A novel role for gag as a cis-acting element regulating RNA structure, dimerization and packaging in HIV-1 lentiviral vectors.

Clinical usage of lentiviral vectors is now established and increasing but remains constrained by vector titer with RNA packaging being a limiting factor. Lentiviral vector RNA is packaged through specific recognition of the packaging signal on the RNA by the viral structural protein Gag. We investigated structurally informed modifications of the 5' leader and gag RNA sequences in which the extended packaging signal lies, to attempt to enhance the packaging process by facilitating vector RNA dimerization, a process closely linked to packaging. We used in-gel SHAPE to study the structures of these mutants in an attempt to derive structure-function correlations that could inform optimized vector RNA design. In-gel SHAPE of both dimeric and monomeric species of RNA revealed a previously unreported direct interaction between the U5 region of the HIV-1 leader and the downstream gag sequences. Our data suggest a structural equilibrium exists in the dimeric viral RNA between a metastable structure that includes a U5-gag interaction and a more stable structure with a U5-AUG duplex. Our data provide clarification for the previously unexplained requirement for the 5' region of gag in enhancing genomic RNA packaging and provide a basis for design of optimized HIV-1 based vectors.

HIV-1 Packaging Visualised by In-Gel SHAPE.

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA-protein complex. Here, we apply the in-gel SHAPE (selective 2'OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag-dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.

Evaluating RNA Structural Flexibility: Viruses Lead the Way.

Our understanding of RNA structure has lagged behind that of proteins and most other biological polymers, largely because of its ability to adopt multiple, and often very different, functional conformations within a single molecule. Flexibility and multifunctionality appear to be its hallmarks. Conventional biochemical and biophysical techniques all have limitations in solving RNA structure and to address this in recent years we have seen the emergence of a wide diversity of techniques applied to RNA structural analysis and an accompanying appreciation of its ubiquity and versatility. Viral RNA is a particularly productive area to study in that this economy of function within a single molecule admirably suits the minimalist lifestyle of viruses. Here, we review the major techniques that are being used to elucidate RNA conformational flexibility and exemplify how the structure and function are, as in all biology, tightly linked.

Duplex formation between the template and the nascent strand in the transcription-regulating sequences is associated with the site of template switching in SARS - CoV-2.

Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis, where the polymerase switches template from a 3' proximal genome body sequence to a 5' untranslated leader sequence. This leads to a fusion between the common 5' leader sequence and a 3' proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesizing the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs. We speculate on the effect of reported variant nucleotide substitutions on our models.

EAP45 association with budding HIV-1: Kinetics and domain requirements.

A number of viruses including HIV use the ESCRT system to bud from the infected cell. We have previously confirmed biochemically that ESCRT-II is involved in this process in HIV-1 and have defined the molecular domains that are important for this. Here, using SNAP-tag fluorescent labelling and both fixed and live cell imaging we show that the ESCRT-II component EAP45 colocalises with the HIV protein Gag at the plasma membrane in a temporal and quantitative manner, similar to that previously shown for ALIX and Gag. We show evidence that a proportion of EAP45 may be packaged within virions, and we confirm the importance of the N terminus of EAP45 and specifically the H0 domain in this process. By contrast, the Glue domain of EAP45 is more critical for recruitment during cytokinesis, emphasising that viruses have ways of recruiting cellular components that may be distinct from those used by some cellular processes. This raises the prospect of selective interference with the pathway to inhibit viral function while leaving cellular functions relatively unperturbed.

Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the Alpha variant B.1.1.7.

We report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of receptor binding motif replacements such as N439K and Y453F, known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that, although ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of spike proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of the H69 and V70 residues in the Alpha variant B.1.1.7 spike (where ΔH69/V70 occurs naturally) impairs spike incorporation and entry efficiency of the B.1.1.7 spike pseudotyped virus. Alpha variant B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild-type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.

Development of a Novel Competitive qRT-PCR Assay to Measure Relative Lentiviral Packaging Efficiency.

Third-generation HIV-1-derived lentiviral vectors are successfully used as therapeutic agents in various clinical applications. To further promote their use, we attempted to enhance vector infectivity by targeting the dimerization and packaging properties of the RNA transfer vector based on the premise that these two processes are tightly linked. We rationally designed mutant vectors to favor the dimeric conformation, potentially enhancing genome packaging. Initial assessments using standard assays generated outputs of variable reproducibility, sometimes with conflicting results. Therefore, we developed a novel competitive qRT-PCR assay in a co-transfection setting to measure the relative packaging efficiencies of wild-type and mutant transfer vectors. Here we report the effect of the dimerization-stabilizing mutations on infectious and physical titers of lentiviral vectors together with their packaging efficiency, measured using our novel assay. Enhancing dimerization did not automatically lead to better vector RNA packaging, suggesting that, for vector functionality, sufficient flexibility of the RNA to adopt different conformations is more important than the dimerization capacity. Our novel competitive qPCR assay enables a more stringent analysis of RNA packaging efficiency, allowing a much more precise understanding of the links between RNA structure, packaging, and infectious titers that will be invaluable for future vector development.

ESCRT-II functions by linking to ESCRT-I in human immunodeficiency virus-1 budding.

Human immunodeficiency virus (HIV) uses the ESCRT (endosomal sorting complexes required for transport) protein pathway to bud from infected cells. Despite the roles of ESCRT-I and -III in HIV budding being firmly established, participation of ESCRT-II in this process has been controversial. EAP45 is a critical component of ESCRT-II. Previously, we utilised a CRISPR-Cas9 EAP45 knockout cell line to assess the involvement of ESCRT-II in HIV replication. We demonstrated that the absence of ESCRT-II impairs HIV budding. Here, we show that virus spread is also defective in physiologically relevant CRISPR/Cas9 EAP45 knockout T cells. We further show reappearance of efficient budding by re-introduction of EAP45 expression into EAP45 knockout cells. Using expression of selected mutants of EAP45, we dissect the domain requirement responsible for this function. Our data show at the steady state that rescue of budding is only observed in the context of a Gag/Pol, but not a Gag expressor, indicating that the size of cargo determines the usage of ESCRT-II. EAP45 acts through the YPXL-ALIX pathway as partial rescue is achieved in a PTAP but not a YPXL mutant virus. Our study clarifies the role of ESCRT-II in the late stages of HIV replication and reinforces the notion that ESCRT-II plays an integral part during this process as it does in sorting ubiquitinated cargos and in cytokinesis.