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.     

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.     

Analysis by in situ hybridization and autoradiography of sites of replication and storage of single- and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells

The distribution in the different compartments of infected nuclei of double-stranded (ds) and single-stranded (ss) adenovirus type 5 (Ad5) DNA and of the sites of viral DNA replication were examined on thin sections of Low-icryl-embedded material. The DNA is visualized with a biotinylated viral probe and immunogold labeling of biotin, and its replication is monitored by high-resolution autoradiography after short pulses with tritiated thymidine. The first detectable sites of viral DNA, named early replicative sites, contained all the ss and ds viral DNA and viral replicative activity. At a later stage of nuclear transformation, they gave rise to two new structures. The compact fibrillar ssDNA accumulation sites enlarged greatly and became transformed functionally to become a transient site of accumulation of large numbers of ss replicative intermediates. Double-stranded viral DNA and its replicative activity shifted primarily into immediately surrounding fibrillogranular peripheral replicative zones. Ad5 DNA replication continues in the ssDNA accumulation sites but it is intermittent, whereas in the peripheral replicative zones it is continuous. Still later in infection, a single, large, centrally located mass of dense fibrils, the viral genome storage site, developed in each nucleus which proved to be the main site of storage of nonreplicating, nonencapsidated, ds viral genomes. We discuss the possible distribution of the various viral DNA replicative intermediates among these virus-induced intranuclear structures.     

Nuclear targeting of SV40 and adenovirus

Import o f viral DNA into the nucleus is essential for the successful replication o f DNA tumour viruses. To achieve this goal, viruses have adapted strategies to traverse the barriers between the plasma membrane and the nucleus o f a host cell. Two DNA tumour viruses, simian virus 40 and adenovirus, achieve the nuclear-entry step in slightly different ways. SV40 DNA enters the nucleus through the nuclear pore complexes (NPCs) in apparently intact virions. By contrast, adenovirus particles dissociate near the NPC before the viral DNA is imported into the nucleus. In both cases, karyophilic protein components o f the viruses appear to mediate nuclear entry o f the viral genomes. In this article, we discuss how an understanding o f the cell biology o f virus entry can help us understand the process o f nuclear transport.     

Analysis by in Situ hybridization and autoradiography of sites of replication and storage of single- and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells

The distribution in the different compartments of infected nuclei of double-stranded (ds) and single-stranded (ss) adenovirus type 5 (Ad5) DNA and of the sites of viral DNA replication were examined on thin sections of Lowicryl-embedded material. The DNA is visualized with a biotinylated viral probe and immunogold labeling of biotin, and its replication is monitored by high-resolution autoradiography after short pulses with tritiated thymidine. The first detectable sites of viral DNA, named early replicative sites, contained all the ss and ds viral DNA and viral replicative activity. At a later stage of nuclear transformation, they gave rise to two new structures. The compact fibrillar ssDNA accumulation sites enlarged greatly and became transformed functionally to become a transient site of accumulation of large numbers of ss replicative intermediates. Double-stranded viral DNA and its replicative activity shifted primarily into immediately surrounding fibrillogranular peripheral replicative zones. Ad5 DNA replication continues in the ssDNA accumulation sites but it is intermittent, whereas in the peripheral replicative zones it is continuous. Still later in infection, a single, large, centrally located mass of dense fibrils, the viral genome storage site, developed in each nucleus which proved to be the main site of storage of nonreplicating, nonencapsidated, ds viral genomes. We discuss the possible distribution of the various viral DNA replicative intermediates among these virus-induced intranuclear structures.     

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.     

Nuclear marginalization of host cell chromatin associated with expansion of two discrete virus-induced subnuclear compartments during baculovirus infection

Chromatin structure is strictly regulated during the cell cycle. DNA viruses occasionally disturb the spatial organization of the host cell chromatin due to formation of the viral DNA replication compartment. To examine chromatin behavior in baculovirus-infected cells, we constructed recombinant plasmids expressing fluorescent protein-tagged histone H4 molecules and visualized the intracellular localization of chromatin by their transient expression in live infected cells. Similar to other DNA viruses, the baculovirus Bombyx mori nucleopolyhedrovirus induced marginal relocation of chromatin within the nuclei of BmN cells, simultaneously with expansion of the viral DNA replication compartment, the virogenic stroma (VS). In the late stage of infection, however, the peristromal region (PR), another virus-induced subnuclear compartment, was also excluded from the chromatin-localizing area. Provided that late-gene products such as PR proteins (e.g., envelope proteins of the occlusion-derived virus) were expressed, blockage of viral DNA synthesis failed to inhibit chromatin relocation, despite abrogation of VS expansion. Instead, chromatin became marginalized concomitantly with PR expansion, suggesting that the PR contributes directly to chromatin replacement. In addition, chromatin was excluded from relatively large subnuclear structures that were induced in uninfected cells by cotransfection with four baculovirus genes, ie1, lef3, p143, and hr. Omission of any of the four genes, however, failed to result in formation of the large structures or chromatin exclusion. This correlation between compartmentalization and chromatin exclusion suggests the possibility that a chromatin-exclusive property of viral molecules, at least in part, supports nuclear compartmentalization of virus-infected cells.     

Nuclear import and export of plant virus proteins and genomes

Nuclear import and export are crucial processes for any eukaryotic cell, as they govern substrate exchange between the nucleus and the cytoplasm. Proteins involved in the nuclear transport network are generally conserved among eukaryotes, from yeast and fungi to animals and plants. Various pathogens, including some plant viruses, need to enter the host nucleus to gain access to its replication machinery or to integrate their DNA into the host genome; the newly replicated viral genomes then need to exit the nucleus to spread between host cells. To gain the ability to enter and exit the nucleus, these pathogens encode proteins that recognize cellular nuclear transport receptors and utilize the host's nuclear import and export pathways. Here, we review and discuss our current knowledge about the molecular mechanisms by which plant viruses find their way into and out of the host cell nucleus.     

Monitoring Insect Transposable Elements in Large Double-Stranded DNA Viruses Reveals Host-to-Virus and Virus-to-Virus Transposition

The mechanisms by which transposable elements (TEs) can be horizontally transferred between animals are unknown, but viruses are possible candidate vectors. Here, we surveyed the presence of host-derived TEs in viral genomes in 35 deep sequencing data sets produced from 11 host–virus systems, encompassing nine arthropod host species (five lepidopterans, two dipterans, and two crustaceans) and six different double-stranded (ds) DNA viruses (four baculoviruses and two iridoviruses). We found evidence of viral-borne TEs in 14 data sets, with frequencies of viral genomes carrying a TE ranging from 0.01% to 26.33% for baculoviruses and from 0.45% to 7.36% for iridoviruses. The analysis of viral populations separated by a single replication cycle revealed that viral-borne TEs originating from an initial host species can be retrieved after viral replication in another host species, sometimes at higher frequencies. Furthermore, we detected a strong increase in the number of integrations in a viral population for a TE absent from the hosts’ genomes, indicating that this TE has undergone intense transposition within the viral population. Finally, we provide evidence that many TEs found integrated in viral genomes (15/41) have been horizontally transferred in insects. Altogether, our results indicate that multiple large dsDNA viruses have the capacity to shuttle TEs in insects and they underline the potential of viruses to act as vectors of horizontal transfer of TEs. Furthermore, the finding that TEs can transpose between viral genomes of a viral species sets viruses as possible new niches in which TEs can persist and evolve.     

Kinesin-1-mediated capsid disassembly and disruption of the nuclear pore complex promote virus infection

Many viruses deliver their genomes into the host cell nucleus for replication. However, the size restrictions of the nuclear pore complex (NPC), which regulates the passage of proteins, nucleic acids, and solutes through the nuclear envelope, require virus capsid uncoating before viral DNA can access the nucleus. We report a microtubule motor kinesin-1-mediated and NPC-supported mechanism of adenovirus uncoating. The capsid binds to the NPC filament protein Nup214 and kinesin-1 light-chain Klc1/2. The nucleoporin Nup358, which is bound to Nup214/Nup88, interacts with the kinesin-1 heavy-chain Kif5c to indirectly link the capsid to the kinesin motor. Kinesin-1 disrupts capsids docked at Nup214, which compromises the NPC and dislocates nucleoporins and capsid fragments into the cytoplasm. NPC disruption increases nuclear envelope permeability as indicated by the nuclear influx of large cytoplasmic?dextran polymers. Thus, kinesin-1 uncoats viral DNA?and compromises NPC integrity, allowing viral genomes nuclear access to promote infection.     

Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.     

Replication initiation complex formation in the absence of nuclear function in Xenopus

In this article, we study how intercalation-induced changes in chromatin and DNA topology affect chromosomal DNA replication using Xenopus egg extracts. Unexpectedly, intercalation by ethidium or doxorubicin prevents formation of a functional nucleus: although nucleosome formation occurs, DNA decondensation is arrested, membranous vesicles accumulate around DNA but do not fuse to form a nuclear membrane, active transport is abolished and lamins are found on chromatin, but do not assemble into a lamina. DNA replication is inhibited at the stage of initiation complex activation, as shown by molecular combing of DNA and by the absence of checkpoint activation. Replication of single-stranded DNA is not prevented. Surprisingly, in spite of the absence of nuclear function, DNA-replication proteins of pre-replication and initiation complexes are loaded onto chromatin. This is a general phenomenon as initiation complexes could also be seen without ethidium in membrane-depleted extracts which do not form nuclei. These results suggest that DNA or chromatin topology is required for generation of a functional nucleus, and activation, but not formation, of initiation complexes.     

The E1 replication protein of bovine papillomavirus type 1 contains an extended nuclear localization signal that includes a p34cdc2 phosphorylation site.

Bovine papillomavirus (BPV) DNA replication occurs in the nucleus of infected cells. Most enzymatic activities are carried out by host cell proteins, with the viral E1 and E2 proteins required for the assembly of an initiation complex at the replication origin. In latently infected cells, viral DNA replication occurs in synchrony with the host cell chromosomes, maintaining a constant average copy number of BPV genomes per infected cell. By analyzing a series of mutants of the amino-terminal region of the E1 protein, we have identified the signal for transport of this protein to the cell nucleus. The E1 nuclear transport motif is highly conserved in the animal and human papillomaviruses and is encoded in a similar region in the related E1 genes. The signal is extended relative to the simple nuclear localization signals and contains two short amino acid sequences which contribute to nuclear transport, located between amino acids 85 and 108 of the BPV-1 E1 protein. Mutations in either basic region reduce nuclear transport of E1 protein and interfere with viral DNA replication. Mutations in both sequences simultaneously prevent any observable accumulation of the protein and reduce replication in transient assays to barely detectable levels. Surprisingly, these mutations had no effect on the ability of viral genomes to morphologically transform cells, although the plasmid DNA in the transformed cells was maintained at a very low copy number. Between these two basic amino acid blocks in the nuclear transport signal, at threonine 102, is a putative site for phosphorylation by the cell cycle regulated kinase p34cdc2. Utilizing an E1 protein purified from either a baculovirus vector system or Escherichia coli, we have shown that the E1 protein is a substrate for this kinase. An E1 gene mutant at threonine 102 encodes for a protein which is no longer a substrate for the p34cdc2 kinase. Mutation of this threonine to isoleucine had no observable effect on either nuclear localization of E1 or DNA replication of the intact viral genome.