HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

Jacques, D.A.; McEwan, William A.; Hilditch, Laura; Price, Amanda J.; Towers, Greg J.; James, Leo C.

doi:10.1038/nature19098

Cited by 204 publications

(320 citation statements)

References 40 publications

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“…This kinetic difference may be related to the observation that E45A cores appear to be more permeable to small molecules than WT cores (24), suggesting that reverse transcription may occur more rapidly owing to enhanced dNTP influx. Recent data suggest that nucleotides are transported into the core through electrostatic channels that were found in CA hexamers (25). It is possible that the increased permeability of the E45A cores is due to changes in these channels.…”

Section: Discussionmentioning

confidence: 99%

Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Rankovic

Varadarajan

Ramalho

et al. 2017

J Virol

100

112

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The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. After cell entry, the core disassembles in a process termed uncoating. Although HIV-1 uncoating has been linked to reverse transcription of the viral genome in target cells, the mechanism by which uncoating is initiated is unknown. Using time-lapse atomic force microscopy, we analyzed the morphology and physical properties of isolated HIV-1 cores during the course of reverse transcription in vitro. We found that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 h. Highresolution mechanical mapping reveals the formation of a stiff coiled filamentous structure underneath the capsid surface. Subsequently, this coiled structure disappears, the stiffness of the capsid drops precipitously to a value below that of a prereverse transcription core, and the capsid undergoes partial or complete rupture near the narrow end of the conical structure. We propose that the transcription of the relatively flexible single-stranded RNA into a more rigid filamentous structure elevates the pressure within the core, which triggers the initiation of capsid disassembly.IMPORTANCE For successful infection, the HIV-1 genome, which is in the form of a single-stranded RNA enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism that triggers uncoating is a pivotal question of long standing. By using atomic force microscopy, we found that during reverse transcription the pressure inside the capsid increases until the internal stress exceeds the strength of the capsid structure and the capsid breaks open. The application of AFM technologies to study purified HIV-1 cores represents a new experimental platform for elucidating additional aspects of capsid disassembly and HIV-1 uncoating.KEYWORDS HIV-1, atomic force microscopy, capsid, reverse transcription, uncoating F ollowing fusion between the HIV-1 membrane and the target cell membrane, the viral core is released into the host cell cytoplasm. The core consists of the singlestranded viral RNA (ssRNA) genome and associated proteins encapsulated within a cone-shaped capsid composed of ϳ250 CA (p24) protein hexamers and 12 CA pentamers (1-3). Successful infection requires reverse transcription of the ssRNA into double-stranded DNA (dsDNA) that is then transported into the nucleus, where it integrates into the target cell genome. Since the intact HIV-1 core is too large to cross the nuclear pore (reviewed in reference 4), the capsid is thought to disassemble prior to nuclear import in a process known as uncoating. This is supported by a study which shows by superresolution microscopy differences in size of reverse transcription complexes between the cytoplasm and the nucleus in infected cells (5). Several studies

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Section: Discussionmentioning

confidence: 99%

Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Rankovic

Varadarajan

Ramalho

et al. 2017

J Virol

100

112

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“…The size of this passage will vary as the protein chains fluctuate. There is a chamber right above that arginine ring as pointed out by Jacques et al 13 , which is about 25Å deep and has a volume of over 3,000Å 3 13 . There is an opening at the top the chamber at the location about 25Å above the arginine ring.…”

Section: 1mentioning

confidence: 93%

“…In a recent work, the capsid, which is composed over 200 hexamers and 12 pentamers, was proposed to import nucleotides for encaspidated DNA synthesis through its hexamer pores 13 . The authors identified a key structure feature, the positively-charge arginine ring (R18), in the bottle neck of the pore.…”

Section: Introductionmentioning

confidence: 99%

Uncover the Mechanism of Nucleotide Import by HIV-1 Capsid

Song

2019

Preprint

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In this work, we carry out a series of Brownian diffusion simulations to elucidate the nucleotide importing process through the hexamer pores of HIV-1 capsid. Our simulations reveal the mechanism by which deoxynucleoside triphosphates (dNTPs) diffuse through the arginine ring, the role of electrostatic potential within the pore in the importing process, and surprisingly, how IP6s and ATPs, though competing with dNTPs for binding at the arginine ring, end up accelerating the nucleotide import rate by thousands of folds so that it is sufficiently high to fuel the encapsidated DNA 1 synthesis. IMPORTANCE: Efficient nucleotide import is critical to fuel the reverse DNA synthesis that takes place within the capsid. However, the mechanism by which capsid imports the nucleotides is presently unclear. The current study through Brownian simulations shed insights into the process. A clear understanding of the mechanism of nucleotide import can be informative in developing drugs that target the process.

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“…The deoxynucleotide triphosphates (dNTP) from the host cell diffuse into the nucleocapsid and reverse transcription begins [44]. Due to the impermeability of the capsid to cellular proteins at this early stage, if A3 enzymes are to access the HIV-1 (-) DNA synthesized by reverse transcriptase, they must be present in the viral capsid (Figure 1.6A).…”

Section: Restriction Of Hiv By Apobec3mentioning

confidence: 99%

“…Maintaining an intact capsid allows the stoichiometry of the viral components to remain high and promotes efficient reverse transcription. The HIV capsid becomes permeable to small macromolecules and the high level of host deoxynucleotide triphosphates in the cytoplasm allows diffusion through the capsid pore to initiate the reverse transcription process (Figure 1.4B) [44].…”

Section: General Hiv Biologymentioning

confidence: 99%

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

2018

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Apolipoprotein B mRNA-editing enzyme-catalytic, polypeptide-like 3 (APOBEC3) enzymes are a family of single-stranded (ss)DNA cytosine deaminases that serve as a host restriction factors for retrotransposons and viruses that contain a ssDNA intermediate. While the APOBEC3 family was originally expanded to restrict endogenous retroelements, the majority of these targets are now inactivated and the family has been found to act on different targets, such as viral ssDNA as a mechanism of viral defense. Some of the family members also catalyze "offtarget" deaminations in human ssDNA and have been implicated in somatic mutagenesis that can lead to cancer.My PhD thesis research investigated the biochemical mechanisms underlying both the benefits and the risks of the A3 family of enzymes. The central hypothesis of this work is that A3 enzymes have evolved distinct biochemical mechanisms to deaminate ssDNA that differentiate A3 restriction of retroelements and retroviruses from A3-induced somatic mutagenesis.Therefore, we investigated the biochemical mechanisms the A3 enzymes utilize during restriction of Human Immunodeficiency Virus (HIV) in both deamination-dependent and deaminationindependent modes, as well as the unique mechanisms employed during mutation of genomic DNA. There are seven APOBEC3 members (A-H, excluding E) and of these, four members (A3D, A3F, A3G, A3H) have been identified to restrict HIV replication in CD4 T+ cells, and currently three members (A3A, A3B and A3H haplotype I) have been implicated in somatic mutagenesis.The APOBEC3 enzymes that restrict HIV replication function optimally in the absence of the HIV viral infectivity factor (Vif). Vif targets APOBEC3 for degradation via the proteasome in HIV infected cells. For APOBEC3s that are able to fortuitously escape Vif mediated degradation and become encapsidated, the enzymes can deaminate cytosines to form uracils in viral (-)DNA. Replication of (-)DNA to (+)DNA causes HIV-1 reverse transcriptase to incorporate adenines opposite uracils which creates C/G→T/A transition mutations. While restriction of HIV most commonly occurs through this deamination-dependent mechanism, APOBEC3s have also been identified to interfere with HIV reverse transcriptase processes in a deamination-independent manner, although this mechanism is secondary to the deaminationdependent mode. In order to restrict retroelements and viruses such as HIV, the A3 enzymes iii require an efficient processive ssDNA scanning mechanism that allows them to search for, and locate cytosines on ssDNA in the finite amount of time the substrate is available during formation of the double-stranded DNA provirus. To understand if this requirement was lost in A3 enzymes unable to restrict HIV, we studied A3C. Human A3C is not able to restrict HIV, in comparison to the more active gorilla and chimpanzee A3C orthologs that can restrict HIV, however an explanation for this difference was lacking. A polymorphism, S188I, in human A3C, which is found in less than 3% of the global population lead...

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HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

Cited by 204 publications

References 40 publications

Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Uncover the Mechanism of Nucleotide Import by HIV-1 Capsid

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

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