Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity.

BACKGROUND: Retroviruses selectively encapsidate two copies of their genomic RNA, the Gag protein binding a specific RNA motif in the 5' UTR of the genome. In human immunodeficiency virus type 2 (HIV-2), the principal packaging signal (Psi) is upstream of the major splice donor and hence is present on all the viral RNA species. Cotranslational capture of the full length genome ensures specificity. HIV-2 RNA dimerisation is thought to occur at the dimer initiation site (DIS) located in stem-loop 1 (SL-1), downstream of the main packaging determinant. However, the HIV-2 packaging signal also contains a palindromic sequence (pal) involved in dimerisation. In this study, we analysed the role of the HIV-2 packaging signal in genomic RNA dimerisation in vivo and its implication in viral replication. RESULTS: Using a series of deletion and substitution mutants in SL-1 and the Psi region, we show that in fully infectious HIV-2, genomic RNA dimerisation is mediated by the palindrome pal. Mutation of the DIS had no effect on dimerisation or viral infectivity, while mutations in the packaging signal severely reduce both processes as well as RNA encapsidation. Electron micrographs of the Psi-deleted virions revealed a significant reduction in the proportion of mature particles and an increase in that of particles containing multiple cores. CONCLUSION: In addition to its role in RNA encapsidation, the HIV-2 packaging signal contains a palindromic sequence that is critical for genomic RNA dimerisation. Encapsidation of a dimeric genome seems required for the production of infectious mature particles, and provides a promising therapeutic target

Fine Structure of Viral dsDNA Encapsidation

In vivo configurations of dsDNA of bacteriophage viruses in a capsid are known to form hexagonal chromonic liquid crystal phases. This article studies the liquid crystal ordering of viral dsDNA in an icosahedral capsid, combining the chromonic model with that of liquid crystals with variable degree of orientation. The scalar order parameter of the latter allows us to distinguish regions of the capsid with well-ordered DNA from the disordered central core. We employ a state-of-the-art numerical algorithm based on the finite element method to find equilibrium states of the encapsidated DNA and calculate the corresponding pressure. With a data-oriented parameter selection strategy, the method yields phase spaces of the pressure and the radius of the disordered core, in terms of relevant dimensionless parameters, rendering the proposed algorithm into a preliminary bacteriophage designing tool. The presence of the order parameter also has the unique role of allowing for non-smooth capsid domains as well as accounting for knot locations of the DNA

Rescue of Synthetic Genomic RNA Analogs of Rabies Virus by Plasmid-Encoded Proteins

Proteins eolirely expressed from cDNA wen used to rescue synthetic RNA genome analogs into infectious defective particles or rabies virus (RV). Synthetic negative-stranded RNAs coßtalning 3' · and S'-terminal RV sequences and tnlßscriptional signal sequences wen transcribed (rom plasmids transfeded into cells expressing 1'7 RNA polymerase (rom recombinant vaccinia virus. After simultaneous expression or RV N, P, and L proteiDS (rom plasmids containing a T7 RNA polymerase promoter, tbe synthetic genomes wen encapsidated. replicated, and transcribed by tbe RV polymerase proteiDS. Insertion or the bac1erial chloramphenicol acetyUransferase gene or l3·galactosidase (IacZ) gene between the 3 ' and 5 ' termini containing transcriptional signal sequenees resulted in transcription of mRNAs and expression of ehloramphenlco

Is HIV-1 RNA dimerization a prerequisite for packaging? Yes, no, probably?

During virus assembly, all retroviruses specifically encapsidate two copies of full-length viral genomic RNA in the form of a non-covalently linked RNA dimer. The absolute conservation of this unique genome structure within the Retroviridae family is strong evidence that a dimerized genome is of critical importance to the viral life cycle. An obvious hypothesis is that retroviruses have evolved to preferentially package two copies of genomic RNA, and that dimerization ensures the proper packaging specificity for such a genome. However, this implies that dimerization must be a prerequisite for genome encapsidation, a notion that has been debated for many years. In this article, we review retroviral RNA dimerization and packaging, highlighting the research that has attempted to dissect the intricate relationship between these two processes in the context of HIV-1, and discuss the therapeutic potential of these putative antiretroviral targets

A Liquid Crystal Model of Viral DNA Encapsidation

A liquid crystal continuum modeling framework for icosahedra bacteriophage viruses is developed and tested. The main assumptions of the model are the chromonic columnar hexagonal structure of confined DNA, the high resistance to bending and the phase transition from solid to fluid-like states as the concentration of DNA in the capsid decreases during infection. The model predicts osmotic pressure inside the capsid and the ejection force of the DNA as well as the size of the isotropic volume at the center of the capsid. Extensions of the model are discussed

Mutagenesis of the conserved 51-nucleotide region of Sindbis virus

We have constructed 25 site-specific mutations in a domain of 51 nucleotides in Sindbis virus that is highly conserved among all alphaviruses sequenced to date. These 51 nucleotides are capable of forming two hairpin structures and are found from nucleotides 155 to 205 in Sindbis virus within the region encoding nsP1. Of the mutations, 21 were silent and did not lead to a change in the amino acid sequence encoded. These silent mutations changed not only the linear sequence but also the stability of the hairpins in most cases. Two double mutants that were constructed led to the replacement of one base pair by another so that the linear sequence was altered but the nature of the hairpins was not. All of the mutants with silent mutations were viable, but 19 of the 21 mutants were severely impaired for growth in both chicken and mosquito cells. Compared with the parental virus, they grew slowly and produced virus at rates of 10(-1) to 10(-4) times the parental rate. Surprisingly, however, the plaques produced by these mutants were indistinguishable from those produced by the parental virus. Two of the silent mutations, found within the first hairpin structure, produced virus at a faster rate than the parental virus. It is clear that the exact sequence of this region is important for some aspect of virus replication. We suggest that one or more proteins, either virus encoded or cellular, bind to the hairpin structures in a sequence-specific fashion in a step that promotes replication of the viral RNA. Of the mutations that resulted in a change of coding, only one of four was viable, suggesting that the amino acid sequence encoded in this domain is essential for virus replication

Electrostatic stability and encapsidation of charged nano-droplets

We investigate electrostatic stability of charged droplets, modeled as permeable, charged spheres, and their encapsidation in thin, arbitrarily charged nano-shells, immersed in a neutralizing asymmetric electrolyte background. The latter consists of a small concentration of mobile multivalent counterions in a bathing solution of monovalent (positive and negative) ions. We use extensive Monte-Carlo simulations to investigate the spatial distribution of multivalent counterions and the electrostatic component of their osmotic pressure on the bounding surface of the spherical nano-shell. The osmotic pressure can be negative (inward pressure), positive (outward pressure) or zero, depending on the system parameters such as the charge density of the droplet, the charge density of the shell, and the electrolyte screening, which thus determine the stability of the nano-container. The counter-intuitive effects of multivalent counterions comprise the increased stability of the charged droplet with larger charge density, increased stability in the case of encapsidating shell of charge density of the same sign as the charged droplet, as well as the possibility to dispense altogether with the encapsidating shell, its confining effect taken over by the multivalent counterions. These dramatic effects are in stark contrast to the conventional mean-field picture, which in particular implies that a more highly charged spherical droplet should be electrostatically less stable because of its larger (repulsive) self-energy.Comment: 10 pages, 7 figure

The effect of RNA stiffness on the self-assembly of virus particles

Under many in vitro conditions, some small viruses spontaneously encapsidate a single stranded (ss) RNA into a protein shell called the capsid. While viral RNAs are found to be compact and highly branched because of long distance base-pairing between nucleotides, recent experiments reveal that in a head-to-head competition between a ssRNA with no secondary or higher order structure and a viral RNA, the capsid proteins preferentially encapsulate the linear polymer! In this paper, we study the impact of genome stiffness on the encapsidation free energy of the complex of RNA and capsid proteins. We show that an increase in effective chain stiffness because of base-pairing could be the reason why under certain conditions linear chains have an advantage over branched chains when it comes to encapsidation efficiency. While branching makes the genome more compact, RNA base-pairing increases the effective Kuhn length of the RNA molecule, which could result in an increase of the free energy of RNA confinement, that is, the work required to encapsidate RNA, and thus less efficient packaging

Effects of RNA branching on the electrostatic stabilization of viruses

Many single-stranded (ss) RNA viruses self assemble from capsid protein subunits and the nucleic acid to form an infectious virion. It is believed that the electrostatic interactions between the negatively charged RNA and the positively charged viral capsid proteins drive the encapsidation, although there is growing evidence that the sequence of the viral RNA also plays a role in packaging. In particular the sequence will determine the possible secondary structures that the ssRNA will take in solution. In this work, we use a mean field theory to investigate how the secondary structure of the RNA combined with electrostatic interactions affects the efficiency of assembly and stability of the assembled virions. We show that the secondary structure of RNA may result in negative osmotic pressures while a linear polymer causes positive osmotic pressures for the same conditions. This may suggest that the branched structure makes the RNA more effectively packaged and the virion more stable