Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly

BACKGROUND: Bluetongue virus (BTV) particles consist of seven structural proteins that are organized into two capsids. In addition, BTV also encodes three non-structural (NS) proteins of which protein 2 (NS2) is the RNA binding protein and is also the major component of virus encoded inclusion bodies (VIBs), which are believed to be virus assembly sites. To investigate the contribution of NS2 in virus replication and assembly we have constructed inducible mammalian cell lines expressing full-length NS2. In addition, truncated NS2 fragments were also generated in an attempt to create dominant negative mutants for NS2 function. RESULTS: Our data revealed that expression of full-length NS2 was sufficient for the formation of inclusion bodies (IBs) that were morphologically similar to the VIBs formed during BTV infection. By using either, individual BTV proteins or infectious virions, we found that while the VP3 of the inner capsid (termed as "core") that surrounds the transcription complex was closely associated with both NS2 IBs and BTV VIBs, the surface core protein VP7 co-localized with NS2 IBs only in the presence of VP3. In contrast to the inner core proteins, the outer capsid protein VP2 was not associated with either IBs or VIBs. Like the core proteins, newly synthesized BTV RNAs also accumulated in VIBs. Unlike full-length NS2, neither the amino-, nor carboxyl-terminal fragments formed complete IB structures and each appeared to interfere in overall virus replication when similarly expressed. CONCLUSION: Together, these data demonstrate that NS2 is sufficient and necessary for IB formation and a key player in virus replication and core assembly. Perturbation of NS2 IB formation resulted in reduced virus synthesis and both the N terminal (NS2-1) and C terminal (NS2-2) fragments act as dominant negative mutants of NS2 function

Defining the structure-function relationships of bluetongue virus helicase protein VP6.

The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6

Assembly and Intracellular Localization of the Bluetongue Virus Core Protein VP3

The bluetongue virus (BTV) core protein VP3 plays a crucial role in the virion assembly and replication process. Although the structure of the protein is well characterized, much less is known about the intracellular processing and localization of the protein in the infected host cell. In BTV-infected cells, newly synthesized viral core particles accumulate in specific locations within the host cell in structures known as virus inclusion bodies (VIBs), which are composed predominantly of the nonstructural protein NS2. However, core protein location in the absence of VIBs remains unclear. In this study, we examined VP3 location and degradation both in the absence of any other viral protein and in the presence of NS2 or the VP3 natural associate protein, VP7. To enable real-time tracking and processing of VP3 within the host cell, a fully functional enhanced green fluorescent protein (EGFP)-VP3 chimera was synthesized, and distribution of the fusion protein was monitored in different cell types using specific markers and inhibitors. In the absence of other BTV proteins, EGFP-VP3 exhibited distinct cytoplasmic focus formation. Further evidence suggested that EGFP-VP3 was targeted to the proteasome of the host cells but was dispersed throughout the cytoplasm when MG132, a specific proteasome inhibitor, was added. However, the distribution of the chimeric EGFP-VP3 protein was altered dramatically when the protein was expressed in the presence of the BTV core protein VP7, a normal partner of VP3 during BTV assembly. Interaction of EGFP-VP3 and VP7 and subsequent assembly of core-like particles was further examined by visualizing fluorescent particles and was confirmed by biochemical analysis and by electron microscopy. These data indicated the correct assembly of EGFP-VP3 subcores, suggesting that core formation could be monitored in real time. When EGFP-VP3 was expressed in BTV-infected BSR cells, the protein was not associated with proteasomes but instead was distributed within the BTV inclusion bodies, where it colocalized with NS2. These findings expand our knowledge about VP3 localization and its fate within the host cell and illustrate the assembly capability of a VP3 molecule with a large amino-terminal extension. This also opens up the possibility of application as a delivery system

Mapping the assembly pathway of Bluetongue virus scaffolding protein VP3.

The structure of the Bluetongue virus (BTV) core and its outer layer VP7 has been solved by X-ray crystallography, but the assembly intermediates that lead to the inner scaffolding VP3 layer have not been defined. In this report, we addressed two key questions: (a) the role of VP3 amino terminus in core assembly and its interaction with the transcription complex (TC) components; and (b) the assembly intermediates involved in the construction of the VP3 shell. To do this, deletion mutants in the amino terminal and decamer-decamer interacting region of VP3 (DeltaDD) were generated, expressed in insect cells using baculovirus expression systems, and their ability to assemble into core-like particles (CLPs) and to incorporate the components of TC were investigated. Deletion of the N-terminal 5 (Delta5N) or 10 (Delta10N) amino acids did not affect the ability to assemble into CLPs in the presence of VP7 although the cores assembled using the 10 residue mutant (Delta10N) deletion were very unstable. Removal of five residues also did not effect incorporation of the internal VP1 RNA polymerase and VP4 mRNA capping enzyme proteins of the TC. Removal of the VP3-VP3 interacting domain (DeltaDD) led to failure to assemble into CLPs yet retained interaction with VP1 and VP4. In solution, purified DeltaDD mutant protein readily multimerized into dimers, pentamers, and decamers, suggesting that these oligomers are the authentic assembly intermediates of the subcore. However, unlike wild-type VP3 protein, the dimerization domain-deleted assembly intermediates were found to have lost RNA binding ability. Our study emphasizes the requirement of the N-terminus of VP3 for binding and encapsidation of the TC components, and defines the role of the dimerization domain in subcore assembly and RNA binding

Impaired imipenem uptake associated with alterations in outer membrane proteins and lipopolysaccharides in imipenem-resistant Shigella dysenteriae

Three imipenem-resistant mutants were obtained from a clinical isolate (C152) of Shigella dysenteriae by selection with increasing concentrations of imipenem. Resistance to imipenem was associated with resistance to several other β-lactam antibiotics. The penicillin-binding protein (PBP) patterns of the resistant and the wild-type strains were comparable. The permeability of the outer membrane proteins (OMPs) of the most resistant mutant, IM16, was lower than that of the parent strain C152 when imipenem and arabinose were used as test solutes. This mutant had lower levels of both the major OMPs of Mr 43,000 and 38,000. There were also differences in the patterns of lipopolysaccharide (LPS) of the mutants and the wild-type strain. The mutant IM16 had less short-chain LPS than the parent C152. Increasing imipenem resistance was also associated with a concomitant decrease in the level of 2-keto-3-deoxyoctonate, a component of the core region of LPS

Alterations in high molecular mass penicillin-binding protein 1 associated with beta-lactam resistance in Shigella dysenteriae

Beta-lactam resistance poses a major problem in the chemotherapy of shigellosis caused by Shigella dysenteriae. Such resistance may arise from alterations in the affinities or amounts of the penicillin-binding proteins (PBPs) for beta-lactams, elaboration of beta-lactamases and reduced permeability across the outer membrane. The mechanisms of resistance in S. dysenteriae have not been studied in depth. This report describes a laboratory mutant, M19 which was characterized by the appearance of two high molecular mass PBPs of 84 (PBP1′) and 82 kDa (PBP1″). M19 was more resistant to cefsulodin and cefoxitin. Resistance could be correlated with lower second order rate constants (k+2/K) of acylation. Moreover there was an overall two-fold increase in the relative amount of PBP1 (i.e. 1′+1″) in the mutant M19 compared to C152. This is the first report which presents evidence of the involvement of altered high molecular mass PBPs in beta-lactam resistance in S. dysenteriae

Biochemical and Biophysical Characterization of a Chimeric TRIM21-TRIM5α Protein▿

The tripartite motif (TRIM) protein, TRIM5α, is an endogenous factor in primates that recognizes the capsids of certain retroviruses after virus entry into the host cell. TRIM5α promotes premature uncoating of the capsid, thus blocking virus infection. Low levels of expression and tendencies to aggregate have hindered the biochemical, biophysical, and structural characterization of TRIM proteins. Here, a chimeric TRIM5α protein (TRIM5Rh-21R) with a RING domain derived from TRIM21 was expressed in baculovirus-infected insect cells and purified. Although a fraction of the TRIM5Rh-21R protein formed large aggregates, soluble fractions of the protein formed oligomers (mainly dimers), exhibited a protease-resistant core, and contained a high percentage of helical secondary structure. Cross-linking followed by negative staining and electron microscopy suggested a globular structure. The purified TRIM5Rh-21R protein displayed E3-ligase activity in vitro and also self-ubiquitylated in the presence of ubiquitin-activating and -conjugating enzymes. The purified TRIM5Rh-21R protein specifically associated with human immunodeficiency virus type 1 capsid-like complexes; a deletion within the V1 variable region of the B30.2(SPRY) domain decreased capsid binding. Thus, the TRIM5Rh-21R restriction factor can directly recognize retroviral capsid-like complexes in the absence of other mammalian proteins