags (FLAG, hemagglutinin [HA], and c-myc) inserted into the N-terminal region of protein 3A. These viruses exhibited growth properties and characteristics very similar to those of the wild-type, untagged virus. Extracts prepared from the infected cells were subjected to immunoaffinity purification of the tagged proteins by adsorption to commercial antibody-linked beads and examined after elution for cellular and other viral proteins that remained bound to 3A sequences during purification. Viral proteins 2C, 2BC, 3D, and 3CD were detected in all three immunopurified 3A samples. Among the cellular proteins previously reported to interact with 3A either directly or indirectly, neither LIS1 nor phosphoinositol-4 kinase (PI4K) were detected in any of the purified tagged 3A samples. However, the guanine nucleotide exchange factor GBF1, which is a key regulator of membrane trafficking in the cellular protein secretory pathway and which has been shown previously to bind enteroviral protein 3A and to be required for viral RNA replication, was readily recovered along with immunoaffinity-purified 3A-FLAG. Surprisingly, we failed to cocapture GBF1 with 3A-HA or 3A-myc proteins. A model for variable binding of these 3A mutant proteins to GBF1 based on amino acid sequence motifs and the resulting practical and functional consequences thereof are discussed.Poliovirus (PV) is a member of the human enterovirus C cluster in the Enterovirus genus of the virus family Picornaviridae. The PV genome encodes a single polyprotein that is proteolytically processed to generate a set of intermediate precursors and final cleavage products that are all required for virus replication. The N-terminal region of the polyprotein (P1) forms the viral capsid proteins, which are dispensable for viral RNA translation and replication but are needed for encapsidation and assembly of infectious particles. The remainder of the polyprotein (P2 and P3 regions) generates proteins that contribute catalytic and structural functions for viral RNA translation and replication, as well as for disruption and/or reorganization of numerous cellular processes and activities that could restrict or combat virus replication. All of the PV noncapsid proteins are essential for viral RNA replication; most manifest multiple activities during the virus replication cycle, and all at least partially localize to large, membraneassociated replication complexes that form from preexisting subcellular organelles after infection. Viral proteins 2B, 2C, and 3A contain hydrophobic trans-membrane regions or amphipathic helices, and these proteins as well as their larger precursor proteins bind membranes directly (7,12,...

Section: Selection Of Viable Viruses Encoding 5-amino-acid Insertionsmentioning

confidence: 99%

Section: Selection Of Viable Viruses Encoding 5-amino-acid Insertionsmentioning

confidence: 99%

Analysis of Poliovirus Protein 3A Interactions with Viral and Cellular Proteins in Infected Cells

Teterina

Pinto

Weaver

et al. 2011

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“…Transposon (Tn)-mediated insertional mutagenesis has been widely used to identify the essential functional domains of many viral proteins (23,35,37,54,69) and cis-acting elements (3,4) and more recently for comprehensive functional analysis of entire viral genomes, such as the hepatitis C virus genome (3,9,44). In comparison to targeted mutagenesis, Tn mutagenesis allows the structure and function of viral proteins to be randomly probed.…”

mentioning

confidence: 99%

“…These have included sequences encoding reporter proteins or small peptide tags to facilitate further characterization of the viral proteins and their interaction partners, as well as to enable imaging of viral replication (1,5,45,70,71). This strategy has been particularly successful for labeling small positive-strand RNA viruses (69,70), for which traditional approaches, such as making insertions in the viral UTRs (51) or targeted viral protein-reporter fusions (11), are challenging. Such approaches are further complicated for MNV due to the compact nature of the genome, the short length of the UTRs, the presence of cis-acting RNA within the coding region, and the little structural information available for the viral proteins.…”

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confidence: 99%

High-Resolution Functional Profiling of the Norovirus Genome

Thorne

Bailey

Goodfellow

2012

Human noroviruses (HuNoV) are a major cause of nonbacterial gastroenteritis worldwide, yet details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. Studies with murine norovirus (MNV), which can be propagated in permissive cells, have begun to probe different aspects of the norovirus life cycle; however, our understanding of the specific functions of the viral proteins lags far behind that of other RNA viruses. Genome-wide functional profiling by insertional mutagenesis can reveal protein domains essential for replication and can lead to generation of tagged viruses, which has not yet been achieved for noroviruses. Here, transposon-mediated insertional mutagenesis was used to create 5 libraries of mutagenized MNV infectious clones, each containing a 15-nucleotide sequence randomly inserted within a defined region of the genome. Infectious virus was recovered from each library and was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCR profiling. Genome-wide profiling of over 2,000 insertions revealed essential protein domains and confirmed known functional motifs. As validation, several insertion sites were introduced into a wild-type clone, successfully allowing the recovery of infectious virus. Screening of a number of reporter proteins and epitope tags led to the generation of the first infectious epitope-tagged noroviruses carrying the FLAG epitope tag in either NS4 or VP2. Subsequent work confirmed that epitope-tagged fully infectious noroviruses may be of use in the dissection of the molecular interactions that occur within the viral replication complex. Human norovirus (HuNoV) is the leading cause of nonbacterial gastroenteritis in developed countries, with initial reports also indicating high incidence and mortality in developing countries (47). Due to the low infectious dose, multiple transmission routes, and high stability of HuNoV in the environment (41, 72), outbreaks typically occur in places with close living conditions, such as hospitals. Infections in hospitals alone cost the United Kingdom National Health Service an estimated £115 million and result in 47,000 lost bed days per year, compromising hospital services (39, 53). The majority of HuNoV infections are acute and self-resolving; however, links with conditions such as inflammatory bowel disease (36), infantile seizures (18), and neonatal necrotizing enterocolitis (65, 74) have been established. There are also increasing reports of persistent infections in the elderly and the immunocompromised, such as transplant recipients and some cancer patients (2,13,20,40,55,56).Details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. The discovery of murine norovirus (MNV) in 2003 has since facilitated studies on norovirus replication. Unlike HuNoV, MNV can be propagated in culture, displaying a tropism for macrophage and dendritic cells (75), and ca...

“…In the case of reporter viruses containing the RLuc gene (933 nucleotides [nt]), deletion commences during the first passage on HeLa cells (37). It should be noted that besides luciferase reporters, other marker proteins have also been engineered into the complete poliovirus polyprotein (59)(60)(61). These chimeras are expressing marker proteins in nonstructural proteins 2A pro and 3A.…”

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confidence: 99%

Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase

Song

Paul

Wimmer

2012

Polioviruses (PVs) carrying a reporter gene are useful tools for studies of virus replication, particularly if the viral chimeras contain the polyprotein that provides all of the proteins necessary for a complete replication cycle. Replication in HeLa cells of a previously constructed poliovirus expressing the gene for Renilla luciferase (RLuc) fused to the N terminus of the polyprotein H 2 NRLuc-P1-P2-P3-COOH (P1, structural domain; P2 and P3, nonstructural domains) led to the deletion of RLuc after only one passage. Here we describe a novel poliovirus chimera that expresses Gaussia luciferase (GLuc) inserted into the polyprotein between P1 and P2 (N 2 H-P1-GLuc-P2-P3-COOH). This chimera, termed PV-GLuc, replicated to 10% of wild-type yield. The reporter signal was fully retained for three passages and then gradually lost. After six passages the signal was barely detectable. On further passages, however, the GLuc signal reappeared, and after eight passages it had reached the same levels observed with the original PV-GLuc at the first passage. We demonstrated that this surprising observation was due to coevolution of defective interfering (DI) particles that had lost part or all of the capsid coding sequence (⌬P1-GLuc-P2-P3) and wild-type-like viruses that had lost the GLuc sequence (P1-P2-P3). When used at low passage, PV-GLuc is an excellent tool for studying aspects of genome replication and morphogenesis. The GLuc protein was secreted from mammalian cells but, in agreement with published data, was not secreted from PV-GLuc-infected cells due to poliovirus-induced inhibition of cellular protein secretion. Published evidence indicates that individual expression of enterovirus polypeptide 3A, 2B, or 2BC in COS-1 cells strongly inhibits host protein secretion. In HeLa cells, however, expression of none of the poliovirus polypeptides, either singly or in pairs, inhibited GLuc secretion. Thus, inhibition of GLuc secretion in PV-infected HeLa cells is likely a result of the interaction between several viral and cellular proteins that are different from those in COS-1 cells.