An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus

Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5′ untranslated region that contains all residues necessary for efficient RNA packaging (Ψ^WT; residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Ψ^WT]₂) binding 12 ± 2 NC molecules with high affinity (K_d = 17 ± 7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Ψ^M), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (∼ 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.     

Host-Cell Repair of Vaccinia Virus and of Double Stranded RNA of Encephalomyocarditis Virus

IN normal human cells DNA which has been damaged by ultraviolet radiation is repaired by excision of thymidine dimers and by repair replication. Patients suffering from xeroderma pigmentosum have a hereditary defect of the excision step and therefore their cells repair ultraviolet-induced lesions in their DNA less efficiently than do normal cells^1–4. An analogous situation has been well characterized in bacteria⁵.     

Purified Membrane-Containing Procapsids of Bacteriophage PRD1 Package the Viral Genome

Icosahedral-tailed double-stranded DNA (dsDNA) bacteriophages and herpesviruses translocate viral DNA into a preformed procapsid in an ATP-driven reaction by a packaging complex that operates at a portal vertex. A similar packaging system operates in the tailless dsDNA phage PRD1 (Tectiviridae family), except that there is an internal membrane vesicle in the procapsid. The unit-length linear dsDNA genome with covalently linked 5′-terminal proteins enters the procapsid through a unique vertex. Two small integral membrane proteins, P20 and P22, provide a conduit for DNA translocation. The packaging machinery also contains the packaging ATPase P9 and the packaging efficiency factor P6. Here we describe a method used to obtain purified packaging-competent PRD1 procapsids. The optimized in vitro packaging system allowed efficient packaging of defined DNA substrates. We determined that the genome terminal protein P8 is necessary for packaging and provided an estimation of the packaging rate.     

Analysis of Herpes Simplex Virus Type 1 DNA Packaging Signal Mutations in the Context of the Viral Genome

The minimal signal required for the cleavage and packaging of replicated concatemeric herpes simplex virus type 1 (HSV-1) DNA corresponds to an approximately 200-bp fragment, Uc-DR1-Ub, spanning the junction of the genomic L and S segments. Uc and Ub occupy positions adjacent to the L and S termini and contain motifs (pac2 and pac1, respectively) that are conserved near the ends of other herpesvirus genomes. We have used homologous Red/ET recombination in Escherichia coli to introduce wild-type and specifically mutated Uc-DR1-Ub fragments into an ectopic site of a cloned HSV-1 genome from which the resident packaging signals had been previously deleted. The resulting constructs were transfected into mammalian cells, and their abilities to replicate and become encapsidated, generate Uc- and Ub-containing terminal fragments, and give rise to progeny virus were assessed. In general, the results obtained agree well with previous observations made using amplicons and confirm roles for the pac2 T element in the initiation of DNA packaging and for the GC-rich motifs flanking the pac1 T element in termination. In contrast to a previous report, the sequence of the DR1 element was also crucial for DNA packaging. Following repair of the resident packaging signals in mammalian cells, recombination occurred at high frequency in progeny virus between the repaired sequences and mutated Uc-DR1-Ub inserts. This restored the ability of mutated Uc-DR1-Ub inserts to generate terminal fragments, although these were frequently larger than expected from simple repair of the original lesion.Herpesviruses possess linear double-stranded DNA genomes that are circularized early after infection and upon replication generate concatemeric structures. During progeny particle assembly, the cleavage of concatemers at specific sites, corresponding to the genomic termini, is tightly coupled to the insertion of the viral DNA into a preformed structure referred to as the procapsid (reviewed in references 2, 4, and 11). In the case of herpes simplex virus type 1 (HSV-1), a terminally redundant region of the genome, known as the a sequence (Fig. ​(Fig.1a),1a), contains all the cis-acting sequences required for DNA packaging (24, 27). This region, which is 250 to 500 bp in length depending on the virus strain, is present as a single copy at the S terminus and as one or more tandem copies at the L terminus. In addition, one or more copies are present in inverted orientation at the junction between the L and S segments (30, 31).Open in a separate windowFIG. 1.Structure of the HSV-1 Uc-DR1-Ub element. (a) Structure of the HSV-1 genome showing the positions and relative orientations (horizontal arrows) of copies of the a sequence. (b) Circularization of linear genomes by direct ligation brings together two copies of the a sequence separated by a single DR1 repeat. The site of ligation, and of cleavage of concatemers, is shown by the vertical arrow. (c) Motifs and regions within the 194-bp Uc-DR1-Ub fragment. To facilitate naming of mutants, component regions of Uc, Ub, and DR1 were also referred to as c1 to c4, b1 to b4, and R, respectively, as indicated in parentheses.The structure of the HSV-1 a sequence is depicted in Fig. ​Fig.1b.1b. Each a sequence is flanked by direct repeats (DR1) of 17 to 20 bp, with single copies of DR1 separating tandem a sequences. Genomic termini are generated by a cleavage event toward one end of DR1, and circularization of infecting genomes restores a complete a sequence. The central portion of the a sequence comprises multiple repeats of one or two other short sequences (DR2 and sometimes DR4), while quasi-unique sequences are located between DR1 and either side of the DR2/DR4 repeats. These regions are termed Ub and Uc, and in virion DNA they lie adjacent to the S and L termini, respectively (6, 17, 18).An approximately 200-bp fragment (Uc-DR1-Ub) spanning the junction between tandem a sequences, such as is generated upon fusion of the genomic ends (Fig. ​(Fig.1b),1b), has been shown to contain all the essential cis-acting sequences necessary for DNA packaging (10, 20). Within the Ub and Uc regions are two domains, pac1 and pac2, respectively, which contain several characteristic sequence motifs that are conserved near the ends of other herpesvirus genomes (3, 8, 15). These motifs, as originally defined by Deiss et al. (8), are illustrated in Fig. ​Fig.1c.1c. It is now recognized that the major conserved motif within the pac1 region comprises the T-rich element flanked on each side by short G tracts (from the proximal and distal GC-rich regions). In the case of pac2, the T-rich element is most highly conserved with a consensus CGCGGCG motif also frequently being present (32).Detailed studies, employing primarily HSV-1 and murine cytomegalovirus (MCMV), have highlighted the roles of the major conserved motifs and suggested the following general mechanism by which concatemers are cleaved and packaged (1, 10, 13, 15, 16, 23, 25, 29, 32). Within Uc the most critical sequence is the pac2 T element, which is essential for cleavage to initiate DNA packaging. Cleavage occurs at a fixed distance from the pac2 T element, and the resulting Uc-containing end is inserted into the procapsid. Additional important cis-acting sequences are present further from the cleavage site, possibly including the pac2 consensus motif. Deletion, but not substitution, of the pac2 GC element and unconserved region impaired DNA packaging, suggesting that the relative spacing of the cleavage site, T element, and distal motifs is crucial. Packaging proceeds from pac2 toward the pac1 terminus, and a second cleavage event terminates DNA packaging. This cleavage appears to be directed by, and occurs at a fixed distance from, a single region comprising the pac1 T element and the flanking G tracts. Surprisingly, substitutions within the highly conserved T element are tolerated, but it remains unclear whether this region functions as a spacer element. The UL28 component of the HSV-1 terminase enzyme binds to a specific conformation adopted by the region comprising the T element and G tracts, and this interaction is likely to be crucial for cleavage.The functional analysis of herpesvirus DNA packaging signals has employed two major approaches. In the first, amplicons (i.e., bacterial plasmids containing a viral DNA replication origin and packaging signal) are transfected into mammalian cells and their ability to be replicated and packaged is assessed following the provision of viral helper functions, either by superinfection with virus particles or by cotransfection of virion DNA (7, 20, 24, 27, 29, 35). The second assay introduces an additional copy of the packaging signal under test at an ectopic site within the viral genome and determines whether it functions as a site for the cleavage of concatemeric DNA and the generation of novel terminal fragments of virion DNA (5, 15, 18, 23, 29, 32). Both these approaches, however, suffer from the disadvantage that recombination occurs between the test packaging signal and the wild-type (wt) signal present either in the helper virus or in its normal location within an ectopic-site recombinant (5, 8, 15, 23, 32). Additionally, concatemers generated following replication of amplicons have a significantly different structure from standard herpesviral genomes in that multiple copies of the packaging signal are present, spaced at regular intervals corresponding to the size of the input plasmid. This raises the possibility that the activity of wt or mutated packaging signals in the amplicon assay may not accurately reflect their behavior in a standard genome.To avoid these difficulties and allow analysis of mutated packaging signals in the context of the viral genome, we have used a cloned full-length HSV-1 genome, fHSVΔpac, which is complete with the exception that all copies of the a sequence have been deleted (22). This molecule is propagated as a bacterial artificial chromosome (BAC), and specific sequences can be inserted via homologous recombination either in mammalian cells or in the bacterial host. We previously demonstrated that a single copy of the minimal packaging signal Uc-DR1-Ub introduced into the viral thymidine kinase (TK) locus of fHSVΔpac by recombination in mammalian cells was sufficient to allow the products of replication to be packaged in mammalian cells and to allow the generation of viable progeny (28). Here, we describe the introduction of the packaging signal into fHSVΔpac by Red/ET recombination in Escherichia coli (19, 34), allowing previously described (10) and new Uc-DR1-Ub mutants to be screened for their ability to direct encapsidation, generate Uc- and Ub-containing terminal fragments, and give rise to progeny virus.     

RNA Sequence Features Are at the Core of Influenza A Virus Genome Packaging

The influenza A virus (IAV), a respiratory pathogen for humans, poses serious medical and economic challenges to global healthcare systems. The IAV genome, consisting of eight single-stranded viral RNA segments, is incorporated into virions by a complex process known as genome packaging. Specific RNA sequences within the viral RNA segments serve as signals that are necessary for genome packaging. Although efficient packaging is a prerequisite for viral infectivity, many of the mechanistic details about this process are still missing. In this review, we discuss the recent advances toward the understanding of IAV genome packaging and focus on the RNA features that play a role in this process.     

Atomic Force Microscopy Imaging of Double Stranded DNA and RNA

Abstract

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Electrostatic Regulation of Genome Packaging in Human Hepatitis B Virus

Hepatitis B virus (HBV) is a contagious human pathogen causing liver diseases such as cirrhosis and hepatocellular carcinoma. An essential step during HBV replication is packaging of a pregenomic (pg) RNA within the capsid of core antigens (HBcAgs) that each contains a flexible C-terminal tail rich in arginine residues. Mutagenesis experiments suggest that pgRNA encapsidation hinges on its strong electrostatic interaction with oppositely charged C-terminal tails of the HBcAgs, and that the net charge of the capsid and C-terminal tails determines the genome size and nucleocapsid stability. Here, we elucidate the biophysical basis for electrostatic regulation of pgRNA packaging in HBV by using a coarse-grained molecular model that explicitly accounts for all nonspecific interactions among key components within the nucleocapsid. We find that for mutants with variant C-terminal length, an optimal genome size minimizes an appropriately defined thermodynamic free energy. The thermodynamic driving force of RNA packaging arises from a combination of electrostatic interactions and molecular excluded-volume effects. The theoretical predictions of the RNA length and nucleocapsid internal structure are in good agreement with available experiments for the wild-type HBV and mutants with truncated HBcAg C-termini.     

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity.     

A Unique HMG-Box Domain of Mouse Maelstrom Binds Structured RNA but Not Double Stranded DNA

Piwi-interacting piRNAs are a major and essential class of small RNAs in the animal germ cells with a prominent role in transposon control. Efficient piRNA biogenesis and function require a cohort of proteins conserved throughout the animal kingdom. Here we studied Maelstrom (MAEL), which is essential for piRNA biogenesis and germ cell differentiation in flies and mice. MAEL contains a high mobility group (HMG)-box domain and a Maelstrom-specific domain with a presumptive RNase H-fold. We employed a combination of sequence analyses, structural and biochemical approaches to evaluate and compare nucleic acid binding of mouse MAEL HMG-box to that of canonical HMG-box domain proteins (SRY and HMGB1a). MAEL HMG-box failed to bind double-stranded (ds)DNA but bound to structured RNA. We also identified important roles of a novel cluster of arginine residues in MAEL HMG-box in these interactions. Cumulatively, our results suggest that the MAEL HMG-box domain may contribute to MAEL function in selective processing of retrotransposon RNA into piRNAs. In this regard, a cellular role of MAEL HMG-box domain is reminiscent of that of HMGB1 as a sentinel of immunogenic nucleic acids in the innate immune response.     

In Vitro Packaging of Adeno-Associated Virus DNA

We have developed an in vitro procedure for packaging of recombinant adeno-associated virus (AAV). By using AAV replicative-form DNA as the substrate, it is possible to synthesize an infectious AAV particle in vitro that can be used to transfer a marker gene to mammalian cells. The packaging procedure requires the presence of both the AAV Rep and capsid proteins. Two kinds of in vitro products can be formed which facilitate DNA transfer. Both are resistant to heat and have a density in cesium chloride gradients that is indistinguishable from that of the in vivo-synthesized wild-type virus. This indicates that the particles formed have the appropriate protein-to-DNA ratio and a structure that shares the heat resistance of mature AAV particles. The two types of particles can be distinguished by their sensitivity to chloroform and DNase I treatment. The chloroform-resistant product is, by several criteria, an authentic AAV particle. In addition to having the correct density and being resistant to treatment with chloroform, DNase I, and heat, this particle is efficiently synthesized only if the AAV genome contains intact terminal repeats, which are known to be required for AAV packaging. It is also precipitated by a monoclonal antibody that recognizes mature virus particles but not bound by an antibody that recognizes monomeric or denatured capsid proteins. The chloroform-resistant species is not made when aphidicolin is present in the reaction mixture, suggesting that active DNA replication is required for in vitro packaging. In contrast, the chloroform-sensitive product has several features that suggest it is an incompletely assembled virus particle. It is sensitive to DNase I, does not require the presence of AAV terminal repeats, and is capable of transferring DNA that is theoretically too large to package. Sucrose gradient centrifugation of the in vitro-synthesized products reveals that the particles have sedimentation values between 60S and 110S, which is consistent with partially assembled and mature AAV particles. The in vitro packaging procedure should be useful for studying the mechanism by which a human icosahedral DNA virus particle is assembled, and it may be useful for producing recombinant AAV for gene therapy. The chloroform-sensitive particle may also be useful for transferring DNA that is too large to be packaged in mature recombinant AAV.     

A Rapid Method for Isolation of Double Stranded RNA

A rapid and simple method for the isolation and purification of dsRNA is presented. The crucial step of this method is the extraction of proteins and DNA with acid phenol. After the extraction, only RNA is left in the aquaeous phase. ssRNA contamination of the RNA preparation can be greatly reduced when ammonium sulfate is present during the extraction.     

A Mathematical Model for Basepair Opening in a DNA Double Helix

In this paper, we consider a mathematical model that draws an analogy between a DNA molecule and a mechanical system consisting of two chains of interconnected pendulums. This model is designed to explore the dynamics of the system determined by rotational movements of nucleobases around a double-stranded pentose phosphate backbone. The workability of this model is assessed with respect to various factors: inhomogeneity of the chain of nucleobases, the properties of bonds in complementary pairs, and the formation of open states. It has been shown that simplified models for averaging the characteristics of the chain of nucleobases or simplification of the type of hydrogen bond in their complementary pairs influence the type of solution significantly, impairing the validity of the results. Therefore, the approach to the solution of rotational DNA molecule dynamics developed here is more consistent with its actual biomechanics. It is shown that the emergence of open states within nucleobase pairs and restoration of the closed structure may occur in the tested mathematical model.     

Virus with a Multipartite Superhelical DNA Genome from the Ichneumonid Parasitoid Campoletis sonorensis

Virus was isolated from the lumen of the calyx region of ovaries in the parasitoid wasp Campoletis sonorensis (Hymenoptera: Ichneumonidae), and the nature of the viral DNA was analyzed. DNA purified from a homogeneous band of virus contained double-stranded superhelical molecules which were polydisperse in molecular weight. At least 25 different covalently closed circles were present, ranging in molecular weight from 4.0 x 10(6) to 13.6 x 10(6). The virus DNA was analyzed with restriction enzymes, and the nature of the genetic complexity was evaluated by Southern blot hybridization of native superhelical and relaxed circular virus DNA and of SalI- and HindIII-digested DNA. The data suggest that most of the variously sized covalently closed DNAs were composed primarily of nonhomologous sequences. The different size classes of covalently closed viral DNAs did not appear to exist in equimolar concentrations. However, there was no evidence from observation of virus particles in the electron microscope or from virus fractionation experiments that a mixture of viruses was present in the calyx fluid. The results from this study suggest' that the virus isolated from C. sonorensis, like those isolated from other endoparasitic hymenoptera, may belong to a new class of DNA viruses in which the genome is multipartite, with each DNA existing as a superhelical molecule.