Nucleic acid packaging in viruses

We review recent literature describing protein nucleic acid interactions and nucleic acid organization in viruses. The nature of the viral genome determines its overall organization and its interactions with the capsid protein. Genomes composed of single strand (ss) RNA and DNA are highly flexible and, in some cases, adapt to the symmetry of the particle-forming protein to show repeated, sequence independent, nucleoprotein interactions. Genomes composed of double-stranded (ds) DNA do not interact strongly with the container due to their intrinsic stiffness, but form well-organized layers in virions. Assembly of virions with ssDNA and ssRNA genomes usually occurs through a cooperative condensation of the protein and genome, while dsDNA viruses usually pump the genome into a preformed capsid with a strong, virally encoded, molecular motor complex. We present data that suggest the packing density of ss genomes and ds genomes are comparable, but the latter exhibit far higher pressures due to their stiffness.     

Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization.

Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occurred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurrence of encapsidation of at least some of the viral genomes while they are still engaged in replication.     

Inducing the replacement of PNA in DNA.PNA duplexes by DNA

The uncharged DNA-analogue peptide nucleic acid (PNA) can invade into dsDNA by displacing the non-complementary DNA strand. The formed strand displacement complexes can create a sterical hindrance to block access of enzymes such as nucleases and polymerases. Due to the high stability of DNA.PNA duplexes it is usually not possible to displace the PNA strand by ssDNA or ssRNA. We herein report that the polycationic, comb-type copolymer alphaPLL-g-Dex can induce such a replacement of PNA in DNA.PNA duplexes by ssDNA. The influence of the copolymer on strand exchange highly depends on the nature of the oligonucleotides. Acceleration has only been observed when both the starting duplex and the single-stranded exchanger strand were negatively charged. The presented approach should allow the withdrawal of PNA induced sterical hindrance of DNA by rehybridisation with ssDNA.     

Marked Variability in the Extent of Protein Disorder within and between Viral Families

Intrinsically disordered regions in eukaryotic proteomes contain key signaling and regulatory modules and mediate interactions with many proteins. Many viral proteomes encode disordered proteins and modulate host factors through the use of short linear motifs (SLiMs) embedded within disordered regions. However, the degree of viral protein disorder across different viruses is not well understood, so we set out to establish the constraints acting on viruses, in terms of their use of disordered protein regions. We surveyed predicted disorder across 2,278 available viral genomes in 41 families, and correlated the extent of disorder with genome size and other factors. Protein disorder varies strikingly between viral families (from 2.9% to 23.1% of residues), and also within families. However, this substantial variation did not follow the established trend among their hosts, with increasing disorder seen across eubacterial, archaebacterial, protists, and multicellular eukaryotes. For example, among large mammalian viruses, poxviruses and herpesviruses showed markedly differing disorder (5.6% and 17.9%, respectively). Viral families with smaller genome sizes have more disorder within each of five main viral types (ssDNA, dsDNA, ssRNA+, dsRNA, retroviruses), except for negative single-stranded RNA viruses, where disorder increased with genome size. However, surveying over all viruses, which compares tiny and enormous viruses over a much bigger range of genome sizes, there is no strong association of genome size with protein disorder. We conclude that there is extensive variation in the disorder content of viral proteomes. While a proportion of this may relate to base composition, to extent of gene overlap, and to genome size within viral types, there remain important additional family and virus-specific effects. Differing disorder strategies are likely to impact on how different viruses modulate host factors, and on how rapidly viruses can evolve novel instances of SLiMs subverting host functions, such as innate and acquired immunity.     

A Phage Single-Stranded DNA (ssDNA) Binding Protein Complements ssDNA Accumulation of a Geminivirus and Interferes with Viral Movement

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.     

Mechanisms for RNA Capture by ssDNA Viruses: Grand Theft RNA

Viruses contain three common types of packaged genomes; double-stranded DNA (dsDNA), RNA (mostly single and occasionally double stranded) and single-stranded DNA (ssDNA). There are relatively straightforward explanations for the prevalence of viruses with dsDNA and RNA genomes, but the evolutionary basis for the apparent success of ssDNA viruses is less clear. The recent discovery of four ssDNA virus genomes that appear to have been formed by recombination between co-infecting RNA and ssDNA viruses, together with the high mutation rate of ssDNA viruses provide possible explanations. RNA–DNA recombination allows ssDNA viruses to access much broader sequence space than through nucleotide substitution and DNA–DNA recombination alone. Multiple non-exclusive mechanisms, all due to the unique replication of ssDNA viruses, are proposed for this unusual RNA capture. RNA capture provides an explanation for the evolutionary success of the ssDNA viruses and may help elucidate the mystery of integrated RNA viruses in viral and cellular DNA genomes.     

Substrate specificities of bacterial and human AlkB proteins

Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.     

Structural and functional analyses of five conserved positively charged residues in the L1 and N-terminal DNA binding motifs of archaeal RADA protein

RecA family proteins engage in an ATP-dependent DNA strand exchange reaction that includes a ssDNA nucleoprotein helical filament and a homologous dsDNA sequence. In spite of more than 20 years of efforts, the molecular mechanism of homology pairing and strand exchange is still not fully understood. Here we report a crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif) and the dsDNA binding N-terminal domain (NTD). L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229) surround a 25 A pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. Biochemical analyses demonstrate that these five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation. We suggest that the overwound right-handed RadA filament represents a functional conformation in the homology search and pairing reaction. A new structural model is proposed for the homologous interactions between a RadA-ssDNA nucleoprotein filament and its dsDNA target.     

Nucleic acid binding activity of pns6 encoded by genome segment 6 of rice ragged stunt oryzavirus

Rice ragged stunt disease, caused by rice ragged stuntoryzavirus (RRSV), was first discovered in 1976–1977 inIndonesia and Philippines [1]. Subsequently the diseasewas found in most rice-growing countries in south-easternand far-eastern Asia [2] and may inflict heavy loss on thecrop. RRSV is the type species of the genus Oryzavirus in thefamily Reoviridae. The virus particle is icosahedral witha diameter of about 65–70 nm and the genome consistsof 10 double stranded RNA (dsRNA) segm…     

Genome packaging within icosahedral capsids and large-scale segmentation in viral genomic sequences

The assembly and maturation of viruses with icosahedral capsids must be coordinated with icosahedral symmetry. The icosahedral symmetry imposes also the restrictions on the cooperative specific interactions between genomic RNA/DNA and coat proteins that should be reflected in quasi-regular segmentation of viral genomic sequences. Combining discrete direct and double Fourier transforms, we studied the quasi-regular large-scale segmentation in genomic sequences of different ssRNA, ssDNA, and dsDNA viruses. The particular representatives included satellite tobacco mosaic virus (STMV) and the strains of satellite tobacco necrosis virus (STNV), STNV-C, STNV-1, STNV-2, Escherichia phages MS2, ?X174, α3, and HK97, and Simian virus 40. In all their genomes, we found the significant quasi-regular segmentation of genomic sequences related to the virion assembly and the genome packaging within icosahedral capsid. We also found good correspondence between our results and available cryo-electron microscopy data on capsid structures and genome packaging in these viruses. Fourier analysis of genomic sequences provides the additional insight into mechanisms of hierarchical genome packaging and may be used for verification of the concepts of 3-fold or 5-fold intermediates in virion assembly. The results of sequence analysis should be taken into account at the choice of models and data interpretation. They also may be helpful for the development of antiviral drugs.     

DNA recognition of a 24-mer peptide derived from RecA protein

A novel 24-residue peptide (L2-G), Ile-Arg-Met-Lys-Ile-Gly-Val-Met-Phe-Gly-Asn-Pro-Glu-Thr-Thr-Thr-Gly-Gly-Asn-Ala-Leu-Lys-Phe-Tyr, derived from RecA can discriminate a single-stranded DNA (ssDNA) from a double-stranded DNA (dsDNA) and a new developed support with this peptide recognizes not dsDNA but ssDNA. The 24-mer peptide with L2 and helix G amino acids of Escherichia coli RecA protein showed the ssDNA binding property with more than 1000 times affinity difference for the dsDNA. However, truncated 15-mer peptide showed no ssDNA binding activity. In the ssDNA binding, L2-G changed its conformation with the perturbation of an alpha-helix structure. The ssDNA binding and the DNA discrimination property of this peptide were due to almost all L2 and helix G amino acids, respectively. This result is useful to design synthetic peptides as functional materials for DNA recognition.     

Characterization of human herpesvirus 6A/B U94 as ATPase,helicase, exonuclease and DNA-binding proteins

Human herpesvirus-6A (HHV-6A) and HHV-6B integrate their genomes into the telomeres of human chromosomes, however, the mechanisms leading to integration remain unknown. HHV-6A/B encode a protein that has been proposed to be involved in integration termed U94, an ortholog of adeno-associated virus type 2 (AAV-2) Rep68 integrase. In this report, we addressed whether purified recombinant maltose-binding protein (MBP)-U94 fusion proteins of HHV-6A/B possess biological functions compatible with viral integration. We could demonstrate that MBP-U94 efficiently binds both dsDNA and ssDNA containing telomeric repeats using gel shift assay and surface plasmon resonance. MBP-U94 is also able to hydrolyze adenosine triphosphate (ATP) to ADP, providing the energy for further catalytic activities. In addition, U94 displays a 3′ to 5′ exonuclease activity on dsDNA with a preference for 3′-recessed ends. Once the DNA strand reaches 8–10 nt in length, the enzyme dissociates it from the complementary strand. Lastly, MBP-U94 compromises the integrity of a synthetic telomeric D-loop through exonuclease attack at the 3′ end of the invading strand. The preferential DNA binding of MBP-U94 to telomeric sequences, its ability to hydrolyze ATP and its exonuclease/helicase activities suggest that U94 possesses all functions required for HHV-6A/B chromosomal integration.     

Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem.     

The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.     

Targeted recombination with single-stranded DNA vectors in mammalian cells.

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells.