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Retroviral capsid assembly: a role for the CA dimer in initiation.

ABSTRACT: In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of capsid assembly and provide insights into underlying mechanisms. CA multimerization was triggered by multivalent anions providing evidence that in vitro assembly is an electrostatically controlled process. In the case of RSV, in vitro assembly was a well-behaved nucleation-driven process that led to the formation of structures with morphologies similar to those found in virions. Isolated RSV dimers, when mixed with monomeric protein, acted as efficient seeds for assembly, eliminating the lag phase characteristic of a monomer-only reaction. This demonstrates for the first time the purification of an intermediate on the assembly pathway. Differences in the intrinsic tryptophan fluorescence of monomeric protein and the assembly-competent dimer fraction suggest the involvement of the NTD in the formation of the functional dimer. Furthermore, in vitro analysis of well-characterized CTD mutants provides evidence for assembly dependence on the second domain and suggests that the establishment of an NTD-CTD interface is a critical step in capsid assembly initiation. Overall, the data provide clear support for a model whereby capsid assembly within the maturing virion is dependent on the formation of a specific nucleating complex that involves a CA dimer and is directed by additional virion constituents.

SUBMITTER: Purdy JG

PROVIDER: S-EPMC2996723 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Retroviral capsid assembly: a role for the CA dimer in initiation.

Purdy John G JG Flanagan John M JM Ropson Ira J IJ Craven Rebecca C RC

Journal of molecular biology 20090408 2

In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of ...[more]

PMID: 19361521

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Project description:Retroviral particles contain a dimeric RNA genome, which serves as template for the generation of double-stranded DNA by reverse transcription. Transfer between RNA strands during DNA synthesis is governed by both sequence similarity between templates and structural features of the dimeric RNA. A kissing hairpin, believed to facilitate intermolecular recognition and dimer formation, was previously found to be a preferred site for recombination. To investigate if hairpin loop-loop-complementarity is the primary determinant for this recombination preference, we have devised a novel 5' leader recombination assay based upon co-packaging of two wild-type or loop-modified murine leukemia virus vector RNAs. We found that insertion of an alternative palindromic loop in one of the two vectors disrupted site-directed template switching, whereas site-specificity was restored between vectors with complementary non-wild-type palindromes. By pairing vector RNAs that contained identical non-palindromic loop motifs and that were unlikely to interact by loop-loop kissing, we found no preference for recombination at the kissing hairpin site. Of vector pairs designed to interact through base pairing of non-palindromic loop motifs, we could in one case restore hairpin-directed template switching, in spite of the reduced sequence identity, whereas another pair failed to support hairpin- directed recombination. However, analyses of in vitro RNA dimerization of all studied vector combinations showed a good correlation between efficient dimer formation between loop-modified viral RNAs and in vivo cDNA transfer at the kissing hairpin. Our findings demonstrate that complementarity between wild-type or non-wild-type hairpin kissing loops is essential but not sufficient for site-specific 5' leader recombination and lend further support to the hypothesis that a specific 'kissing' loop-loop interaction is guided by complementary sequences and maintained within the mature dimeric RNA of retroviruses.

Project description:Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity (KD of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity (KD of ∼10 μM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition.IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5α protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5α lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5α SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5α RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5α, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits much more tightly through its cyclophilin A domain and therefore was thought to act independently of higher-order assembly. Here, we show that TRIMCyp shares the assembly properties of TRIM5α and that both forms of TRIM5 use the same mechanism of hexagonal lattice formation to promote viral recognition and restriction.

Dataset Information

Retroviral capsid assembly: a role for the CA dimer in initiation.

Publications

Retroviral capsid assembly: a role for the CA dimer in initiation.

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