Constraints on the assembly of spherical virus particles

112

160

The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a crucial role in both assembly and maturation of the virion. Numerous recent studies have focused on either the soluble form of CA or the polymer end product of in vitro CA assembly. The CA polymer, in particular, has been used to study CA-CA interactions because it is a good model for the CA interactions within the virion core. However, analysis of the process of in vitro CA assembly can yield valuable insights into CA-CA interactions and the mechanism of core assembly. We describe here a method for the analysis of CA assembly kinetics wherein the progress of assembly is monitored by using turbidity. At pH 7.0 the addition of either of the isolated CA domains (i.e., the N or the C domain) to an assembly reaction caused a decrease in the assembly rate by competing for binding to the full-length CA protein. At pH 8.0 the addition of the isolated C domain had a similar inhibitory affect on CA assembly. However, at pH 8.0 the isolated N domain had no affect on the rate of CA assembly but, when mixed with the C domain, it alleviated the C-domain inhibition. These data provide biochemical evidence for a pH-sensitive homotypic N-domain interaction, as well as for an N-and C-domain interaction.

Section: Discussionmentioning

confidence: 87%

Kinetic Analysis of the Role of Intersubunit Interactions in Human Immunodeficiency Virus Type 1 Capsid Protein Assembly In Vitro

Lanman

Sexton

Sakalian

et al. 2002

112

160

“…5B. If we assume, as other researchers have done (9,15,36,37), that each Q␤ coat protein dimer binds a single RNA hairpin, then the K a of the wild-type 77-base fragment is 510 mol Ϫ1 . This corresponds well to the K a of 570 mol Ϫ1 previously reported for a 29-base RNA fragment (53).…”

Section: Construction Of the Expression Vectormentioning

confidence: 99%

Evolution of Bacteriophage in Continuous Culture: a Model System To Test Antiviral Gene Therapies for the Emergence of Phage Escape Mutants

Lindemann

Klug²,

Schwienhorst

2002

The emergence of viral escape mutants is usually a highly undesirable phenomenon. This phenomenon is frequently observed in antiviral drug applications for the treatment of viral infections and can undermine long-term therapeutic success. Here, we propose a strategy for evaluating a given antiviral approach in terms of its potential to provoke the appearance of resistant virus mutants. By use of Q␤ RNA phage as a model system, the effect of an antiviral gene therapy, i.e., a virus-specific repressor protein expressed by a recombinant Escherichia coli host, was studied over the course of more than 100 generations. In 13 experiments carried out in parallel, 12 phage populations became resistant and 1 became extinct. Sequence analysis revealed that only two distinct phage mutants emerged in the 12 surviving phage populations. For both escape mutants, sequence variations located in the repressor binding site of the viral genomic RNA, which decrease affinity for the repressor protein, conferred resistance to translational repression. The results clearly suggest the feasibility of the proposed strategy for the evaluation of antiviral approaches in terms of their potential to allow resistant mutants to appear. In addition, the strategy proved to be a valuable tool for observing virus-specific molecular targets under the impact of antiviral drugs.Ideally, antiviral strategies should be highly virus specific and should not affect the host organism. In addition, they should exhibit a minimal potential to allow viral escape mutants to appear. In this paper, we propose a general strategy to evaluate a given antiviral approach with regard to resistance phenomena that may occur due to the emergence of escape mutants. Utilizing RNA phage Q␤ as a model system, we investigated the potential of virus-specific translational repressor proteins to suppress viral propagation. Among the mechanisms that control gene expression, translational regulation is certainly one of the most specific and therefore should be a valuable target mechanism for antiviral strategies.First observed in the RNA phages (29), translational repression has since been discovered in many bacteriophages (47), prokaryotes (11,16,45), and eukaryotes (46) as well as in several mammalian viruses (14,17,35,44). Within the closely related RNA phage species R17, MS2, F2, fr, and Q␤, two proteins, namely, the coat protein and the replicase, are translational regulators of each other. Shortly after single-stranded viral RNA enters the host cell, replicase protein is translated from the proximal end of the phage genome, leading to high levels of replicated phage RNA.However, it has been shown that replication cannot occur when phage RNA is being translated (25,47). Therefore, strict translational regulation of the remaining cistrons, namely, A1/ coat protein and A2, from the distal portion of the Q␤ RNA genome is essential for the replication. Hence, blocking the translation of the coat protein, which is produced abundantly during the late phase of the viral infection cycl...

“…Spherical viruses possess icosahedral symmetry, the simplest form of which contains 60 subunits arranged as 12 pentamers (9,26). Larger capsids contain subtriangulated faces in which subunits are positioned in nonequivalent bonding environments (9,26).…”

mentioning

confidence: 99%

“…Larger capsids contain subtriangulated faces in which subunits are positioned in nonequivalent bonding environments (9,26). Remarkably, many viral capsid proteins are capable of conformational switching during assembly to allow for subtriangulation with a single protein.…”

mentioning

confidence: 99%

“…Remarkably, many viral capsid proteins are capable of conformational switching during assembly to allow for subtriangulation with a single protein. This variability in conformational states renders capsid assembly pathway dependent, and so information needs to be imparted to the incoming subunits during assembly to ensure that the proper global icosahedral symmetry is obtained (26). To accomplish this, most double-stranded DNA (dsDNA) bacteriophage as well as herpesviruses first assemble a procapsid devoid of nucleic acid by using catalytic scaffold protein to guide the assembly process (10).…”

mentioning

confidence: 99%

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A P22 Scaffold Protein Mutation Increases the Robustness of Head Assembly in the Presence of Excess Portal Protein

Moore

Prevelige

2002

Bacteriophage with linear, double-stranded DNA genomes package DNA into preassembled protein shells called procapsids. Located at one vertex in the procapsid is a portal complex composed of a ring of 12 subunits of portal protein. The portal complex serves as a docking site for the DNA packaging enzymes, a conduit for the passage of DNA, and a binding site for the phage tail. An excess of the P22 portal protein alters the assembly pathway of the procapsid, giving rise to defective procapsid-like particles and aberrant heads. In the present study, we report the isolation of escape mutant phage that are able to replicate more efficiently than wild-type phage in the presence of excess portal protein. The escape mutations all mapped to the same phage genome segment spanning the portal, scaffold, coat, and open reading frame 69 genes. The mutations present in five of the escape mutants were determined by DNA sequencing. Interestingly, each mutant contained the same mutation in the scaffold gene, which changes the glycine at position 287 to glutamate. This mutation alone conferred an escape phenotype, and the heads assembled by phage harboring only this mutation had reduced levels of portal protein and exhibited increased head assembly fidelity in the presence of excess portal protein.Because this mutation resides in a region of scaffold protein necessary for coat protein binding, these findings suggest that the P22 scaffold protein may define the portal vertices in an indirect manner, possibly by regulating the fidelity of coat protein polymerization.