RNA Encapsidation Research Articles

The coat protein leads the way: an update on basic and applied studies with the Brome mosaic virus coat protein.

The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.

Novel Functions of Prototype Foamy Virus Gag Glycine- Arginine-Rich Boxes in Reverse Transcription and Particle Morphogenesis

Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

Three-dimensional structure of the hepatitis B core antigen particle truncated at residue 154

The three-dimensional structure of recombinant hepatitis B core antigen (HBcAg) particles truncated at residue 154 (HBcAg-154) was determined to 7.8 Å resolution by cryo-electron microscopy (cryoEM) and computer reconstruction. The capsid of HBcAg-154 is mainly constituted by α-helical folds, highly similar to that of HBcAg-149. The C-terminal region between residues 155 and 183 of the core protein is more crucial to the encapsidation of RNA, and the short C-terminal tail of HBcAg-154 results in a nearly empty capsid.

Genetic Dissection of Interferon-Antagonistic Functions of Rabies Virus Phosphoprotein: Inhibition of Interferon Regulatory Factor 3 Activation Is Important for Pathogenicity

The rabies virus (RV) phosphoprotein (P) is a type I interferon (IFN) antagonist preventing both transcriptional induction of IFN and IFN-mediated JAK/STAT signaling. In addition, P is an essential cofactor of the viral polymerase and is required for encapsidation of viral RNA into nucleoprotein during replication. By site-directed mutagenesis, we have identified a domain of P required for efficient inhibition of IFN induction. Phosphoproteins lacking amino acids (aa) 176 to 181, 182 to 186, or 176 to 186 were severely compromised in counteracting phosphorylation of IRF3 and IRF7 by TBK1 or IKKi while retaining the full capacity of preventing nuclear import of activated STATs and of supporting virus transcription and replication. Recombinant RV carrying the mutated phosphoproteins (the SAD ΔInd1, SAD ΔInd2, and SAD ΔInd1/2 viruses) activated IRF3 and beta IFN (IFN-β) transcription in infected cells but still blocked STAT-mediated expression of IFN-stimulated genes. Due to a somewhat higher transcription rate, the SAD ΔInd1 virus activated IRF3 more efficiently than the SAD ΔInd2 virus. After intracerebral injection into mouse brains at high doses, the SAD ΔInd1 virus was completely apathogenic for wild-type (wt) mice, while the SAD ΔInd2 virus was partially attenuated and caused a slower progression of lethal rabies than wt RV. Neurovirulence of IFN-resistant RV thus correlates with the capacity of the virus to prevent activation of IRF3 and IRF7.

The Arginine Clusters of the Carboxy-Terminal Domain of the Core Protein of Hepatitis B Virus Make Pleiotropic Contributions to Genome Replication

The carboxy-terminal domain (CTD) of the core protein of hepatitis B virus is not necessary for capsid assembly. However, the CTD does contribute to encapsidation of pregenomic RNA (pgRNA). The contribution of the CTD to DNA synthesis is less clear. This is the case because some mutations within the CTD increase the proportion of spliced RNA to pgRNA that are encapsidated and reverse transcribed. The CTD contains four clusters of consecutive arginine residues. The contributions of the individual arginine clusters to genome replication are unknown. We analyzed core protein variants in which the individual arginine clusters were substituted with either alanine or lysine residues. We developed assays to analyze these variants at specific steps throughout genome replication. We used a replication template that was not spliced in order to study the replication of only pgRNA. We found that alanine substitutions caused defects at both early and late steps in genome replication. Lysine substitutions also caused defects, but primarily during later steps. These findings demonstrate that the CTD contributes to DNA synthesis pleiotropically and that preserving the charge within the CTD is not sufficient to preserve function.

RNA Aptamers Directed to Human Immunodeficiency Virus Type 1 Gag Polyprotein Bind to the Matrix and Nucleocapsid Domains and Inhibit Virus Production

Gag orchestrates the assembly and release of human immunodeficiency virus type 1 (HIV-1) particles. We explored here the potential of anti-Gag RNA aptamers to inhibit HIV-1 replication. In vitro, RNA aptamers raised against an HIV-1 Gag protein, lacking the N-terminal myristate and the C-terminal p6 (DP6-Gag), could bind to matrix protein (MA), nucleocapsid protein (NC), or entire DP6-Gag protein. Upon cotransfection with pNL4-3.Luc molecular clone into 293T cells, six of the aptamers caused mild inhibition (2- to 3-fold) in the extracellular capsid levels, and one aptamer displayed 20-fold inhibition. The reduction was not due to a release defect but reflected Gag mRNA levels. We hypothesized that the aptamers influence genomic RNA levels via perturbation of specific Gag-genomic RNA interactions. Binding studies revealed that the "NC-binders" specifically compete with the packaging signal (ψ) of HIV-1 for binding to DP6-Gag. Therefore, we tested the ability of two NC-binders to inhibit viruses containing ψ-region deletions (ΔSL1 or ΔSL3) and found that the NC-binders were no longer able to inhibit Gag synthesis. The inability of these aptamers to inhibit ψ-deleted viruses correlated with the absence of competition with the corresponding ψ transcripts lacking SL1 or SL3 for binding DP6-Gag in vitro. These results indicate that the NC-binding aptamers disrupt Gag-genomic RNA interaction and negatively affect genomic RNA transcription, processing, or stability. Our results reveal an essential interaction between HIV-1 Gag and the ψ-region that may be distinct from that which occurs during the encapsidation of genomic RNA. Thus, anti-Gag aptamers can be an effective tool to perturb Gag-genomic RNA interactions.

Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles

Triatoma virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1–3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0–VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.

Human importin alpha and RNA do not compete for binding to influenza A virus nucleoprotein

Influenza virus has a segmented genome composed of eight negative stranded RNA segments. Each segment is covered with NP forming ribonucleoproteins (vRNPs) and carries a copy of the heterotrimeric polymerase complex. As a rare phenomenon among the RNA viruses, the viral replication occurs in the nucleus and therefore implies interactions between host and viral factors, such as between importin alpha and nucleoprotein. In the present study we report that through binding with the human nuclear receptor importin α5 (Impα5), the viral NP is no longer oligomeric but maintained as a monomer inside the complex. In this regard, Impα5 acts as a chaperone until NP is delivered in the nucleus for viral RNA encapsidation. Moreover, we show that the association of NP with the host transporter does not impair the binding of NP to RNA. The complex human Impα5-NP binds RNA with the same affinity as wt NP alone, whereas engineered monomeric NP through point mutations binds RNA with a strongly reduced affinity.

Amino acid changes within the Bunyamwera virus nucleocapsid protein differentially affect the mRNA transcription and RNA replication activities of assembled ribonucleoprotein templates

The genome of Bunyamwera virus (BUNV) comprises three RNA segments that are encapsidated by the virus-encoded nucleocapsid (N) protein to form ribonucleoprotein (RNP) complexes. These RNPs are the functional templates for RNA synthesis by the virus-encoded RNA-dependent RNA polymerase (RdRp). We investigated the roles of conserved positively charged N-protein amino acids in RNA binding, in oligomerization to form model RNPs and in generating RNP templates active for both RNA replication and mRNA transcription. We identified several residues that performed important roles in RNA binding, and furthermore showed that a single amino acid change can differentially affect the ability of the resulting RNP templates to regulate the transcription and replication activities of the RdRp. These results indicate that the BUNV N protein possesses functions outside of its primary role of RNA encapsidation.

Extensive Phosphorylation Flanking the C-Terminal Functional Domains of the Measles Virus Nucleoprotein

The measles virus nucleoprotein (vNP) is the first and most abundant protein in infected cells. It plays numerous important roles including the encapsidation of genomic viral RNA and the transcription of viral proteins. Intricate interactions with host cell proteins rely on the structural integrity of its functional domains. Although some of these functional domains are known, their structural features are still poorly understood. Here we identified multiple isoforms of measles vNP by two-dimensional differential gel electrophoresis (2D-DIGE) and 2D Western blot. These isoforms were further analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)/TOF using MS (PMF) and MSMS (PSD) and electrospray ionization (ESI)-ion trap using LC-ESI-ion trap MS(1), MS(2) (neutral loss), MS(3) (phosphosite). Both recombinant NP (rNP) and vNP were α-acetylated at the N-terminus. After tryptic or chymotryptic digestion, phosphopeptides were enriched and nine phosphorylation sites were identified and localized in the rNP, seven of which were also phosphorylated in vNP, probably by casein kinase 2. The phosphosites were all found within the intrinsically unstructured C-terminal domain. They clustered around functional domains involved in transcription and replication, as well as in sequences interacting with host-cell proteins. This underlines the importance of these post-translational modifications.

Direct Interaction between Two Viral Proteins, the Nonstructural Protein 2CATPase and the Capsid Protein VP3, Is Required for Enterovirus Morphogenesis

In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2CATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel “reporter virus”, we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2CATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2CATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2CATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2CATPase is an essential component of the replication complex, and (iv) 2CATPase has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex.

Nuclear Magnetic Resonance Structure of the Prohead RNA E-Loop Hairpin,

The Bacillus subtilis phage phi29 packaging motor requires prohead RNA for genome encapsidation. The nuclear magnetic resonance structure of the prohead RNA E-loop hairpin, r(5'AUUGAGUU), is presented and compared to predictions from MC-SYM. The prohead RNA E-loop hairpins contain sequences similar to rRNA hairpins. Comparison of predicted and experimentally determined prohead and ribosomal hairpin structures reveals that sequence similarity is a stronger determinant of hairpin structural similarity than grouping similar types of RNA. All the hairpins contain a U-turn motif but differ in the first noncanonical pair and backbone orientation. These structures provide benchmarks for further improvements in RNA structure predictions from sequence.

A G–C-Rich Palindromic Structural Motif and a Stretch of Single-Stranded Purines Are Required for Optimal Packaging of Mason–Pfizer Monkey Virus (MPMV) Genomic RNA

During retroviral RNA packaging, two copies of genomic RNA are preferentially packaged into the budding virus particles whereas the spliced viral RNAs and the cellular RNAs are excluded during this process. Specificity towards retroviral RNA packaging is dependent upon sequences at the 5′ end of the viral genome, which at times extend into Gag sequences. It has earlier been suggested that the Mason–Pfizer monkey virus (MPMV) contains packaging sequences within the 5′ untranslated region (UTR) and Gag. These studies have also suggested that the packaging determinants of MPMV that lie in the UTR are bipartite and are divided into two regions both upstream and downstream of the major splice donor. However, the precise boundaries of these discontinuous regions within the UTR and the role of the intervening sequences between these dipartite sequences towards MPMV packaging have not been investigated. Employing a combination of genetic and structural prediction analyses, we have shown that region “A”, immediately downstream of the primer binding site, is composed of 50 nt, whereas region “B” is composed of the last 23 nt of UTR, and the intervening 55 nt between these two discontinuous regions do not contribute towards MPMV RNA packaging. In addition, we have identified a 14-nt G–C-rich palindromic sequence (with 100% autocomplementarity) within region A that has been predicted to fold into a structural motif and is essential for optimal MPMV RNA packaging. Furthermore, we have also identified a stretch of single-stranded purines (ssPurines) within the UTR and 8 nt of these ssPurines are duplicated in region B. The native ssPurines or its repeat in region B when predicted to refold as ssPurines has been shown to be essential for RNA packaging, possibly functioning as a potential nucleocapsid binding site. Findings from this study should enhance our understanding of the steps involved in MPMV replication including RNA encapsidation process.

Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation

Rift Valley fever virus (RVFV) is a negative-sense RNA virus (genus Phlebovirus, family Bunyaviridae) that infects livestock and humans and is endemic to sub-Saharan Africa. Like all negative-sense viruses, the segmented RNA genome of RVFV is encapsidated by a nucleocapsid protein (N). The 1.93-A crystal structure of RVFV N and electron micrographs of ribonucleoprotein (RNP) reveal an encapsidated genome of substantially different organization than in other negative-sense RNA virus families. The RNP polymer, viewed in electron micrographs of both virus RNP and RNP reconstituted from purified N with a defined RNA, has an extended structure without helical symmetry. N-RNA species of approximately 100-kDa apparent molecular weight and heterogeneous composition were obtained by exhaustive ribonuclease treatment of virus RNP, by recombinant expression of N, and by reconstitution from purified N and an RNA oligomer. RNA-free N, obtained by denaturation and refolding, has a novel all-helical fold that is compact and well ordered at both the N and C termini. Unlike N of other negative-sense RNA viruses, RVFV N has no positively charged surface cleft for RNA binding and no protruding termini or loops to stabilize a defined N-RNA oligomer or RNP helix. A potential protein interaction site was identified in a conserved hydrophobic pocket. The nonhelical appearance of phlebovirus RNP, the heterogeneous approximately 100-kDa N-RNA multimer, and the N fold differ substantially from the RNP and N of other negative-sense RNA virus families and provide valuable insights into the structure of the encapsidated phlebovirus genome.

Expanding Knowledge of P3 Proteins in the Poliovirus Lifecycle

Poliovirus is the most extensively studied member of the order Picornavirales, which contains numerous medical, veterinary and agricultural pathogens. The picornavirus genome encodes a single polyprotein that is divided into three regions: P1, P2 and P3. P3 proteins are known to participate more directly in genome replication, for example by containing the viral RNA-dependent RNA polymerase (RdRp or 3Dpol), among several other proteins and enzymes. We will review recent data that provide new insight into the structure, function and mechanism of P3 proteins and their complexes, which are required for initiation of genome replication. Replication of poliovirus genomes occurs within macromolecular complexes, containing viral RNA, viral proteins and host-cell membranes, collectively referred to as replication complexes. P2 proteins clearly contribute to interactions with the host cell that are required for virus multiplication, including formation of replication complexes. We will discuss recent data that suggest a role for P3 proteins in formation of replication complexes. Among the least understood steps of the poliovirus lifecycle is encapsidation of genomic RNA. We will also describe data that suggest a role for P3 proteins in this step.

A highly basic KGKKGK sequence in the RNA-binding domain of the Cucumber necrosis virus coat protein is associated with encapsidation of full-length CNV RNA during infection

The Cucumber necrosis virus particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. The N-terminal 58 aa residue segment of the CP R domain is believed to bind viral RNA within virions and during assembly. We report results of in vivo experiments that examine the role of the R domain in assembly. Deletion analyses identified 3 conserved 5-10 aa regions as playing critical roles. A highly basic KGKKGK sequence was found to be both necessary and sufficient for encapsidation of the full-length genome and polymorphic virions were produced in mutants lacking the KGKKGK sequence. The amount of full-length RNA present in virions was substantially reduced in R domain mutants where 2 of the 4 lysine residues were substituted with alanine, whereas substitution of 4 lysines by arginine had only a modest effect. The potential role of the R domain in formation of a scaffold for particle assembly is discussed.

Molecular biology of the hepatitis B virus and role of the X gene

The hepatitis B virus (HBV) is a widespread human pathogen and a major health problem in many countries. Molecular cloning and sequencing of the viral DNA genome has demonstrated a small and compact structure organized into four overlapping reading frames that encode the viral proteins. Besides structural proteins of the core and the envelope, HBV encodes a DNA polymerase with reverse transcriptase activity, a secreted antigen of unknown function, and a transcriptional activator that is essential for viral replication. Major steps of the viral life cycle have been unraveled, including transcription of all viral RNAs from nuclear covalently closed circular DNA (cccDNA), followed by encapsidation of pregenomic RNA, a more-than-genome length transcript, and reverse transcription of pregenomic RNA leading to asymmetric synthesis of the DNA strands. Although HBV has been recognized as a human tumor virus, no direct transforming activity could be evidenced in different cellular and animal models. However, the transcriptional regulatory protein HBx encoded by the X gene is endowed with weak oncogenic activity. HBx harbors pleiotropic activities and plays a major role in HBV pathogenesis and in liver carcinogenesis.

Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA

Retroviral Gag polyproteins coopt host factors to traffic from cytosolic ribosomes to the plasma membrane, where virions are released. Before membrane transport, the multidomain Gag protein of Rous sarcoma virus (RSV) undergoes importin-mediated nuclear import and CRM1-dependent nuclear export, an intrinsic step in the assembly pathway. Transient nuclear trafficking of Gag is required for efficient viral RNA (vRNA) encapsidation, suggesting that Gag:vRNA binding might occur in the nucleus. Here, we show that Gag is imported into the nucleus through direct interactions of the Gag NC domain with importin-alpha (imp-alpha) and the MA domain with importin-11 (imp-11). The vRNA packaging signal, known as psi, inhibited imp-alpha binding to Gag, indicating that the NC domain does not bind to imp-alpha and vRNA simultaneously. Unexpectedly, vRNA binding also prevented the association of imp-11 with both the MA domain alone and with Gag, suggesting that the MA domain may bind to the vRNA genome. In contrast, direct binding of Gag to the nuclear export factor CRM1, via the CRM1-RanGTP heterodimer, was stimulated by psiRNA. These findings suggest a model whereby the genomic vRNA serves as a switch to regulate the ordered association of host import/export factors that mediate Gag nucleocytoplasmic trafficking for virion assembly. The Gag:vRNA interaction appears to serve multiple critical roles in assembly: specific selection of the vRNA genome for packaging, stimulating the formation of Gag dimers, and triggering export of viral ribonucleoprotein complexes from the nucleus.

Nuclear RNA Export and Packaging Functions of HIV-1 Rev Revisited

Although the viral Rev protein is necessary for HIV replication, its main function in the viral replication cycle has been controversial. Reinvestigating the effect of Rev on the HIV-1 RNA distribution in various cell lines and primary cells revealed that Rev enhanced cytoplasmic levels of the unspliced HIV-1 RNA, mostly 3- to 12-fold, while encapsidation of the RNA and viral infectivity could be stimulated >1,000-fold. Although this clearly questions the general notion that the nuclear export of viral RNAs is the major function of Rev, mechanistically encapsidation seems to be linked to nuclear export, since the tethering of the nuclear export factor TAP to the HIV-1 RNA also enhanced encapsidation. Interference with the formation of an inhibitory ribonucleoprotein complex in the nucleus could lead to enhanced accessibility of the cytoplasmic HIV-1 RNA for translation and encapsidation. This might explain why Rev and tethered TAP exert the same pattern of pleiotropic effects.

RNA nanoparticle vaccines: combining cytokine activation with antigen and CTL peptide delivery to improve vaccine efficacy (131.4)

Abstract We have used an RNA nanoparticle system to test the hypothesis that co-expression of a tumor antigen-cytokine fusion, with or without a co-delivered tumor CTL epitope, can improve anti-tumor immunity to a self antigen. Our initial studies demonstrated that a Semliki Forest Virus (SFV) bGal RNA expression vector could be modified to allow for in vitro coat protein RNA encapsidation by a distant alphavirus family member, Tobacco Mosaic Virus (TMV). Encapsidated RNA stimulated robust targeted immune cell uptake and activation, including dendritic cells. In response to vaccination, encapsidation significantly improved humoral and cellular immune responses compared to naked RNA, including IFNg activation , and protection from challenge with a lethal dose of bGal expressing tumor. More recently, our goal is to test efficacy of encapsidated RNA in a Non-hodgkin’s murine lymphoma tumor self-antigen system using the A20 expressing tumor. We have constructed a variety of SFV A20 scFv antigen-cytokine fusions, and are co-developing an A20 CTL surface antigen fusion. In vitro expression will be confirmed, and mice will be immunized and tested by ELISA and ELISpot for functional B and T cell immune activation. This nanoparticle RNA system generates vaccine compositions that are easy to customize, easy to produce, and facilitates intracellular delivery of unique nucleic acid/protein antigen combinations that should improve vaccine efficacy against a weakly immunogenic tumor.