Research Article: Toward the “unravelling” of HIV: Host cell factors involved in HIV-1 core uncoating

Date Published: October 4, 2018

Publisher: Public Library of Science

Author(s): Daniel J. Rawle, David Harrich, Matthew J. Evans.

http://doi.org/10.1371/journal.ppat.1007270

Abstract

Partial Text

Early HIV-1 replication requires fusion of viral and cellular membranes that release a fullerene-shaped viral core structure, formed by a lattice of primarily hexameric capsid (CA) rings, into the cellular cytoplasm. The core contains positive-polarity single-strand viral genomic RNA and enzymes that synthesize viral double-strand DNA that is integrated into a host chromosome. Only HIV-1 that release intact cores into the cytoplasm are infectious [1], and disassembly of the core occurs by a regulated process called uncoating. For the purpose of this article, we define uncoating as any dissociation of CA from the viral core. However, the precise mechanism, timing, and location of uncoating is contentious. Recent live-cell single-particle tracking of infectious HIV-1 cores has provided unprecedented insight into uncoating kinetics [2]. One such study found that core integrity, measured by “leakiness” of a core-trapped fluorescent marker, was lost in the cytoplasm approximately 30 min after fusion (after first-strand transfer of reverse transcription) and that this was required for productive infection [3]. However, any such loss of core integrity must not allow innate cellular sensors, such as cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS), to identify and restrict retroviral DNA in the cytoplasm [4]. Another recent study found that a small amount of CA dissociates gradually from the core post-entry before the remaining core structure docks at the nuclear pore complex (NPC), leading to accelerated CA loss, nuclear entry, integration of viral DNA into host DNA, and productive infection [5]. As these two studies measure different events and are not mutually exclusive, it is our view, consistent with that expressed in the review by Francis and Melikyan [2], that HIV-1 uncoating may begin by the loss of core integrity and gradual CA dissociation in the cytoplasm, followed by CA-dependent nuclear docking and accelerated CA loss at the NPC. An alternative model of uncoating is that an intact core arrives at the NPC before uncoating [1]. Here, we discuss the host cell factors subverted by HIV-1 to regulate ordered uncoating in cells (Fig 1), with the caveat that the spatiotemporal specifics of where and when host factors interact with the core and affect CA uncoating requires further investigation.

The cellular cytoskeleton is made up of filamentous actin, intermediate filaments, and microtubules, which function to maintain cell structural integrity and transport cargo within the cell. McDonald and colleagues used HIV-1 with green fluorescent protein (GFP) fused to the viral protein R (Vpr) to monitor HIV-1 trafficking from the cytoplasmic periphery to the nucleus. This revealed that replication complexes were associated with microtubules and accumulated near microtubule-organizing centers (MTOC) at the nuclear periphery [6]. In addition to facilitating cytoplasmic–nuclear trafficking of the core, the engagement of the host cytoskeleton by HIV-1 is important for regulated uncoating.

Eukaryotic translation elongation factor 1A (eEF1A) is a highly abundant protein primarily involved in delivering aa-tRNAs to the ribosomes during translation elongation, and our work has shown that eEF1A also binds tightly and directly with HIV-1 reverse transcriptase (RT) and is important for HIV-1 reverse transcription [16]. We showed that a point mutation of a single strictly conserved HIV-1 RT surface-exposed residue, E300R, reduced HIV-1 RT interaction with eEF1A, resulting in a delay in reverse transcription and uncoating kinetics in cells [17]. A panel of RT mutations was used to show that uncoating kinetics were not solely dependent on reverse transcription kinetics but instead correlated with the mutant RT interaction with eEF1A [17]. While the mechanism is unclear, we speculate that there are two possibilities: 1) virion-associated eEF1A [18] stimulates post-entry uncoating or 2) eEF1A in an infected cell binds RT in a partially uncoated core and stimulates further downstream CA dissociation up to two hours post-infection (hpi). However, our earlier study found the level of CA in cell fractions containing reverse transcription complexes (RTCs) at four hpi was reduced when cells were treated with an eEF1A-binding drug called didemnin B, indicating eEF1A stabilizes CA association with the RTCs later in infection [16]. The link between the efficiency of HIV-1 reverse transcription and the kinetics of core uncoating [19] suggests that multiple host cofactors may work in concert to coregulate reverse transcription and uncoating, and this is likely to be a fruitful research area. It is notable that eEF1A is also a cytoskeletal regulator, and the uncoating regulators Dia1 and Dia2 have an eEF1A-binding site [20], but it is not known if an eEF1A interaction with Dia1/2 is important for HIV-1 uncoating.

Peptidyl-prolyl isomerases (PPIases) are involved in protein folding by isomerising cis and trans isomers of proline residues in polypeptides. PPIase A, also known as cyclophilin A (CypA), is a multifunctional CA-binding host protein that is packaged into HIV-1 virions [21]. Several studies have found that CypA stabilizes HIV-1 cores in vitro [22–24], and cryogenic electron microscopy (cryoEM) shows that it does this by binding two CA molecules from adjacent hexamers [24]. This enhanced core stability prevents premature uncoating of HIV-1 cores in human CD4 expressing T cells and enables productive infection [22], possibly by preventing premature innate sensing of retroviral DNA in the cytoplasm.

Evidence is emerging that a small amount of HIV-1 CA remains associated with the viral replication complex for post-nuclear entry stages of replication. Host cell proteins that bind CA near or within the nucleus are required for nuclear import and may also be involved in disassembly of the CA that remains associated. The CA remaining with the pre-integration complex (PIC) can interact with proteins of the nuclear pore complex nucleoporin 358 and 153 (Nup358 and Nup153), and this is important for nuclear import of the PIC and final stages of uncoating regulation. Specifically, Nup358 has been shown to facilitate uncoating in cooperation with kinesin-1 in the cytoplasm [29], and Nup153 has been shown to prolong CA association with PICs in the nucleus [30].

Research investigating the mechanisms of host cell factor facilitation of viral replication is intensifying to better understand how viruses with limited genomic capacities can successfully complete their complex replication processes, and this may translate to novel therapeutic targets in the future. It is becoming clearer that HIV-1 uncoating is a complex process that is regulated by several host cell proteins, and investigating the regulatory mechanisms of these host proteins may provide insight into the elusive specificities of uncoating spatiotemporal regulation. Therapies that block or enhance these virus–host interactions may cause premature or delayed uncoating and have potential to be a new addition to the combination antiretroviral therapy for HIV-1 patients.

 

Source:

http://doi.org/10.1371/journal.ppat.1007270