Modulation of HIV-like particle assembly<i>in vitro</i>by inositol phosphates

Campbell, Stephen J.; Fisher, Robert J.; Towler, Eric M.; Fox, Stephen D.; Issaq, Haleem J.; Wolfe, Tracy L.; Phillips, L.; Rein, Alan

doi:10.1073/pnas.191224698

Cited by 195 publications

(216 citation statements)

References 23 publications

(19 reference statements)

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“…Several explanations include our preliminary data using Gag⌬p6 and CA-NC, showing a lower affinity for the 1/6 probe than mGag. Others have employed full-length MA sequences that do not acquire proper conformation in a prokaryotic environment (68,69). In addition, others have compared the binding activities of both proteins with a 178/383 probe comprising SL1, 2, 3, and 4, and we show here that sequences downstream of nucleotide 383 are crucial for the differential binding of mGag (39).…”

Section: Discussionmentioning

confidence: 58%

In Vitro Identification and Characterization of an Early Complex Linking HIV-1 Genomic RNA Recognition and Pr55Gag Multimerization

Roldán

Russell

Marchand

et al. 2004

Journal of Biological Chemistry

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The minimal protein requirements that drive viruslike particle formation of human immunodeficiency virus type 1 (HIV-1) have been established. The C-terminal domain of capsid (CTD-CA) and nucleocapsid (NC) are the most important domains in a so-called minimal Gag protein (mGag). The CTD is essential for Gag oligomerization. NC is known to bind and encapsidate HIV-1 genomic RNA. The spacer peptide, SP1, located between CA and NC is important for the multimerization process, viral maturation and recognition of HIV-1 genomic RNA by NC. In this study, we show that NC in the context of an mGag protein binds HIV-1 genomic RNA with almost 10-fold higher affinity. The protein region encompassing the 11th ␣-helix of CA and the proposed ␣-helix in the CA/SP1 boundary region play important roles in this increased binding capacity. Furthermore, sequences downstream from stem loop 4 of the HIV-1 genomic RNA are also important for this RNA-protein interaction. In gel shift assays using purified mGag and a model RNA spanning the region from ؉223 to ؉506 of HIV-1 genomic RNA, we have identified an early complex (EC) formation between 2 proteins and 1 RNA molecule. This EC was not present in experiments performed with a mutant mGag protein, which contains a CTD dimerization mutation (M318A). These data suggest that the dimerization interface of the CTD plays an important role in EC formation, and, as a consequence, in RNA-protein association and multimerization. We propose a model for the RNA-protein interaction, based on previous results and those presented in this study.

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Section: Discussionmentioning

confidence: 58%

In Vitro Identification and Characterization of an Early Complex Linking HIV-1 Genomic RNA Recognition and Pr55Gag Multimerization

Roldán

Russell

Marchand

et al. 2004

Journal of Biological Chemistry

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“…To a first approximation, all of the information necessary for retroviral particle assembly resides in the Gag polypeptide. For example, Gag alone can form extracellular virus-like particles in the absence of other viral proteins [11] and Gag molecules can spontaneously assemble into spherical, immature viruslike particles in vitro [12][13][14]. Nevertheless, although Gag itself encodes the necessary tertiary and quaternary interactions, it must be emphasized that assembly requires nonspecific RNA interactions both in vivo and in vitro, and is assisted by host factors in vivo, including trafficking factors, assembly chaperones, and the ESCRT budding pathway, as reviewed elsewhere [15][16][17][18].…”

Section: Gag As An Assembly Machinementioning

confidence: 99%

The structural biology of HIV assembly

Ganser‐Pornillos

Yeager

Sundquist

2008

Current Opinion in Structural Biology

409

422

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HIV assembly and replication proceed through formation of morphologically distinct immature and mature viral capsids that are organized by the Gag polyprotein (immature) and by the fully processed CA protein (mature). The Gag polyprotein is composed of three folded polypeptides (MA, CA, and NC) and three smaller peptides (SP1, SP2, and p6) that function together to coordinate membrane binding and Gag-Gag lattice interactions in immature virions. Following budding, HIV maturation is initiated by proteolytic processing of Gag, which induces conformational changes in the CA domain and results in assembly of the distinctive conical capsid. Retroviral capsids are organized following the principles of fullerene cones, and the hexagonal CA lattice is stabilized by three distinct interfaces. Recently identified inhibitors of viral maturation act by disrupting the final stage of Gag processing, or by inhibiting formation of a critical intermolecular CA-CA interface in the mature capsid. Following release into a new host cell, the capsid disassembles and host cell factors can potently restrict this stage of retroviral replication. Here, we review the structures of immature and mature HIV virions, focusing on recent studies that have defined the global organization of the immature Gag lattice, identified sites likely to undergo conformational changes during maturation, revealed the molecular structure of the mature capsid lattice, demonstrated that capsid architectures are conserved, identified the first capsid assembly inhibitors, and begun to uncover the remarkable biology of the mature capsid.

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“…122,123 During the life cycle of the virus, NC acts as both a nucleic acid binding domain and a nucleic acid chaperone. [124][125][126][127][128][129][130][131][132][133][134][135][136] This chaperone activity, facilitating restructuring of the nucleic acid complex, has been attributed to both an aggregating ability of NC and a facility for duplex destabilization. 125,131,[137][138][139][140][141][142][143][144][145] This destabilization, however, is weak, 143,144,146,147 and will not completely melt DNA without a complementary strand.…”

Section: Figure 11mentioning

confidence: 99%

Mechanisms of DNA binding determined in optical tweezers experiments

McCauley¹,

Williams²

2006

Biopolymers

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The last decade has seen rapid development in single molecule manipulation of RNA and DNA. Measuring the response force for a particular manipulation has allowed the free energies of various nucleic acid structures and configurations to be determined. Optical tweezers represent a class of single molecule experiments that allows the energies and structural dynamics of DNA to be probed up to and beyond the transition from the double helix to its melted single strands. These experiments are capable of high force resolution over a wide dynamic range. Additionally, these investigations may be compared with results obtained when the nucleic acids are in the presence of proteins or other binding ligands. These ligands may bind into the major or minor groove of the double helix, intercalate between bases or associate with an already melted single strand of DNA. By varying solution conditions and the pulling dynamics, energetic and dynamic information may be deduced about the mechanisms of binding to nucleic acids, providing insight into the function of proteins and the utility of drug treatments. © 2006 Wiley Periodicals, Inc. Biopolymers 85:154–168, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Modulation of HIV-like particle assemblyin vitroby inositol phosphates

Cited by 195 publications

References 23 publications

In Vitro Identification and Characterization of an Early Complex Linking HIV-1 Genomic RNA Recognition and Pr55Gag Multimerization

In Vitro Identification and Characterization of an Early Complex Linking HIV-1 Genomic RNA Recognition and Pr55Gag Multimerization

The structural biology of HIV assembly

Mechanisms of DNA binding determined in optical tweezers experiments

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