Differential distribution of non-structural proteins of foot-and-mouth disease virus in BHK-21 cells

A poliovirus (PV) mutant (termed GG), which is incapable of producing 3AB, VPg, and 3CD proteins due to a defective cleavage site between the 3B and 3C proteins, replicated, producing 3BC-linked RNA rather than the VPg-linked RNA produced by the wild type (WT). GG PV RNA is quasi-infectious. The yield of infectious GG PV relative to replicated RNA is reduced by almost 5 logs relative to that of WT PV. Proteolytic activity required for polyprotein processing is normal for the GG mutant. 3BC-linked RNA can be encapsidated as efficiently as VPg-linked RNA. However, a step after genome replication but preceding virus assembly that is dependent on 3CD and/or 3AB proteins limits production of infectious GG PV. This step may involve release of replicated genomes from replication complexes. A pseudorevertant (termed EG) partially restored cleavage at the 3B-3C cleavage site. The reduced rate of formation of 3AB and 3CD caused corresponding reductions in the observed rate of genome replication and infectious virus production by EG PV without impacting the final yield of replicated RNA or infectious virus relative to that of WT PV. Using EG PV, we showed that genome replication and encapsidation were distinct steps in the multiplication cycle. Ectopic expression of 3CD protein reversed the genome replication phenotype without alleviating the infectious-virus production phenotype. This is the first report of a trans-complementable function for 3CD for any picornavirus. This observation supports an interaction between 3CD protein and viral and/or host factors that is critical for genome replication, perhaps formation of replication complexes.

supporting

confidence: 68%

Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

Pathak

Goodfellow

et al. 2009

“…The correct orientation and sequence of the resulting plasmids were confirmed by sequencing with primers 3A-F and 3A-R (Table 2), as well as primers 3A-1 and 3ABB-2 (30). For transient expression, plasmid pRSV3Awt (30) and derivatives of pRSV/L in which the luciferase gene was replaced by the 3A coding sequence of the different mutants described above were used. To this end, 3A-containing sequences were amplified from the corresponding pMT28 derivatives using oligonucleotides 3A-1 and 3A-2 (30), which included restriction sites for cloning into pRSV (HindIII and KpnI).…”

Section: Methodsmentioning

confidence: 99%

Mutations That Hamper Dimerization of Foot-and-Mouth Disease Virus 3A Protein Are Detrimental for Infectivity

González-Magaldi

Postigo

Torre

et al. 2012

Self Cite

Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, virulence, and host range. In other picornaviruses, homodimerization of 3A has been shown to be relevant for its biological activity. In this work, FMDV 3A homodimerization was evidenced by an in situ protein fluorescent ligation assay. A molecular model of the FMDV 3A protein, derived from the nuclear magnetic resonance (NMR) structure of the poliovirus 3A protein, predicted a hydrophobic interface spanning residues 25 to 44 as the main determinant for 3A dimerization. Replacements L38E and L41E, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, reduced the dimerization signal in the protein ligation assay and prevented the detection of dimer/multimer species in both transiently expressed 3A proteins and in synthetic peptides reproducing the N terminus of 3A. These replacements also led to production of infective viruses that replaced the acidic residues introduced (E) by nonpolar amino acids, indicating that preservation of the hydrophobic interface is essential for virus replication. Replacements that favored (Q44R) or impaired (Q44D) the polar interactions predicted between residues Q44 and D32 did not abolish dimer formation of transiently expressed 3A, indicating that these interactions are not critical for 3A dimerization. Nevertheless, while Q44R led to recovery of viruses that maintained the mutation, Q44D resulted in selection of infective viruses with substitution D44E with acidic charge but with structural features similar to those of the parental virus, suggesting that Q44 is involved in functions other than 3A dimerization.

“…Importantly, our study also delineates the likely temporal course of events in HRV16 infection, with 2A pro action preceding 3C pro action. The presence of 3CD in the nuclei of infected cells has been noted for a number of picornaviruses (6,27,28), but the mechanism by which this large protein enters the nucleus has been unclear. Together with other recent studies (29)(30)(31), the results here suggest that a unique mechanism regulates nuclear localization of picornavirus 3CD/3D, with protease activity being key.…”

Section: Discussionmentioning

confidence: 99%

“…Results for FMDV also suggest an important role for 3D pol nuclear localization in virus infection, whereby NLS mutations that inhibit it result in attenuated FMDV infection (28), although a link to splicing factor interaction has not been assessed. Although no nuclear function has as yet been assigned to HRV 3D pol , it seems likely that it plays roles similar to those of the proteins from these other viruses.…”

Section: Discussionmentioning

confidence: 99%

“…[see references 34 and 35]), the NLS may contribute to HRV 3CD nuclear localization. Interestingly, a recent study (31) showed increased nuclear localization of coxsackievirus B3 (CVB3) enhanced GFP (EGFP)-tagged 3D pol above that of EGFP alone, even though no NLS-like sequence can be identified within the CVB3 3D pol amino acid sequence, while the nuclear localization of FMDV 3D pol (28), in contrast to HRV and poliovirus proteins, appears to be dependent on an N-terminal sequence (MRKTKLAPT 24 ) based on mutagenic analysis (28). Clearly, observations with respect to one genus within the Picornaviridae family cannot be assumed to hold true for other genera and, indeed, for serotypes within the same species (16,36).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

Walker

Jensen

Croft

et al. 2016

The human rhinovirus (HRV) 3C and 2A proteases (3C pro and 2A pro , respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3C pro is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2A pro activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3C pro , 3D, and mutant derivatives thereof, we show that 3C pro is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3C pro activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2A pro fusion proteins, we demonstrate formally that 2A pro activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3C pro is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2A pro activity and not 3C pro activity, which is observed only later in infection. The results thus define the temporal activities of 2A pro and 3CD/3C pro activities in HRV serotype16 infection. IMPORTANCEThe human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection and defines the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.