Toward a Taxonomy of Virus-ncRNAs Interactions in an RNA World for Disentangling Some Tiny Secrets of Dengue Virus

In recent years, the role of non-coding RNAs (ncRNAs) in regulating cell physiology has begun to be better understood. Recent discoveries in viral molecular biology have revealed that such cellular functions are disturbed during viral infections mainly due to host cell ncRNAs, cellular factors, and virus-derived ncRNAs. Apart from the interplay between those molecules, other interactions derive from the specific folding of RNA virus genomes. These fulfill canonical regulation functions such as replication, translation, and viral packaging. In some cases, folds serve as precursors of small viral RNAs whose biogenesis is not yet clearly understood. Since ncRNAs and RNA viral genomes modulate complex molecular and cellular processes in viral infections, a new taxonomy is being proposed here overarching three main categories, considering the current information about ncRNA interactions in some well-known viral infections. The first category shows examples of host ncRNAs associated with the trigger of the immune response under viral infections. The second category describes interactions between the virus and host ncRNAs. The last category shows how the shape of the RNA viral genome is essential in processing RNAs derived from viruses. Finally, we introduce evidence of how these three categories can also work as a framework in order to organize known interactions of ncRNAs and cellular factors under DENV infection. This new taxonomy of interactions provides a comprehensive framework for organizing the ncRNA regulatory roles in the context of viral interactions and an RNA world.

Wild-type HIV infection after treatment with lentiviral gene therapy for β-thalassemia

Betibeglogene autotemcel (beti-cel) gene therapy (GT) for patients with transfusion-dependent β-thalassemia uses autologous CD34+ cells transduced with BB305 lentiviral vector (LVV), which encodes a modified β-globin gene. BB305 LVV also contains select HIV sequences for viral packaging, reverse transcription, and integration. This case report describes a patient successfully treated with beti-cel in a phase 1/2 study (HGB-204; #NCT01745120) and subsequently diagnosed with wild-type (WT) HIV infection. From 3.5 to 21 months postinfusion, the patient stopped chronic red blood cell transfusions; total hemoglobin (Hb) and GT-derived HbAT87Q levels were 6.6 to 9.5 and 2.8 to 3.8 g/dL, respectively. At 21 months postinfusion, the patient resumed transfusions for anemia that coincided with an HIV-1 infection diagnosis. Quantitative polymerase chain reaction assays detected no replication-competent lentivirus. Next-generation sequencing confirmed WT HIV sequences. Six months after starting antiretroviral therapy, total Hb and HbAT87Q levels recovered to 8.6 and 3.6 g/dL, respectively, and 3.5 years postinfusion, 13.4 months had elapsed since the patient’s last transfusion. To our knowledge, this is the first report of WT HIV infection in an LVV-based GT recipient and demonstrates persistent long-term hematopoiesis after treatment with beti-cel and the ability to differentiate between WT HIV and BB305-derived sequences.

Viral packaging ATPases utilize a glutamate switch to couple ATPase activity and DNA translocation

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors.

Viral Packaging ATPases Utilize a Glutamate Switch to Couple ATPase Activity and DNA Translocation

SummaryMany viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in apo, ATP-bound, and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an inactive to an active pose upon ATP binding, and that a residue assigned as the glutamate switch is necessary for regulating the transition. Further, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental data. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the predicted structural coupling from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway in viral DNA packaging motors that ensures coordination between chemical and mechanical events involved in viral DNA packaging.

CRISPR-guided programmable self-assembly of artificial virus-like nucleocapsids

Designer virus-inspired proteins drive the manufacturing of more effective and safer gene-delivery systems as well as simpler models to study viral assembly. However, the self-assembly of engineered viromimetic proteins on specific nucleic acid templates, a distinctive viral property, has proved difficult. Inspired by viral packaging signals, we harness the programmability of CRISPR-Cas12a to direct the nucleation and growth of a self-assembling synthetic polypeptide into virus-like particles (VLP) on specific DNA molecules. Positioning up to ten nuclease-dead Cas12a (dCas12a) proteins along a 48.5 kbp DNA template triggers particle growth and full DNA encapsidation at limiting polypeptide concentrations. Particle growth rate was further increased when dCas12a was dimerized with a polymerization silk-like domain. Such improved self-assembly efficiency allows for discrimination between cognate versus non-cognate DNA templates by the synthetic polypeptide. Our CRISPR-guided VLPs could help develop programmable bio-inspired nanomaterials with applications in biotechnology as well as viromimetic scaffolds to improve our understanding of viral self-assembly.

Evolving Infection Paradox of SARS-CoV-2: Fitness Costs Virulence?

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is continuously spreading worldwide at an unprecedented scale in 2020. Within the first six months of the COVID-19 pandemic, it has evolved into six clades according to GISAID where three (G, GH, and GR) are now globally prevalent (>75%). Here we report the prevalence of these dominant clades, both individually and in combination, with disease progression and death-case scenario that leads to infer fitness of the SARS-CoV-2 by compromising its virulence. Unlike G or GH clades, the GR clade strains represent a significant negative association with the death-case ratio (R= -0.558, p=0.019). Docking analysis revealed the molecular scenario behind more infectiousness of S protein D614G mutation and reasoned more favorable binding of G614 with the elastase-2. Viral RNA-dependent-RNA-polymerase (RdRp) mutation p.P323L facilitated significantly higher (p<0.0001) genome-wide mutations because more flexible RdRp (mutant)-NSP8 interaction may accelerate replication. Superior RNA stability and structural variation at NSP3:C241T might change the protein’s conformation with a speculated impact on 5'UTR, nucleocapsid, and replication complex interactions. Another silent 5'UTR:C241T mutation might affect translational efficiency and viral packaging. These G-featured coevolving mutations might together increase the viral load, quicker cell death, and potentially a stronger immune response within the host, hence can modulate intra-host genomic plasticity. In addition, viroporin ORF3a:p.Q57H mutation of GH-clade prevents ion permeability by constricting the channel pore more tightly due to additional ionic interaction with the cysteine (C81) of transmembrane-domain-2, which possibly reduces viral release and immune response. GR strains (four G clade mutations with N:p.RG203-204KR) would have maintained more stability with stronger RNA interaction, a flexible linker region, and the molecular effect of hypo-phosphorylation at SR-stretch. These empirical assumptions need further retrospective and prospective studies to understand detailed molecular and evolutionary events featuring the fitness and virulence of SARS-CoV-2.

Generation of a neutralizing antibody against RD114-pseudotyped viral vectors

The feline endogenous RD114 glycoprotein has proved to be an attractive envelope to pseudotype both retroviral and lentiviral vectors. As a surface protein, its detection on packaging cells as well as viral particles would be useful in different fields of its use. To address this, we generated a monoclonal antibody against RD114 by immunization of rats, termed 22F10. Once seroconversion was confirmed, purified 22F10 was cloned into murine Fc and characterized with a binding affinity of 10nM. The antibody was used to detect RD114 and its variant envelopes on different stable viral packaging cell lines (FLYRD18 and WinPac-RD). 22F10 was also shown to prevent the infections of different strains of RD-pseudotyped vectors but not related envelope glycoproteins by blocking cell surface receptor binding. We are the first to report the neutralization of viral particles by a monoclonal αRD114 antibody.

Competition between social cheater viruses is driven by mechanistically different cheating strategies

Cheater viruses, also known as defective interfering viruses, cannot replicate on their own yet replicate faster than the wild type upon coinfection. While there is growing interest in using cheaters as antiviral therapeutics, the mechanisms underlying cheating have been rarely explored. During experimental evolution of MS2 phage, we observed the parallel emergence of two independent cheater mutants. The first, a point deletion mutant, lacked polymerase activity but was advantageous in viral packaging. The second synonymous mutant cheater displayed a completely different cheating mechanism, involving an altered RNA structure. Continued evolution revealed the demise of the deletion cheater and rise of the synonymous cheater. A mathematical model inferred that while a single cheater is expected to reach an equilibrium with the wild type, cheater demise arises from antagonistic interactions between coinfecting cheaters. These findings highlight layers of parasitism: viruses parasitizing cells, cheaters parasitizing intact viruses, and cheaters may parasitize other cheaters.

A pVII positioning code determines the dynamics of Adenoviral nucleoprotein structure and primes it for early gene activation

SUMMARYInside the capsid adenovirus DNA is associated with the structural protein pVII. However, the viral DNA organisation, pVII positioning and the dynamic changes of viral packaging upon infection, to form a transcriptional active genome, are not known. We combined MNase-Seq and single genome imaging during early infection to provide the structure and time resolved dynamics of viral chromatin changes, correlated with gene transcription. pVII complexes form nucleosome-like arrays, being precisely positioned on DNA, creating a defined and unique adenoviral nucleoprotein-architecture. The structure renders the viral genome transcription competent with lower pVII densities at early gene loci, correlating with viral chromatin de-condensation upon infection. Nucleosomes specifically replace pVII at transcription start sites of early genes, preceding transcriptional activation. Our study suggests an underlying regulatory pVII nucleoprotein-architecture, required for the dynamic changes during early infection, including transcription related nucleosome assembly. We suggest that our study provides a basis for the development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy.

Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

A mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5’-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5’-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.