Mechanistic Insights Into an Ancient Adenovirus Precursor Protein VII Show Multiple Nuclear Import Receptor Pathways.

Adenoviral pVII proteins are multifunctional, highly basic, histone-like proteins that can bind to and transport the viral genome into the host cell nucleus. Despite the identification of several nuclear localization signals (NLSs) in the pVII protein of human adenovirus (HAdV)2, the mechanistic details of nuclear transport are largely unknown. Here we provide a full characterization of the nuclear import of precursor (Pre-) pVII protein from an ancient siadenovirus, frog siadenovirus 1 (FrAdV1), using a combination of structural, functional, and biochemical approaches. Two strong NLSs (termed NLSa and NLSd) interact with importin (IMP)ß1 and IMPα, respectively, and are the main drivers of nuclear import. A weaker NLS (termed NLSb) also contributes, together with an additional signal (NLSc) which we found to be important for nucleolar targeting and intranuclear binding. Expression of wild-type and NLS defective derivatives Pre-pVII in the presence of selective inhibitors of different nuclear import pathways revealed that, unlike its human counterpart, FrAdV1 Pre-pVII nuclear import is dependent on IMPα/ß1 and IMPß1, but not on transportin-1 (IMPß2). Clearly, AdVs evolved to maximize the nuclear import pathways for the pVII proteins, whose subcellular localization is the result of a complex process. Therefore, our results pave the way for an evolutionary comparison of the interaction of different AdVs with the host cell nuclear transport machinery.

Single-Molecule Visualization of Bunyavirus Genome Segments Using Fluorescence In Situ Hybridization.

The genome of most bunyaviruses is divided over three (S, M, and L) single-stranded RNA segments of negative polarity. The three viral RNA segments are essential to establish a productive infection. RNA fluorescence in situ hybridization (FISH) enables the detection, localization, and quantification of RNA molecules at single-molecule resolution. This chapter describes an RNA FISH method to directly visualize individual segment-specific bunyavirus RNAs in fixed infected cells and in mature virus particles, using Rift Valley fever virus as an example. Imaging of bunyavirus RNA segments is a valuable experimental tool to investigate fundamental aspects of the bunyavirus life cycle, such as virus replication, genome packaging, and virion assembly, among others.

Rotavirus NSP2: A Master Orchestrator of Early Viral Particle Assembly.

Rotaviruses (RVs) are 11-segmented, double-stranded (ds) RNA viruses and important causes of acute gastroenteritis in humans and other animal species. Early RV particle assembly is a multi-step process that includes the assortment, packaging and replication of the 11 genome segments in close connection with capsid morphogenesis. This process occurs inside virally induced, cytosolic, membrane-less organelles called viroplasms. While many viral and cellular proteins play roles during early RV assembly, the octameric nonstructural protein 2 (NSP2) has emerged as a master orchestrator of this key stage of the viral replication cycle. NSP2 is critical for viroplasm biogenesis as well as for the selective RNA-RNA interactions that underpin the assortment of 11 viral genome segments. Moreover, NSP2's associated enzymatic activities might serve to maintain nucleotide pools for use during viral genome replication, a process that is concurrent with early particle assembly. The goal of this review article is to summarize the available data about the structures, functions and interactions of RV NSP2 while also drawing attention to important unanswered questions in the field.

Involvement of ribosomal protein L17 and Y-box binding protein 1 in the assembly of hepatitis C virus potentially via their interaction with the 3' untranslated region of the viral genome.

The 3' untranslated region (3'UTR) of the hepatitis C virus (HCV) RNA genome, which contains a highly conserved 3' region named the 3'X-tail, plays an essential role in RNA replication and promotes viral IRES-dependent translation. Although our previous work has found a cis-acting element for genome encapsidation within 3'X, there is limited information on the involvement of the 3'UTR in particle formation. In this study, proteomic analyses identified host cell proteins that bind to the 3'UTR containing the 3'X region but not to the sequence lacking the 3'X. Further characterization showed that RNA-binding proteins, ribosomal protein L17 (RPL17), and Y-box binding protein 1 (YBX1) facilitate the efficient production of infectious HCV particles in the virus infection cells. Using small interfering RNA (siRNA)-mediated gene silencing in four assays that distinguish between the various stages of the HCV life cycle, RPL17 and YBX1 were found to be most important for particle assembly in the trans-packaging assay with replication-defective subgenomic RNA. In vitro assays showed that RPL17 and YBX1 bind to the 3'UTR RNA and deletion of the 3'X region attenuates their interaction. Knockdown of RPL17 or YBX1 resulted in reducing the amount of HCV RNA co-precipitating with the viral Core protein by RNA immunoprecipitation and increasing the relative distance in space between Core and double-stranded RNA by confocal imaging, suggesting that RPL17 and YBX1 potentially affect HCV RNA-Core interaction, leading to efficient nucleocapsid assembly. These host factors provide new clues to understanding the molecular mechanisms that regulate HCV particle formation. IMPORTANCE: Although basic research on the HCV life cycle has progressed significantly over the past two decades, our understanding of the molecular mechanisms that regulate the process of particle formation, in particular encapsidation of the genome or nucleocapsid assembly, has been limited. We present here, for the first time, that two RNA-binding proteins, RPL17 and YBX1, bind to the 3'X in the 3'UTR of the HCV genome, which potentially acts as a packaging signal, and facilitates the viral particle assembly. Our study revealed that RPL17 and YBX1 exert a positive effect on the interaction between HCV RNA and Core protein, suggesting that the presence of both host factors modulate an RNA structure or conformation suitable for packaging the viral genome. These findings help us to elucidate not only the regulatory mechanism of the particle assembly of HCV but also the function of host RNA-binding proteins during viral infection.

Single-Virion Analysis: A Method to Visualize HIV-1 Particle Content Using Fluorescence Microscopy.

HIV-1 virions incorporate viral RNA, cellular RNAs, and proteins during the assembly process. Some of these components, such as the viral RNA genome and viral proteins, are essential for viral replication, whereas others, such as host innate immune proteins, can inhibit virus replication. Therefore, analyzing the virion content is an integral part of studying HIV-1 replication. Traditionally, virion contents have been examined using biochemical assays, which can provide information on the presence or absence of the molecule of interest but not its distribution in the virion population. Here, we describe a method, single-virion analysis, that directly examines the presence of molecules of interest in individual viral particles using fluorescence microscopy. Thus, this method can detect both the presence and the distribution of molecules of interest in the virion population. Single-virion analysis was first developed to study HIV-1 RNA genome packaging. In this assay, HIV-1 unspliced RNA is labeled with a fluorescently tagged RNA-binding protein (protein A) and some of the Gag proteins are labeled with a different fluorescent protein (protein B). Using fluorescence microscopy, HIV-1 particles can be identified by the fluorescent protein B signal and the presence of unspliced HIV-1 RNA can be identified by the fluorescent protein A signal. Therefore, the proportions of particles that contain unspliced RNA can be determined by the fraction of Gag particles that also have a colocalized RNA signal. By tagging the molecule of interest with fluorescent proteins, single-virion analysis can be easily adapted to study the incorporation of other viral or host cell molecules into particles. Indeed, this method has been adapted to examine the proportion of HIV-1 particles that contain APOBEC3 proteins and the fraction of particles that contain a modified Gag protein. Therefore, single-virion analysis is a flexible method to study the nucleic acid and protein content of HIV-1 particles.

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.

Mapping of the influenza A virus genome RNA structure and interactions reveals essential elements of viral replication.

Influenza A virus (IAV) represents a constant public health threat. The single-stranded, segmented RNA genome of IAV is replicated in host cell nuclei as a series of 8 ribonucleoprotein complexes (vRNPs) with RNA structures known to exert essential function to support viral replication. Here, we investigate RNA secondary structures and RNA interactions networks of the IAV genome and construct an in vivo structure model for each of the 8 IAV genome segments. Our analyses reveal an overall in vivo and in virio resemblance of the IAV genome conformation but also wide disparities among long-range and intersegment interactions. Moreover, we identify a long-range RNA interaction that exerts an essential role in genome packaging. Disrupting this structure displays reduced infectivity, attenuating virus pathogenicity in mice. Our findings characterize the in vivo RNA structural landscape of the IAV genome and reveal viral RNA structures that can be targeted to develop antiviral interventions.

Decoding cellular mechanism of recombinant adeno-associated virus (rAAV) and engineering host-cell factories toward intensified viral vector manufacturing.

Recombinant adeno-associated virus (rAAV) is one of the prominent gene delivery vehicles that has opened promising opportunities for novel gene therapeutic approaches. However, the current major viral vector production platform, triple transfection in mammalian cells, may not meet the increasing demand. Thus, it is highly required to understand production bottlenecks from the host cell perspective and engineer the cells to be more favorable and tolerant to viral vector production, thereby effectively enhancing rAAV manufacturing. In this review, we provided a comprehensive exploration of the intricate cellular process involved in rAAV production, encompassing various stages such as plasmid entry to the cytoplasm, plasmid trafficking and nuclear delivery, rAAV structural/non-structural protein expression, viral capsid assembly, genome replication, genome packaging, and rAAV release/secretion. The knowledge in the fundamental biology of host cells supporting viral replication as manufacturing factories or exhibiting defending behaviors against viral production is summarized for each stage. The control strategies from the perspectives of host cell and materials (e.g., AAV plasmids) are proposed as our insights based on the characterization of molecular features and our existing knowledge of the AAV viral life cycle, rAAV and other viral vector production in the Human embryonic kidney (HEK) cells.

Bipartite viral RNA genome heterodimerization influences genome packaging and virion thermostability.

Multi-segmented viruses often multimerize their genomic segments to ensure efficient and stoichiometric packaging of the correct genetic cargo. In the bipartite Nodaviridae family, genome heterodimerization is also observed and conserved among different species. However, the nucleotide composition and biological function for this heterodimer remain unclear. Using Flock House virus as a model system, we developed a next-generation sequencing approach ("XL-ClickSeq") to probe heterodimer site sequences. We identified an intermolecular base-pairing site which contributed to heterodimerization in both wild-type and defective virus particles. Mutagenic disruption of this heterodimer site exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNAs. Furthermore, the disruption of this intermolecular interaction directly impacts the thermostability of the mature virions. These results demonstrate that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus have an important role on virus particle integrity and thus may impact its transmission to a new host.IMPORTANCEFlock House virus is a member of Nodaviridae family of viruses, which provides a well-studied model virus for non-enveloped RNA virus assembly, cell entry, and replication. The Flock House virus genome consists of two separate RNA molecules, which can form a heterodimer upon heating of virus particles. Although similar RNA dimerization is utilized by other viruses (such as retroviruses) as a packaging mechanism and is conserved among Nodaviruses, the role of heterodimerization in the Nodavirus replication cycle is unclear. In this research, we identified the RNA sequences contributing to Flock House virus genome heterodimerization and discovered that such RNA-RNA interaction plays an essential role in virus packaging efficiency and particle integrity. This provides significant insight into how the interaction of packaged viral RNA may have a broader impact on the structural and functional properties of virus particles.

Structural and computational studies of HIV-1 RNA.

Viruses remain a global threat to animals, plants, and humans. The type 1 human immunodeficiency virus (HIV-1) is a member of the retrovirus family and carries an RNA genome, which is reverse transcribed into viral DNA and further integrated into the host-cell DNA for viral replication and proliferation. The RNA structures from the HIV-1 genome provide valuable insights into the mechanisms underlying the viral replication cycle. Moreover, these structures serve as models for designing novel therapeutic approaches. Here, we review structural data on RNA from the HIV-1 genome as well as computational studies based on these structural data. The review is organized according to the type of structured RNA element which contributes to different steps in the viral replication cycle. This is followed by an overview of the HIV-1 transactivation response element (TAR) RNA as a model system for understanding dynamics and interactions in the viral RNA systems. The review concludes with a description of computational studies, highlighting the impact of biomolecular simulations in elucidating the mechanistic details of various steps in the HIV-1's replication cycle.

Packing up the genome.

Nucleotide and force-dependent mechanisms control how the viral genome of lambda bacteriophage is inserted into capsids.

The genome of a bunyavirus cannot be defined at the level of the viral particle but only at the scale of the viral population.

Bunyaviruses are enveloped negative or ambisense single-stranded RNA viruses with a genome divided into several segments. The canonical view depicts each viral particle packaging one copy of each genomic segment in one polarity named the viral strand. Several opposing observations revealed nonequal ratios of the segments, uneven number of segments per virion, and even packaging of viral complementary strands. Unfortunately, these observations result from studies often addressing other questions, on distinct viral species, and not using accurate quantitative methods. Hence, what RNA segments and strands are packaged as the genome of any bunyavirus remains largely ambiguous. We addressed this issue by first investigating the virion size distribution and RNA content in populations of the tomato spotted wilt virus (TSWV) using microscopy and tomography. These revealed heterogeneity in viral particle volume and amount of RNA content, with a surprising lack of correlation between the two. Then, the ratios of all genomic segments and strands were established using RNA sequencing and qRT-PCR. Within virions, both plus and minus strands (but no mRNA) are packaged for each of the three L, M, and S segments, in reproducible nonequimolar proportions determined by those in total cell extracts. These results show that virions differ in their genomic content but together build up a highly reproducible genetic composition of the viral population. This resembles the genome formula described for multipartite viruses, with which some species of the order Bunyavirales may share some aspects of the way of life, particularly emerging properties at a supravirion scale.

Disruption of influenza virus packaging signals results in various misassembled genome complexes.

IMPORTANCE: The influenza A virus genome consists of eight distinct viral RNAs (vRNAs) that are typically packaged into a single virion as an octameric complex. How this genome complex is assembled and incorporated into the virion is poorly understood, but previous research suggests a coordinative role for packaging signals present in all vRNAs. Here, we show that disruption of two packaging signals in a model H7N7 influenza A virus results in a mixture of virions with unusual vRNA content, including empty virions, virions with one to four vRNAs, and virions with octameric complexes composed of vRNA duplicates. Our results suggest that (i) the assembly of error-free octameric complexes proceeds through a series of defined vRNA sub-complexes and (ii) virions can bud without incorporating complete octameric complexes.

Packaging defects in pestiviral NS4A can be compensated by mutations in NS2 and NS3.

The non-structural (NS) proteins of the Flaviviridae members play a dual role in genome replication and virion morphogenesis. For pestiviruses, like bovine viral diarrhea virus, the NS2-3 region and its processing by the NS2 autoprotease is of particular importance. While uncleaved NS2-3 in complex with NS4A is essential for virion assembly, it cannot replace free NS3/4A in the viral replicase. Furthermore, surface interactions between NS3 and the C-terminal cytosolic domain of NS4A were shown to serve as a molecular switch between RNA replication and virion morphogenesis. To further characterize the functionality of NS4A, we performed an alanine-scanning mutagenesis of two NS4A regions, a short highly conserved cytoplasmic linker downstream of the transmembrane domain and the C-terminal domain. NS4A residues critical for polyprotein processing, RNA replication, and/or virion morphogenesis were identified. Three double-alanine mutants, two in the linker region and one close to the C-terminus of NS4A, showed a selective effect on virion assembly. All three packaging defective mutants could be rescued by a selected set of two second-site mutations, located in NS2 and NS3, respectively. This phenotype was additionally confirmed by complementation studies providing the NS2-3/4A packaging molecules containing the rescue mutations in trans. This indicates that the linker region and the cytosolic C-terminal part of NS4A are critical for the formation of protein complexes required for virion morphogenesis. The ability of the identified sets of second-site mutations in NS2-3 to compensate for diverse NS4A defects highlights a surprising functional flexibility for pestiviral NS proteins. IMPORTANCE Positive-strand RNA viruses have a limited coding capacity due to their rather small genome size. To overcome this constraint, viral proteins often exhibit multiple functions that come into play at different stages during the viral replication cycle. The molecular basis for this multifunctionality is often unknown. For the bovine viral diarrhea virus, the non-structural protein (NS) 4A functions as an NS3 protease cofactor, a replicase building block, and a component in virion morphogenesis. Here, we identified the critical amino acids of its C-terminal cytosolic region involved in those processes and show that second-site mutations in NS2 and NS3 can compensate for diverse NS4A defects in virion morphogenesis. The ability to evolve alternative functional solutions by gain-of-function mutations highlights the astounding plasticity of the pestiviral system.

The curious case of genome packaging and assembly in RNA viruses infecting plants.

Genome packaging is the crucial step for maturation of plant viruses containing an RNA genome. Viruses exhibit a remarkable degree of packaging specificity, despite the probability of co-packaging cellular RNAs. Three different types of viral genome packaging systems are reported so far. The recently upgraded type I genome packaging system involves nucleation and encapsidation of RNA genomes in an energy-dependent manner, which have been observed in most of the plant RNA viruses with a smaller genome size, while type II and III packaging systems, majorly discovered in bacteriophages and large eukaryotic DNA viruses, involve genome translocation and packaging inside the prohead in an energy-dependent manner, i.e., utilizing ATP. Although ATP is essential for all three packaging systems, each machinery system employs a unique mode of ATP hydrolysis and genome packaging mechanism. Plant RNA viruses are serious threats to agricultural and horticultural crops and account for huge economic losses. Developing control strategies against plant RNA viruses requires a deep understanding of their genome assembly and packaging mechanism. On the basis of our previous studies and meticulously planned experiments, we have revealed their molecular mechanisms and proposed a hypothetical model for the type I packaging system with an emphasis on smaller plant RNA viruses. Here, in this review, we apprise researchers the technical breakthroughs that have facilitated the dissection of genome packaging and virion assembly processes in plant RNA viruses.

Identification of a 1.4-kb-Long Sequence Located in the nsp12 and nsp13 Coding Regions of SARS-CoV-2 Genomic RNA That Mediates Efficient Viral RNA Packaging.

The specific packaging of the viral RNA genome into virus particles is an essential step in the replication cycle of coronaviruses (CoVs). Using a single-cycle, replicable severe acute respiratory syndrome CoV-2 (SARS-CoV-2) mutant, we demonstrated the preferential packaging of the SARS-CoV-2 genomic RNA into purified virus particles. Furthermore, based on the sequence of an efficiently packaged defective interfering RNA of SARS-CoV, a closely related CoV, that was generated after serial passages of SARS-CoV in cell culture, we designed a series of replication-competent SARS-CoV-2 minigenome RNAs to identify the specific viral RNA region that is important for SARS-CoV-2 RNA packaging into virus particles. We showed that a 1.4-kb-long sequence, derived from the nsp12 and nsp13 coding regions of the SARS-CoV-2 genomic RNA, is required for the efficient packaging of SARS-CoV-2 minigenome RNA into SARS-CoV-2 particles. In addition, we also showed that the presence of possibly the entire 1.4-kb-long sequence is important for the efficient packaging of SARS-CoV-2 RNA. Our findings highlight the differences between the RNA packaging sequence identified in SARS-CoV-2, a Sarbecovirus, and the packaging signal of mouse hepatitis virus (MHV), an Embecovirus, which is a 95-nt-long sequence located at the nsp15 coding region of MHV genomic RNA. Collectively, our data imply that both the location and the sequence/structural features of the RNA element(s) that drives the selective and efficient packaging of viral genomic RNA are not conserved among the subgenera Embecovirus and Sarbecovirus within the Betacoronavirus genus. IMPORTANCE Elucidating the mechanism of SARS-CoV-2 RNA packaging into virus particles is important for the rational design of antiviral drugs that inhibit this vital step in the replication cycle of CoVs. However, our knowledge about the RNA packaging mechanism in SARS-CoV-2, including the identification of the viral RNA region important for SARS-CoV-2 RNA packaging, is limited, primarily due to the logistical challenges of handing SARS-CoV-2 in biosafety level 3 (BSL3) facilities. Our study, using a single-cycle, replicable SARS-CoV-2 mutant, which can be handled in a BSL2 lab, demonstrated the preferential packaging of full-length SARS-CoV-2 genomic RNA into virus particles and identified a specific 1.4-kb-long RNA region in SARS-CoV-2 genomic RNA that is required for the efficient packaging of SARS-CoV-2 RNA into virus particles. The information generated in our study could be valuable for clarifying the mechanisms of SARS-CoV-2 RNA packaging and for the development of targeted therapeutics against SARS-CoV-2 and other related CoVs.

HIV-1 usurps transcription start site heterogeneity of host RNA polymerase II to maximize replication fitness.

HIV-1 relies on host RNA polymeraseII (Pol II) to transcribe its genome and uses multiple transcription start sites (TSS), including three consecutive guanosines located near the U3-R junction, to generate transcripts containing three, two, and one guanosine at the 5' end, referred to as 3G, 2G, and 1G RNA, respectively. The 1G RNA is preferentially selected for packaging, indicating that these 99.9% identical RNAs exhibit functional differences and highlighting the importance of TSS selection. Here, we demonstrate that TSS selection is regulated by sequences between the CATA/TATA box and the beginning of R. Furthermore, we have generated two HIV-1 mutants with distinct 2-nucleotide modifications that predominantly express 3G RNA or 1G RNA. Both mutants can generate infectious viruses and undergo multiple rounds of replication in T cells. However, both mutants exhibit replication defects compared to the wild-type virus. The 3G-RNA-expressing mutant displays an RNA genome-packaging defect and delayed replication kinetics, whereas the 1G-RNA-expressing mutant exhibits reduced Gag expression and a replication fitness defect. Additionally, reversion of the latter mutant is frequently observed, consistent with sequence correction by plus-strand DNA transfer during reverse transcription. These findings demonstrate that HIV-1 maximizes its replication fitness by usurping the TSS heterogeneity of host RNA Pol II to generate unspliced RNAs with different specialized roles in viral replication. The three consecutive guanosines at the junction of U3 and R may also maintain HIV-1 genome integrity during reverse transcription. These studies reveal the intricate regulation of HIV-1 RNA and complex replication strategy.

Mechanisms of bunyavirus morphogenesis and egress.

Unlike many segmented negative-sense RNA viruses, most members of the Bunyavirales bud at Golgi membranes, as opposed to the plasma membrane. Central players in this assembly process are the envelope glycoproteins, Gn and Gc, which upon translation undergo proteolytic processing, glycosylation and trafficking to the Golgi, where they interact with ribonucleoprotein genome segments and bud into Golgi-derived compartments. The processes involved in genome packaging during virion assembly can lead to the generation of reassorted viruses, if a cell is co-infected with two different bunyaviruses, due to mismatching of viral genome segment packaging. This can lead to viruses with high pathogenic potential, as demonstrated by the emergence of Schmallenberg virus. This review focuses on the assembly pathways of tri-segmented bunyaviruses, highlighting some areas in need of further research to understand these important pathogens with zoonotic potential.

CryoEM of Viral Ribonucleoproteins and Nucleocapsids of Single-Stranded RNA Viruses.

Single-stranded RNA viruses (ssRNAv) are characterized by their biological diversity and great adaptability to different hosts; traits which make them a major threat to human health due to their potential to cause zoonotic outbreaks. A detailed understanding of the mechanisms involved in viral proliferation is essential to address the challenges posed by these pathogens. Key to these processes are ribonucleoproteins (RNPs), the genome-containing RNA-protein complexes whose function is to carry out viral transcription and replication. Structural determination of RNPs can provide crucial information on the molecular mechanisms of these processes, paving the way for the development of new, more effective strategies to control and prevent the spread of ssRNAv diseases. In this scenario, cryogenic electron microscopy (cryoEM), relying on the technical and methodological revolution it has undergone in recent years, can provide invaluable help in elucidating how these macromolecular complexes are organized, packaged within the virion, or the functional implications of these structures. In this review, we summarize some of the most prominent achievements by cryoEM in the study of RNP and nucleocapsid structures in lipid-enveloped ssRNAv.

Modulation of Capsid Dynamics in Bromoviruses by the Host and Heterologous Viral Replicase.

The three genomic and a single subgenomic RNA of Cowpea chlorotic mottle virus (CCMV), which is pathogenic to plants, is packaged into three morphologically indistinguishable icosahedral virions with T=3 symmetry. The two virion types, C1V and C2V, package genomic RNAs 1 (C1) and 2 (C2), respectively. The third virion type, C3+4V, copackages genomic RNA3 and its subgenomic RNA (RNA4). In this study, we sought to evaluate how the alteration of native capsid dynamics by the host and viral replicase modulate the general biology of the virus. The application of a series of biochemical, molecular, and biological assays revealed the following. (i) Proteolytic analysis of the three virion types of CCMV assembled individually in planta revealed that, while retaining the structural integrity, C1V and C2V virions released peptide regions encompassing the N-terminal arginine-rich RNA binding motif. In contrast, a minor population of the C3+4V virion type was sensitive to trypsin-releasing peptides encompassing the entire capsid protein region. (ii) The wild-type CCMV virions purified from cowpea are highly susceptible to trypsin digestion, while those from Nicotiana benthamiana remained resistant, and (iii) finally, the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis evaluated the relative dynamics of C3+4V and B3+4V virions assembled under the control of the homologous versus heterologous replicase. The role of viral replicase in modulating the capsid dynamics was evident by the differential sensitivity to protease exhibited by B3+4V and C3+4V virions assembled under the homologous versus heterologous replicase. Our results collectively conclude that constant modulation of capsid dynamics by the host and viral replicase is obligatory for successful infection. IMPORTANCE Infectious virus particles or virions are considered static structures and undergo various conformational transitions to replicate and infect many eukaryotic cells. In viruses, conformational changes are essential for establishing infection and evolution. Although viral capsid fluctuations, referred to as dynamics or breathing, have been well studied in RNA viruses pathogenic to animals, such information is limited among plant viruses. The primary focus of this study is to address how capsid dynamics of plant-pathogenic RNA viruses, namely, Cowpea chlorotic mottle (CCMV) and Brome mosaic virus (BMV), are modulated by the host and viral replicase. The results presented have improved and transformed our understanding of the functional relationship between capsid dynamics and the general biology of the virus. They are likely to provide stimulus to extend similar studies to viruses pathogenic to eukaryotic organisms.