MMTV RNA packaging requires an extended long-range interaction for productive Gag binding to packaging signals.

The packaging of genomic RNA (gRNA) into retroviral particles relies on the specific recognition by the Gag precursor of packaging signals (Psi), which maintain a complex secondary structure through long-range interactions (LRIs). However, it remains unclear whether the binding of Gag to Psi alone is enough to promote RNA packaging and what role LRIs play in this process. Using mouse mammary tumor virus (MMTV), we investigated the effects of mutations in 4 proposed LRIs on gRNA structure and function. Our findings revealed the presence of an unsuspected extended LRI, and hSHAPE revealed that maintaining a wild-type-like Psi structure is crucial for efficient packaging. Surprisingly, filter-binding assays demonstrated that most mutants, regardless of their packaging capability, exhibited significant binding to Pr77Gag, suggesting that Gag binding to Psi is insufficient for efficient packaging. Footprinting experiments indicated that efficient RNA packaging is promoted when Pr77Gag binds to 2 specific sites within Psi, whereas binding elsewhere in Psi does not lead to efficient packaging. Taken together, our results suggest that the 3D structure of the Psi/Pr77Gag complex regulates the assembly of viral particles around gRNA, enabling effective discrimination against other viral and cellular RNAs that may also bind Gag efficiently.

A specific domain within the 3' untranslated region of Usutu virus confers resistance to the exonuclease ISG20.

Usutu virus (USUV) and West Nile virus (WNV) are two closely related emerging mosquito-borne flaviviruses. Their natural hosts are wild birds, but they can also cause severe neurological disorders in humans. Both viruses are efficiently suppressed by type I interferon (IFN), which interferes with viral replication, dissemination, pathogenesis and transmission. Here, we show that the replication of USUV and WNV are inhibited through a common set of IFN-induced genes (ISGs), with the notable exception of ISG20, which USUV is resistant to. Strikingly, USUV was the only virus among all the other tested mosquito-borne flaviviruses that demonstrated resistance to the 3'-5' exonuclease activity of ISG20. Our findings highlight that the intrinsic resistance of the USUV genome, irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a unique sequence present in its 3' untranslated region. Importantly, this genomic region alone can confer ISG20 resistance to a susceptible flavivirus, without compromising its infectivity, suggesting that it could be acquired by other flaviviruses. This study provides new insights into the strategy employed by emerging flaviviruses to overcome host defense mechanisms.

Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments.

Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.

Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging.

The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous regions within the 5' end of the gRNA that assumes a higher order structure harboring several structural motifs. To better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ sequences using genetic, biochemical, and structure-function relationship approaches. Our mutational analysis revealed that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput selective 2' hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences, with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained nucleotides 80-92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA-Pr50Gag interactions. Delineating sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of FIV-based vectors for human gene therapy.

The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses.

Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.

Structural maturation of the HIV-1 RNA 5' untranslated region by Pr55Gag and its maturation products.

Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55Gag precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55Gag or its maturation products (NCp15, NCp9 and NCp7) and the 5' gRNA region and their structural consequences, in vitro. We show that Pr55Gag and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55Gag maturation products on the gRNA structural maturation.

Chemical and Enzymatic Probing of Viral RNAs: From Infancy to Maturity and Beyond.

RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques.

A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag.

Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem-loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.

Identification of Pr78Gag Binding Sites on the Mason-Pfizer Monkey Virus Genomic RNA Packaging Determinants.

How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.

Role of Purine-Rich Regions in Mason-Pfizer Monkey Virus (MPMV) Genomic RNA Packaging and Propagation.

A distinguishing feature of the Mason-Pfizer monkey virus (MPMV) packaging signal RNA secondary structure is a single-stranded purine-rich sequence (ssPurines) in close vicinity to a palindromic stem loop (Pal SL) that functions as MPMV dimerization initiation site (DIS). However, unlike other retroviruses, MPMV contains a partially base-paired repeat sequence of ssPurines (bpPurines) in the adjacent region. Both purine-rich sequences have earlier been proposed to act as potentially redundant Gag binding sites to initiate the process of MPMV genomic RNA (gRNA) packaging. The objective of this study was to investigate the biological significance of ssPurines and bpPurines in MPMV gRNA packaging by systematic mutational and biochemical probing analyses. Deletion of either ssPurines or bpPurines individually had no significant effect on MPMV gRNA packaging, but it was severely compromised when both sequences were deleted simultaneously. Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) analysis of the mutant RNAs revealed only mild effects on structure by deletion of either ssPurines or bpPurines, while the structure was dramatically affected by the two simultaneous deletions. This suggests that ssPurines and bpPurines play a redundant role in MPMV gRNA packaging, probably as Gag binding sites to facilitate gRNA capture and encapsidation. Interestingly, the deletion of bpPurines revealed an additional severe defect on RNA propagation that was independent of the presence or absence of ssPurines or the gRNA structure of the region. These findings further suggest that the bpPurines play an additional role in the early steps of MPMV replication cycle that is yet to be identified.