Label-free and amplification-free viral RNA quantification from primate biofluids using a trapping-assisted optofluidic nanopore platform.

Viral outbreaks can cause widespread disruption, creating the need for diagnostic tools that provide high performance and sample versatility at the point of use with moderate complexity. Current gold standards such as PCR and rapid antigen tests fall short in one or more of these aspects. Here, we report a label-free and amplification-free nanopore sensor platform that overcomes these challenges via direct detection and quantification of viral RNA in clinical samples from a variety of biological fluids. The assay uses an optofluidic chip that combines optical waveguides with a fluidic channel and integrates a solid-state nanopore for sensing of individual biomolecules upon translocation through the pore. High specificity and low limit of detection are ensured by capturing RNA targets on microbeads and collecting them by optical trapping at the nanopore location where targets are released and rapidly detected. We use this device for longitudinal studies of the viral load progression for Zika and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections in marmoset and baboon animal models, respectively. The up to million-fold trapping-based target concentration enhancement enables amplification-free RNA quantification across the clinically relevant concentration range down to the assay limit of RT-qPCR as well as cases in which PCR failed. The assay operates across all relevant biofluids, including semen, urine, and whole blood for Zika and nasopharyngeal and throat swab, rectal swab, and bronchoalveolar lavage for SARS-CoV-2. The versatility, performance, simplicity, and potential for full microfluidic integration of the amplification-free nanopore assay points toward a unique approach to molecular diagnostics for nucleic acids, proteins, and other targets.

Significance of hepatitis B virus capsid dephosphorylation via polymerase.

BACKGROUND: It is generally believed that hepatitis B virus (HBV) core protein (HBc) dephosphorylation (de-P) is important for viral DNA synthesis and virion secretion. HBV polymerase contains four domains for terminal protein, spacer, reverse transcriptase, and RNase H activities. METHODS: HBV Polymerase mutants were transfected into HuH-7 cells and assayed for replication and HBc de-P by the Phos-tag gel analysis. Infection assay was performed by using a HepG2-NTCP-AS2 cell line. RESULTS: Here, we show that a novel phosphatase activity responsible for HBc de-P can be mapped to the C-terminal domain of the polymerase overlapping with the RNase H domain. Surprisingly, while HBc de-P is crucial for viral infectivity, it is essential for neither viral DNA synthesis nor virion secretion. The potential origin, significance, and mechanism of this polymerase-associated phosphatase activity are discussed in the context of an electrostatic homeostasis model. The Phos-tag gel analysis revealed an intriguing pattern of "bipolar distribution" of phosphorylated HBc and a de-P HBc doublet. CONCLUSIONS: It remains unknown if such a polymerase-associated phosphatase activity can be found in other related biosystems. This polymerase-associated phosphatase activity could be a druggable target in clinical therapy for hepatitis B.

Viroids, Satellite RNAs and Prions: Folding of Nucleic Acids and Misfolding of Proteins.

Theodor ("Ted") Otto Diener (* 28 February 1921 in Zürich, Switzerland; † 28 March 2023 in Beltsville, MD, USA) pioneered research on viroids while working at the Plant Virology Laboratory, Agricultural Research Service, USDA, in Beltsville. He coined the name viroid and defined viroids' important features like the infectivity of naked single-stranded RNA without protein-coding capacity. During scientific meetings in the 1970s and 1980s, viroids were often discussed at conferences together with other "subviral pathogens". This term includes what are now called satellite RNAs and prions. Satellite RNAs depend on a helper virus and have linear or, in the case of virusoids, circular RNA genomes. Prions, proteinaceous infectious particles, are the agents of scrapie, kuru and some other diseases. Many satellite RNAs, like viroids, are non-coding and exert their function by thermodynamically or kinetically controlled folding, while prions are solely host-encoded proteins that cause disease by misfolding, aggregation and transmission of their conformations into infectious prion isoforms. In this memorial, we will recall the work of Ted Diener on subviral pathogens.

Deciphering the Role of Post-Translational Modifications and Cellular Location of Hepatitis Delta Virus (HDV) Antigens in HDV-Mediated Liver Damage in Mice.

Hepatitis D virus (HDV) infection represents the most severe form of chronic viral hepatitis. We have shown that the delivery of HDV replication-competent genomes to the hepatocytes using adeno-associated virus (AAV-HDV) as gene delivery vehicles offers a unique platform to investigate the molecular aspects of HDV and associated liver damage. For the purpose of this study, we generated HDV genomes modified by site-directed mutagenesis aimed to (i) prevent some post-translational modifications of HDV antigens (HDAgs) such as large-HDAg (L-HDAg) isoprenylation or short-HDAg (S-HDAg) phosphorylation; (ii) alter the localization of HDAgs within the subcellular compartments; and (iii) inhibit the right conformation of the delta ribozyme. First, the different HDV mutants were tested in vitro using plasmid-transfected Huh-7 cells and then in vivo in C57BL/6 mice using AAV vectors. We found that Ser177 phosphorylation and ribozymal activity are essential for HDV replication and HDAg expression. Mutations of the isoprenylation domain prevented the formation of infectious particles and increased cellular toxicity and liver damage. Furthermore, altering HDAg intracellular localization notably decreased viral replication, though liver damage remained unchanged versus normal HDAg distribution. In addition, a mutation in the nuclear export signal impaired the formation of infectious viral particles. These findings contribute valuable insights into the intricate mechanisms of HDV biology and have implications for therapeutic considerations.

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.

A Balancing Act: The Viral-Host Battle over RNA Binding Proteins.

A defining feature of a productive viral infection is the co-opting of host cell resources for viral replication. Despite the host repertoire of molecular functions and biological counter measures, viruses still subvert host defenses to take control of cellular factors such as RNA binding proteins (RBPs). RBPs are involved in virtually all steps of mRNA life, forming ribonucleoprotein complexes (mRNPs) in a highly ordered and regulated process to control RNA fate and stability in the cell. As such, the hallmark of the viral takeover of a cell is the reshaping of RNA fate to modulate host gene expression and evade immune responses by altering RBP interactions. Here, we provide an extensive review of work in this area, particularly on the duality of the formation of RNP complexes that can be either pro- or antiviral. Overall, in this review, we highlight the various ways viruses co-opt RBPs to regulate RNA stability and modulate the outcome of infection by gathering novel insights gained from research studies in this field.

Host-like RNA Elements Regulate Virus Translation.

Viruses are obligate, intracellular parasites that co-opt host cell machineries for propagation. Critical among these machineries are those that translate RNA into protein and their mechanisms of control. Most regulatory mechanisms effectuate their activity by targeting sequence or structural features at the RNA termini, i.e., at the 5' or 3' ends, including the untranslated regions (UTRs). Translation of most eukaryotic mRNAs is initiated by 5' cap-dependent scanning. In contrast, many viruses initiate translation at internal RNA regions at internal ribosome entry sites (IRESs). Eukaryotic mRNAs often contain upstream open reading frames (uORFs) that permit condition-dependent control of downstream major ORFs. To offset genome compression and increase coding capacity, some viruses take advantage of out-of-frame overlapping uORFs (oORFs). Lacking the essential machinery of protein synthesis, for example, ribosomes and other translation factors, all viruses utilize the host apparatus to generate virus protein. In addition, some viruses exhibit RNA elements that bind host regulatory factors that are not essential components of the translation machinery. SARS-CoV-2 is a paradigm example of a virus taking advantage of multiple features of eukaryotic host translation control: the virus mimics the established human GAIT regulatory element and co-opts four host aminoacyl tRNA synthetases to form a stimulatory binding complex. Utilizing discontinuous transcription, the elements are present and identical in all SARS-CoV-2 subgenomic RNAs (and the genomic RNA). Thus, the virus exhibits a post-transcriptional regulon that improves upon analogous eukaryotic regulons, in which a family of functionally related mRNA targets contain elements that are structurally similar but lacking sequence identity. This "thrifty" virus strategy can be exploited against the virus since targeting the element can suppress the expression of all subgenomic RNAs as well as the genomic RNA. Other 3' end viral elements include 3'-cap-independent translation elements (3'-CITEs) and 3'-tRNA-like structures. Elucidation of virus translation control elements, their binding proteins, and their mechanisms can lead to novel therapeutic approaches to reduce virus replication and pathogenicity.

Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein.

The coordinated packaging of the segmented genome of the influenza A virus (IAV) into virions is an essential step of the viral life cycle. This process is controlled by the interaction of packaging signals present in all eight viral RNA (vRNA) segments and the viral nucleoprotein (NP), which binds vRNA via a positively charged binding groove. However, mechanistic models of how the packaging signals and NP work together to coordinate genome packaging are missing. Here, we studied genome packaging in influenza A/SC35M virus mutants that carry mutated packaging signals as well as specific amino acid substitutions at the highly conserved lysine (K) residues 184 and 229 in the RNA-binding groove of NP. Because these lysines are acetylated and thus neutrally charged in infected host cells, we replaced them with glutamine to mimic the acetylated, neutrally charged state or arginine to mimic the non-acetylated, positively charged state. Our analysis shows that the coordinated packaging of eight vRNAs is influenced by (i) the charge state of the replacing amino acid and (ii) its location within the RNA-binding groove. Accordingly, we propose that lysine acetylation induces different charge states within the RNA-binding groove of NP, thereby supporting the activity of specific packaging signals during coordinated genome packaging. IMPORTANCE: Influenza A viruses (IAVs) have a segmented viral RNA (vRNA) genome encapsidated by multiple copies of the viral nucleoprotein (NP) and organized into eight distinct viral ribonucleoprotein complexes. Although genome segmentation contributes significantly to viral evolution and adaptation, it requires a highly sophisticated genome-packaging mechanism. How eight distinct genome complexes are incorporated into the virion is poorly understood, but previous research suggests an essential role for both vRNA packaging signals and highly conserved NP amino acids. By demonstrating that the packaging process is controlled by charge-dependent interactions of highly conserved lysine residues in NP and vRNA packaging signals, our study provides new insights into the sophisticated packaging mechanism of IAVs.

Biochemical analysis of the host factor activity of ZCCHC14 in hepatitis A virus replication.

Relatively little is known of the mechanisms underlying hepatitis A virus (HAV) genome replication. Unlike other well-studied picornaviruses, HAV RNA replication requires the zinc finger protein ZCCHC14 and non-canonical TENT4 poly(A) polymerases with which it forms a complex. The ZCCHC14-TENT4 complex binds to a stem-loop located within the internal ribosome entry site (IRES) in the 5' untranslated RNA (5'UTR) and is essential for viral RNA synthesis, but the underlying mechanism is unknown. Here, we describe how different ZCCHC14 domains contribute to its RNA-binding, TENT4-binding, and HAV host factor activities. We show that the RNA-binding activity of ZCCHC14 requires both a sterile alpha motif (SAM) and a downstream unstructured domain (D4) and that ZCCHC14 contains two TENT4-binding sites: one at the N-terminus and the other around D4. Both RNA-binding and TENT4-binding are required for HAV host factor activity of ZCCHC14. We also demonstrate that the location of the ZCCHC14-binding site within the 5'UTR is critical for its function. Our study provides a novel insight into the function of ZCCHC14 and helps elucidate the mechanism of the ZCCHC14-TENT4 complex in HAV replication.IMPORTANCEThe zinc finger protein ZCCHC14 is an essential host factor for both hepatitis A virus (HAV) and hepatitis B virus (HBV). It recruits the non-canonical TENT4 poly(A) polymerases to viral RNAs and most likely also a subset of cellular mRNAs. Little is known about the details of these interactions. We show here the functional domains of ZCCHC14 that are involved in binding to HAV RNA and interactions with TENT4 and describe previously unrecognized peptide sequences that are critical for the HAV host factor activity of ZCCHC14. Our study advances the understanding of the ZCCHC14-TENT4 complex and how it functions in regulating viral and cellular RNAs.

Challenges to mapping and defining m6A function in viral RNA.

Viral RNA molecules contain multiple layers of regulatory information. This includes features beyond the primary sequence, such as RNA structures and RNA modifications, including N6-methyladenosine (m6A). Many recent studies have identified the presence and location of m6A in viral RNA and have found diverse regulatory roles for this modification during viral infection. However, to date, viral m6A mapping strategies have limitations that prevent a complete understanding of the function of m6A on individual viral RNA molecules. While m6A sites have been profiled on bulk RNA from many viruses, the resulting m6A maps of viral RNAs described to date present a composite picture of m6A across viral RNA molecules in the infected cell. Thus, for most viruses, it is unknown if unique viral m6A profiles exist throughout infection, nor if they regulate specific viral life cycle stages. Here, we describe several challenges to defining the function of m6A in viral RNA molecules and provide a framework for future studies to help in the understanding of how m6A regulates viral infection.

Bioinformatics Insights on Viral Gene Expression Transactivation: From HIV-1 to SARS-CoV-2.

Viruses provide vital insights into gene expression control. Viral transactivators, with other viral and cellular proteins, regulate expression of self, other viruses, and host genes with profound effects on infected cells, underlying inflammation, control of immune responses, and pathogenesis. The multifunctional Tat proteins of lentiviruses (HIV-1, HIV-2, and SIV) transactivate gene expression by recruiting host proteins and binding to transacting responsive regions (TARs) in viral and host RNAs. SARS-CoV-2 nucleocapsid participates in early viral transcription, recruits similar cellular proteins, and shares intracellular, surface, and extracellular distribution with Tat. SARS-CoV-2 nucleocapsid interacting with the replication-transcription complex might, therefore, transactivate viral and cellular RNAs in the transcription and reactivation of self and other viruses, acute and chronic pathogenesis, immune evasion, and viral evolution. Here, we show, by using primary and secondary structural comparisons, that the leaders of SARS-CoV-2 and other coronaviruses contain TAR-like sequences in stem-loops 2 and 3. The coronaviral nucleocapsid C-terminal domains harbor a region of similarity to TAR-binding regions of lentiviral Tat proteins, and coronaviral nonstructural protein 12 has a cysteine-rich metal binding, dimerization domain, as do lentiviral Tat proteins. Although SARS-CoV-1 nucleocapsid transactivated gene expression in a replicon-based study, further experimental evidence for coronaviral transactivation and its possible implications is warranted.

Knockout of SlDCL2b attenuates the resistance of tomato to potato spindle tuber viroid infection.

RNA interference, or RNA silencing, is an important defence mechanism against viroid infection in plants. Plants encode multiple DICER-LIKE (DCL) proteins that are key components of the RNA silencing pathway. However, the roles of different DCLs in defence responses against viroid infection remain unclear. Here, we determined the function of tomato DCL2b (SlDCL2b) in defence responses against potato spindle tuber viroid (PSTVd) infection using SlDCL2b loss-of-function tomato mutant plants. Compared with wild-type plants, mutant plants were more susceptible to PSTVd infection, developing more severe symptoms earlier and accumulating higher levels of PSTVd RNAs. Moreover, we verified the feedback mechanism for the regulation of SlDCL2b expression by miR6026. Functional blocking of tomato miR6026, by expressing its target mimics, can enhance resistance to PSTVd infection in tomato plants. These findings deepen the current understanding of RNAi-based resistance against viroid infection and provide a potentially new strategy for viroid control.

Neurofilament light chain and glial fibrillary acid protein levels are elevated in post-mild COVID-19 or asymptomatic SARS-CoV-2 cases.

Given the huge impact of the COVID-19 pandemic, it appears of paramount importance to assess the cognitive effects on the population returning to work after COVID-19 resolution. Serum levels of neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) represent promising biomarkers of neuro-axonal damage and astrocytic activation. In this cohort study, we explored the association between sNfL and sGFAP concentrations and cognitive performance in a group of 147 adult workers with a previous asymptomatic SARS-CoV-2 infection or mild COVID-19, one week and, in 49 of them, ten months after SARS-Cov2 negativization and compared them to a group of 82 age and BMI-matched healthy controls (HCs). sNfL and sGFAP concentrations were assessed using SimoaTM assay Neurology 2-Plex B Kit. COVID-19 patients were interviewed one-on-one by trained physicians and had to complete a list of questionnaires, including the Cognitive Failure Questionnaire (CFQ). At the first assessment (T0), sNfL and sGFAP levels were significantly higher in COVID-19 patients than in HCs (p < 0.001 for both). The eleven COVID-19 patients with cognitive impairment had significantly higher levels of sNfL and sGFAP than the others (p = 0.005 for both). At the subsequent follow-up (T1), sNfL and sGFAP levels showed a significant decrease (median sNfL 18.3 pg/mL; median sGFAP 77.2 pg/mL), although they were still higher than HCs (median sNfL 7.2 pg/mL, median sGFAP 63.5 pg/mL). Our results suggest an ongoing damage involving neurons and astrocytes after SARS-Cov2 negativization, which reduce after ten months even if still evident compared to HCs.

Loxapine inhibits replication of hepatitis A virus in vitro and in vivo by targeting viral protein 2C.

No antiviral drugs currently are available for treatment of infection by hepatitis A virus (HAV), a causative agent of acute hepatitis, a potentially life-threatening disease. Chemical screening of a small-compound library using nanoluciferase-expressing HAV identified loxapine succinate, a selective dopamine receptor D2 antagonist, as a potent inhibitor of HAV propagation in vitro. Loxapine succinate did not inhibit viral entry nor internal ribosome entry site (IRES)-dependent translation, but exhibited strong inhibition of viral RNA replication. Blind passage of HAV in the presence of loxapine succinate resulted in the accumulation of viruses containing mutations in the 2C-encoding region, which contributed to resistance to loxapine succinate. Analysis of molecular dynamics simulations of the interaction between 2C and loxapine suggested that loxapine binds to the N-terminal region of 2C, and that resistant mutations impede these interactions. We further demonstrated that administration of loxapine succinate to HAV-infected Ifnar1-/- mice (which lack the type I interferon receptor) results in decreases in the levels of fecal HAV RNA and of intrahepatic HAV RNA at an early stage of infection. These findings suggest that HAV protein 2C is a potential target for antivirals, and provide novel insights into the development of drugs for the treatment of hepatitis A.

Molecular and biological characterization of a novel partitivirus from Talaromyces pinophilus.

Talaromyces spp. have a worldwide distribution, are ecologically diverse and have been isolated from numerous different substrates. Talaromyces spp. are considered biotechnologically important due to their ability to produce a range of enzymes and pigments. Talaromyces pinophilus, belonging to genus Talaromyces and family Trichocomaceae, is known for producing several important bioactive metabolites. Here we report the isolation and characterisation of a partitivirus from T. pinophilus which we have nominated Talaromyces pinophilus partitivirus-1 (TpPV-1). TpPV-1 possesses a genome consisting of three double stranded (ds) RNA segments i.e., dsRNAs1-3, 1824 bp, 1638 bp and 1451 bp respectively, which are encapsidated in icosahedral particles 35 nm in diameter. Both dsRNA1 and dsRNA2 contain a single open reading frame (ORF) encoding respectively a 572 amino acid (aa) protein of 65 kDa and a 504 aa protein of 50 kDa. The third segment (dsRNA3) is potentially a satellite RNA. Phylogenetic analysis revealed that the TpPV-1 belongs to the family Partitiviridae in the proposed genus Zetapartitivirus. TpPV-1 infection decreases the mycelial growth rate of the host fungus and alters pigmentation as indicated by time course experiments performed on a range of different solid media comparing virus-infected and virus-free isogenic lines. This is the first report of mycovirus infection in T. pinophilus and may provide insights into understanding the effect of the mycovirus on the production of enzymes and pigments by the host fungus.

Hepatitis B virus RNAs co-opt ELAVL1 for stabilization and CRM1-dependent nuclear export.

Hepatitis B virus (HBV) chronically infects 296 million people worldwide, posing a major global health threat. Export of HBV RNAs from the nucleus to the cytoplasm is indispensable for viral protein translation and genome replication, however the mechanisms regulating this critical process remain largely elusive. Here, we identify a key host factor embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) that binds HBV RNAs and controls their nuclear export. Using an unbiased quantitative proteomics screen, we demonstrate direct binding of ELAVL1 to the HBV pregenomic RNA (pgRNA). ELAVL1 knockdown inhibits HBV RNAs posttranscriptional regulation and suppresses viral replication. Further mechanistic studies reveal ELAVL1 recruits the nuclear export receptor CRM1 through ANP32A and ANP32B to transport HBV RNAs to the cytoplasm via specific AU-rich elements, which can be targeted by a compound CMLD-2. Moreover, ELAVL1 protects HBV RNAs from DIS3+RRP6+ RNA exosome mediated nuclear RNA degradation. Notably, we find HBV core protein is dispensable for HBV RNA-CRM1 interaction and nuclear export. Our results unveil ELAVL1 as a crucial host factor that regulates HBV RNAs stability and trafficking. By orchestrating viral RNA nuclear export, ELAVL1 is indispensable for the HBV life cycle. Our study highlights a virus-host interaction that may be exploited as a new therapeutic target against chronic hepatitis B.

Contrasting functions of ATP hydrolysis by MDA5 and LGP2 in viral RNA sensing.

Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiation-associated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-ß responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes.

HIV-1 RNA genome packaging: it's G-rated.

A member of the Retroviridae, human immunodeficiency virus type 1 (HIV-1), uses the RNA genome packaged into nascent virions to transfer genetic information to its progeny. The genome packaging step is a highly regulated and extremely efficient process as a vast majority of virus particles contain two copies of full-length unspliced HIV-1 RNA that form a dimer. Thus, during virus assembly HIV-1 can identify and selectively encapsidate HIV-1 unspliced RNA from an abundant pool of cellular RNAs and various spliced HIV-1 RNAs. Several "G" features facilitate the packaging of a dimeric RNA genome. The viral polyprotein Gag orchestrates virus assembly and mediates RNA genome packaging. During this process, Gag preferentially binds unpaired guanosines within the highly structured 5' untranslated region (UTR) of HIV-1 RNA. In addition, the HIV-1 unspliced RNA provides a scaffold that promotes Gag:Gag interactions and virus assembly, thereby ensuring its packaging. Intriguingly, recent studies have shown that the use of different guanosines at the junction of U3 and R as transcription start sites results in HIV-1 unspliced RNA species with 99.9% identical sequences but dramatically distinct 5' UTR conformations. Consequently, one species of unspliced RNA is preferentially packaged over other nearly identical RNAs. These studies reveal how conformations affect the functions of HIV-1 RNA elements and the complex regulation of HIV-1 replication. In this review, we summarize cis- and trans-acting elements critical for HIV-1 RNA packaging, locations of Gag:RNA interactions that mediate genome encapsidation, and the effects of transcription start sites on the structure and packaging of HIV-1 RNA.

Visualization of Early RNA Replication Kinetics of SARS-CoV-2 by Using Single Molecule RNA-FISH Combined with Immunofluorescence.

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19.

Genomic characterization of a novel cytorhabdovirus infecting Ixeris denticulata in China.

Ixeris denticulata is a perennial herbal plant with important medical and economic value. In this study, a novel rhabdovirus from I. denticulata with leaf curling and mottle symptoms was identified through next-generation sequencing and molecular cloning approaches. Based on the host species and properties of this virus, it was tentatively named "Ixeris denticulata-associated rhabdovirus" (IdaRV). IdaRV has a negative-sense RNA genome that is 12,705 nucleotides in length and has five open reading frames (ORFs) in the order 3'-nucleoprotein -phosphoprotein -movement protein -matrix protein -large RNA-dependent RNA polymerase-5'. Pairwise sequence comparisons showed that IdaRV had 42.2-53.0% sequence identity to members of the genera Cytorhabdovirus, Varicosavirus, Betanucleorhabdovirus, Gammanucleorhabdovirus, Dichorhavirus, and Alphanucleorhabdovirus in the subfamily Betarhabdovirinae. BLASTp searches indicated that putative products of ORF1, ORF2, ORF3, ORF4, and ORF5 of IdaRV are most closely related to those of rudbeckia virus 1 (RudV1, GenBank accession number ON185810), with 32.1%, 21.3%, 52.4%, 37.6%, and 57.1% amino acid sequence identity, respectively, at the protein level. Phylogenetic analysis showed that IdaRV forms a smaller branch with RudV1, which belongs to the genus Cytorhabdovirus. These results establish IdaRV as a novel rhabdovirus in the genus Cytorhabdovirus of the family Rhabdoviridae.