Understanding viral replication and transcription using single-molecule techniques.

DNA and RNA viruses depend on one or more enzymes to copy and transcribe their genome, such as a polymerase, helicase, or exonuclease. Because of the important role of these enzymes in the virus replication cycle, they are key targets for antiviral development. To better understand the function of these enzymes and their interactions with host and viral factors, biochemical, structural and single-molecule approaches have been used to study them. Each of these techniques has its own strengths, and single-molecule methods have proved particularly powerful in providing insight into the step-sizes of motor proteins, heterogeneity of enzymatic activities, transient conformational changes, and force-sensitivity of reactions. Here we will discuss how single-molecule FRET, magnetic tweezers, optical tweezers, atomic force microscopy and flow stretching approaches have revealed novel insights into polymerase fidelity, the mechanism of action of antivirals, and the protein choreography within replication complexes.

50th anniversary of the discovery of reverse transcriptase.

The simultaneous discovery in 1970 of reverse transcriptase in virions of retroviruses by Howard Temin and David Baltimore was perhaps the most dramatic scientific moment of the second half of the 20th century. Ten years previously, Temin's observation of cells transformed by Rous Sarcoma virus led him to the conclusion that retroviruses replicate through a DNA intermediate he called the provirus. This heretical hypothesis was greeted with derision by fellow scientists; Temin and Baltimore performed a simple experiment, rapidly reproduced, and convincing to all. Its result was a major paradigm shift-reversal of the central dogma of molecular biology. It immediately grabbed the attention of both the scientific and lay press. It also came at a key time for cancer research, at the start of the "War on Cancer." As a theoretical base and fundamental molecular tool, it enabled a decade of (largely fruitless) search for human oncogenic retroviruses but laid the foundation for the discovery of HIV 13 years later, leading to the development of effective therapy. I had the good fortune, as a student in Temin's lab, to witness these events. I am honored to be able to share my recollection on the occasion of their 50th anniversary.

Diversity and evolution of B-family DNA polymerases.

B-family DNA polymerases (PolBs) represent the most common replicases. PolB enzymes that require RNA (or DNA) primed templates for DNA synthesis are found in all domains of life and many DNA viruses. Despite extensive research on PolBs, their origins and evolution remain enigmatic. Massive accumulation of new genomic and metagenomic data from diverse habitats as well as availability of new structural information prompted us to conduct a comprehensive analysis of the PolB sequences, structures, domain organizations, taxonomic distribution and co-occurrence in genomes. Based on phylogenetic analysis, we identified a new, widespread group of bacterial PolBs that are more closely related to the catalytically active N-terminal half of the eukaryotic PolEpsilon (PolEpsilonN) than to Escherichia coli Pol II. In Archaea, we characterized six new groups of PolBs. Two of them show close relationships with eukaryotic PolBs, the first one with PolEpsilonN, and the second one with PolAlpha, PolDelta and PolZeta. In addition, structure comparisons suggested common origin of the catalytically inactive C-terminal half of PolEpsilon (PolEpsilonC) and PolAlpha. Finally, in certain archaeal PolBs we discovered C-terminal Zn-binding domains closely related to those of PolAlpha and PolEpsilonC. Collectively, the obtained results allowed us to propose a scenario for the evolution of eukaryotic PolBs.

Rational design of an XNA ligase through docking of unbound nucleic acids to toroidal proteins.

Xenobiotic nucleic acids (XNA) are nucleic acid analogues not present in nature that can be used for the storage of genetic information. In vivo XNA applications could be developed into novel biocontainment strategies, but are currently limited by the challenge of developing XNA processing enzymes such as polymerases, ligases and nucleases. Here, we present a structure-guided modelling-based strategy for the rational design of those enzymes essential for the development of XNA molecular biology. Docking of protein domains to unbound double-stranded nucleic acids is used to generate a first approximation of the extensive interaction of nucleic acid processing enzymes with their substrate. Molecular dynamics is used to optimise that prediction allowing, for the first time, the accurate prediction of how proteins that form toroidal complexes with nucleic acids interact with their substrate. Using the Chlorella virus DNA ligase as a proof of principle, we recapitulate the ligase's substrate specificity and successfully predict how to convert it into an XNA-templated XNA ligase.

Targeting the Viral Polymerase of Diarrhea-Causing Viruses as a Strategy to Develop a Single Broad-Spectrum Antiviral Therapy.

Viral gastroenteritis is an important cause of morbidity and mortality worldwide, being particularly severe for children under the age of five. The most common viral agents of gastroenteritis are noroviruses, rotaviruses, sapoviruses, astroviruses and adenoviruses, however, no specific antiviral treatment exists today against any of these pathogens. We here discuss the feasibility of developing a broad-spectrum antiviral treatment against these diarrhea-causing viruses. This review focuses on the viral polymerase as an antiviral target, as this is the most conserved viral protein among the diverse viral families to which these viruses belong to. We describe the functional and structural similarities of the different viral polymerases, the antiviral effect of reported polymerase inhibitors and highlight common features that might be exploited in an attempt of designing such pan-polymerase inhibitor.

The dUTPase of white spot syndrome virus assembles its active sites in a noncanonical manner.

dUTPases are essential enzymes for maintaining genome integrity and have recently been shown to play moonlighting roles when containing extra sequences. Interestingly, the trimeric dUTPase of white spot syndrome virus (wDUT) harbors a sequence insert at the position preceding the C-terminal catalytic motif V (pre-V insert), rarely seen in other dUTPases. However, whether this extra sequence endows wDUT with additional properties is unknown. Herein, we present the crystal structures of wDUT in both ligand-free and ligand-bound forms. We observed that the pre-V insert in wDUT forms an unusual ß-hairpin structure in the domain-swapping region and thereby facilitates a unique orientation of the adjacent C-terminal segment, positioning the catalytic motif V onto the active site of its own subunit instead of a third subunit. Consequently, wDUT employs two-subunit active sites, unlike the widely accepted paradigm that the active site of trimeric dUTPase is contributed by all three subunits. According to results from local structural comparisons, the active-site configuration of wDUT is similar to that of known dUTPases. However, we also found that residues in the second-shell region of the active site are reconfigured in wDUT as an adaption to its unique C-terminal orientation. We also show that deletion of the pre-V insert significantly reduces wDUT's enzymatic activity and thermal stability. We hypothesize that this rare structural arrangement confers additional functionality to wDUT. In conclusion, our study expands the structural diversity in the conserved dUTPase family and illustrates how sequence insertion and amino acid substitution drive protein evolution cooperatively.

Phaeocystis globosa Virus DNA Polymerase X: a "Swiss Army knife", Multifunctional DNA polymerase-lyase-ligase for Base Excision Repair.

Phaeocystis globosa virus 16T is a giant virus that belongs to the so-called nucleo-cytoplasmic large DNA virus (NCLDV) group. Its linear dsDNA genome contains an almost full complement of genes required to participate in viral base excision repair (BER). Among them is a gene coding for a bimodular protein consisting of an N-terminal Polß-like core fused to a C-terminal domain (PgVPolX), which shows homology with NAD+-dependent DNA ligases. Analysis of the biochemical features of the purified enzyme revealed that PgVPolX is a multifunctional protein that could act as a "Swiss army knife" enzyme during BER since it is endowed with: 1) a template-directed DNA polymerization activity, preferentially acting on DNA structures containing gaps; 2) 5'-deoxyribose-5-phosphate (dRP) and abasic (AP) site lyase activities; and 3) an NAD+-dependent DNA ligase activity. We show how the three activities act in concert to efficiently repair BER intermediates, leading us to suggest that PgVPolX may constitute, together with the viral AP-endonuclease, a BER pathway. This is the first time that this type of protein fusion has been demonstrated to be functional.

Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure.

The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size.

Lausannevirus Encodes a Functional Dihydrofolate Reductase Susceptible to Proguanil.

Lausannevirus belongs to the family Marseilleviridae within the group of nucleocytoplasmic large DNA viruses (NCLDVs). These giant viruses exhibit unique features, including a large genome, ranging from 100 kb to 2.5 Mb and including from 150 to more than 2,500 genes, as well as the presence of genes coding for proteins involved in transcription and translation. The large majority of Lausannevirus open reading frames have unknown functions. Interestingly, a bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is encoded in the Lausannevirus genome. The enzyme plays central roles in DNA precursor biosynthesis. DHFR is the pharmacological target of antifolates, such as trimethoprim, pyrimethamine, and proguanil. First, the functionality of Lausannevirus DHFR-TS was demonstrated by the successful complementation of a DHFR-deficient Saccharomyces cerevisiae strain with a plasmid expressing the heterologous gene. Additionally, using this heterologous expression system, we demonstrated the in vitro susceptibility of Lausannevirus DHFR-TS to proguanil and its resistance to pyrimethamine and trimethoprim. Proguanil may provide a unique and useful treatment if Lausannevirus proves to be a human pathogen. To our knowledge, this is the first time that a DHFR-TS has been described and characterized in an NCLDV.

Development of Potent Antiviral Drugs Inspired by Viral Hexameric DNA-Packaging Motors with Revolving Mechanism.

The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance necessitate the development of new potent antiviral drugs. Recently, a method for developing potent inhibitory drugs by targeting biological machines with high stoichiometry and a sequential-action mechanism was described. Inspired by this finding, we reviewed the development of antiviral drugs targeting viral DNA-packaging motors. Inhibiting multisubunit targets with sequential actions resembles breaking one bulb in a series of Christmas lights, which turns off the entire string. Indeed, studies on viral DNA packaging might lead to the development of new antiviral drugs. Recent elucidation of the mechanism of the viral double-stranded DNA (dsDNA)-packaging motor with sequential one-way revolving motion will promote the development of potent antiviral drugs with high specificity and efficiency. Traditionally, biomotors have been classified into two categories: linear and rotation motors. Recently discovered was a third type of biomotor, including the viral DNA-packaging motor, beside the bacterial DNA translocases, that uses a revolving mechanism without rotation. By analogy, rotation resembles the Earth's rotation on its own axis, while revolving resembles the Earth's revolving around the Sun (see animations at http://rnanano.osu.edu/movie.html). Herein, we review the structures of viral dsDNA-packaging motors, the stoichiometries of motor components, and the motion mechanisms of the motors. All viral dsDNA-packaging motors, including those of dsDNA/dsRNA bacteriophages, adenoviruses, poxviruses, herpesviruses, mimiviruses, megaviruses, pandoraviruses, and pithoviruses, contain a high-stoichiometry machine composed of multiple components that work cooperatively and sequentially. Thus, it is an ideal target for potent drug development based on the power function of the stoichiometries of target complexes that work sequentially.

Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase.

UNLABELLED: Sulfolobus turreted icosahedral virus (STIV), an archaeal virus that infects the hyperthermoacidophile Sulfolobus solfataricus, is one of the most well-studied viruses of the domain Archaea. STIV shares structural, morphological, and sequence similarities with viruses from other domains of life, all of which are thought to belong to the same viral lineage. Several of these common features include a conserved coat protein fold, an internal lipid membrane, and a DNA-packaging ATPase. B204 is the ATPase encoded by STIV and is thought to drive packaging of viral DNA during the replication process. Here, we report the crystal structure of B204 along with the biochemical analysis of B204 mutants chosen based on structural information and sequence conservation patterns observed among members of the same viral lineage and the larger FtsK/HerA superfamily to which B204 belongs. Both in vitro ATPase activity assays and transfection assays with mutant forms of B204 confirmed the essentiality of conserved and nonconserved positions. We also have identified two distinct particle morphologies during an STIV infection that differ in the presence or absence of the B204 protein. The biochemical and structural data presented here are not only informative for the STIV replication process but also can be useful in deciphering DNA-packaging mechanisms for other viruses belonging to this lineage. IMPORTANCE: STIV is a virus that infects a host from the domain Archaea that replicates in high-temperature, acidic environments. While STIV has many unique features, there exist several striking similarities between this virus and others that replicate in different environments and infect a broad range of hosts from Bacteria and Eukarya. Aside from structural features shared by viruses from this lineage, there exists a significant level of sequence similarity between the ATPase genes carried by these different viruses; this gene encodes an enzyme thought to provide energy that drives DNA packaging into the virion during infection. The experiments described here highlight the elements of this enzyme that are essential for proper function and also provide supporting evidence that B204 is present in the mature STIV virion.

Evolution of Eukaryotic DNA Polymerases via Interaction Between Cells and Large DNA Viruses.

B-family DNA-directed DNA polymerases are DNA replication enzymes found in Eukaryota, Archaea, large DNA viruses, and in some, but not all, bacteria. Several polymerase domains are conserved among the B-family DNA polymerases from these organisms, suggesting that the B-family DNA polymerases evolved from a common ancestor. Eukaryotes retain at least three replicative B-family DNA polymerases, DNA polymerase α, δ, and ε, and one translesion B-family DNA polymerase, DNA polymerase ζ. Here, we present molecular evolutionary evidence that suggests DNA polymerase genes evolved through horizontal gene transfer between the viral and archaeal-eukaryotic lineages. Molecular phylogenetic analyses of the B-family DNA polymerases from nucleo-cytoplasmic large DNA viruses (NCLDVs), eukaryotes, and archaea suggest that different NCLDV lineages such as Poxviridae and Mimiviridae were involved in the evolution of different DNA polymerases (pol-α-, δ-, ε-, and ζ-like genes) in archaeal-eukaryotic cell lineages, putatively through horizontal gene transfer. These results support existing theories that link the evolution of NCLDVs and the origin of the eukaryotic nucleus.

Sulfur oxidation genes in diverse deep-sea viruses.

Viruses are the most abundant biological entities in the oceans and a pervasive cause of mortality of microorganisms that drive biogeochemical cycles. Although the ecological and evolutionary effects of viruses on marine phototrophs are well recognized, little is known about their impact on ubiquitous marine lithotrophs. Here, we report 18 genome sequences of double-stranded DNA viruses that putatively infect widespread sulfur-oxidizing bacteria. Fifteen of these viral genomes contain auxiliary metabolic genes for the α and γ subunits of reverse dissimilatory sulfite reductase (rdsr). This enzyme oxidizes elemental sulfur, which is abundant in the hydrothermal plumes studied here. Our findings implicate viruses as a key agent in the sulfur cycle and as a reservoir of genetic diversity for bacterial enzymes that underpin chemosynthesis in the deep oceans.

Repair of base damage and genome maintenance in the nucleo-cytoplasmic large DNA viruses.

Among the DNA viruses, the so-called nucleo-cytoplasmic large DNA viruses (NCLDV) constitute a monophyletic group that currently consists of seven families of viruses infecting a very broad variety of eukaryotes, from unicellular marine protists to humans. Many recent papers have analyzed the sequence and structure of NCLDV genomes and their phylogeny, providing detailed analysis about their genomic structure and evolutionary history and proposing their inclusion in a new viral order named Megavirales that, according to some authors, should be considered as a fourth domain of life, aside from Bacteria, Archaea and Eukarya. The maintenance of genetic information protected from environmental attacks and mutations is essential not only for the survival of cellular organisms but also viruses. In cellular organisms, damaged DNA bases are removed in two major repair pathways: base excision repair (BER) and nucleotide incision repair (NIR) that constitute the major pathways responsible for repairing most endogenous base lesions and abnormal bases in the genome by precise repair procedures. Like cells, many NCLDV encode proteins that might constitute viral DNA repair pathways that would remove damages through BER/NIR pathways. However, the molecular mechanisms and, specially, the biological roles of those viral repair pathways have not been deeply addressed in the literature so far. In this paper, we review viral-encoded BER proteins and the genetic and biochemical data available about them. We propose and discuss probable viral-encoded DNA repair mechanisms and pathways, as compared with the functional and molecular features of known homologs proteins.

AAA ATPase p529 of Acidianus two-tailed virus ATV and host receptor recognition.

The two structural domains of p529, a predicted AAA ATPase of Acidianus two-tailed virus (ATV), were expressed and purified. The N-terminal domain was demonstrated by loss-of-function mutations to carry ATPase activity with a temperature optimum of 60°C. This domain also showed DNA binding activity that was stronger for the whole protein and was weakened in the presence of ATP. The C-terminal domain exhibits Mg(2+)-dependent endonuclease activity that was eliminated by site-directed mutagenesis at a conserved catalytic PD…D/ExK motif. p529 pull-down experiments with cell extracts of Sulfolobus solfataricus demonstrated a specific interaction with Sso1273, corresponding to OppA(Ss), an N-linked glycoprotein that specifically binds oligopeptides. The sso1273 gene lies in an operon encoding an oligopeptide/dipeptide ABC transporter system. It is proposed that p529 is involved in ATV-host cell receptor recognition and possibly the endonuclease activity is required for cleavage of the circular viral DNA prior to cell entry.

Computational analysis of DNA replicases in double-stranded DNA viruses: relationship with the genome size.

Genome duplication in free-living cellular organisms is performed by DNA replicases that always include a DNA polymerase, a DNA sliding clamp and a clamp loader. What are the evolutionary solutions for DNA replicases associated with smaller genomes? Are there some general principles? To address these questions we analyzed DNA replicases of double-stranded (ds) DNA viruses. In the process we discovered highly divergent B-family DNA polymerases in phiKZ-like phages and remote sliding clamp homologs in Ascoviridae family and Ma-LMM01 phage. The analysis revealed a clear dependency between DNA replicase components and the viral genome size. As the genome size increases, viruses universally encode their own DNA polymerases and frequently have homologs of DNA sliding clamps, which sometimes are accompanied by clamp loader subunits. This pattern is highly non-random. The absence of sliding clamps in large viral genomes usually coincides with the presence of atypical polymerases. Meanwhile, sliding clamp homologs, not accompanied by clamp loaders, have an elevated positive electrostatic potential, characteristic of non-ring viral processivity factors that bind the DNA directly. Unexpectedly, we found that similar electrostatic properties are shared by the eukaryotic 9-1-1 clamp subunits, Hus1 and, to a lesser extent, Rad9, also suggesting the possibility of direct DNA binding.

Functional dissection of the DNA interface of the nucleotidyltransferase domain of chlorella virus DNA ligase.

Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive ß-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3'-OH side of the nick. Here we performed a structure-guided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3'-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3'-OH nucleoside in the catalysis of DNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig.

Viral serine/threonine protein kinases.

Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets.

Evolution of DNA ligases of nucleo-cytoplasmic large DNA viruses of eukaryotes: a case of hidden complexity.

BACKGROUND: Eukaryotic Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) encode most if not all of the enzymes involved in their DNA replication. It has been inferred that genes for these enzymes were already present in the last common ancestor of the NCLDV. However, the details of the evolution of these genes that bear on the complexity of the putative ancestral NCLDV and on the evolutionary relationships between viruses and their hosts are not well understood. RESULTS: Phylogenetic analysis of the ATP-dependent and NAD-dependent DNA ligases encoded by the NCLDV reveals an unexpectedly complex evolutionary history. The NAD-dependent ligases are encoded only by a minority of NCLDV (including mimiviruses, some iridoviruses and entomopoxviruses) but phylogenetic analysis clearly indicated that all viral NAD-dependent ligases are monophyletic. Combined with the topology of the NCLDV tree derived by consensus of trees for universally conserved genes suggests that this enzyme was represented in the ancestral NCLDV. Phylogenetic analysis of ATP-dependent ligases that are encoded by chordopoxviruses, most of the phycodnaviruses and Marseillevirus failed to demonstrate monophyly and instead revealed an unexpectedly complex evolutionary trajectory. The ligases of the majority of phycodnaviruses and Marseillevirus seem to have evolved from bacteriophage or bacterial homologs; the ligase of one phycodnavirus, Emiliana huxlei virus, belongs to the eukaryotic DNA ligase I branch; and ligases of chordopoxviruses unequivocally cluster with eukaryotic DNA ligase III. CONCLUSIONS: Examination of phyletic patterns and phylogenetic analysis of DNA ligases of the NCLDV suggest that the common ancestor of the extant NCLDV encoded an NAD-dependent ligase that most likely was acquired from a bacteriophage at the early stages of evolution of eukaryotes. By contrast, ATP-dependent ligases from different prokaryotic and eukaryotic sources displaced the ancestral NAD-dependent ligase at different stages of subsequent evolution. These findings emphasize complex routes of viral evolution that become apparent through detailed phylogenomic analysis but not necessarily in reconstructions based on phyletic patterns of genes. REVIEWERS: This article was reviewed by: Patrick Forterre, George V. Shpakovski, and Igor B. Zhulin.