The Structure of the Cyprinid herpesvirus 3 ORF112-Zα·Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L, a Poxvirus Inhibitor of Interferon Response.

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.

2'-O methylation of the viral mRNA cap evades host restriction by IFIT family members.

Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2'-O positions of the 5' guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2'-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2'-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2'-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2'-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2'-O methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2'-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.

Diverse energetic effects of charge reversal mutations of poxvirus topoisomerase IB.

A key aspect of the reaction mechanism of type IB topoisomerases is the controlled unwinding of DNA supercoils while the enzyme is transiently bound to one strand of the DNA duplex via a phosphotyrosyl linkage. In this complex, the mobile segment of the bound DNA downstream from the site of cleavage must rotate around the helical axis, requiring that interactions with the enzyme must break and re-form multiple times during the course of removing supercoils. A crystal structure of variola virus type IB topoisomerase (vTopo) bound to DNA shows several positively charged side chains that interact with the downstream mobile and upstream rigid segments, suggesting that these groups may play a role in catalysis, including the processive unwinding of supercoils. We have mutated three such residues, R67, K35, and K271, to Ala and Glu and determined the energetic effects of these mutations at each point along the reaction coordinate of vTopo. R67 interacts with a phosphate group in the rigid DNA segment across from the site of DNA strand cleavage. The ~30-fold damaging effects of the R67A and R67E mutations were primarily on the phosphoryl transfer step, with little effect on enzyme-DNA binding, or the processivity of supercoil unwinding. Removal of the K35 interaction shows mutational effects similar to those of R67, even though this residue interacts with the mobile segment 3 bp from the cleavage site. The two mutations of K271, which interacts with the mobile region even further from the site of covalent linkage, show significant effects not only on phosphoryl transfer but also on downstream DNA strand positioning. Moreover, supercoil unwinding measurements indicate that the K271A and K271E mutations increase the average number of supercoils that are removed during the lifetime of the covalent complex, enhancing the processivity of supercoil unwinding. These measurements support the proposal that the processivity of supercoil unwinding can be regulated by electrostatic interactions between the enzyme and the mobile DNA phosphate backbone.

Cell cycle deregulation by a poxvirus partial mimic of anaphase-promoting complex subunit 11.

The anaphase-promoting complex (APC), or cyclosome, is a ubiquitin ligase with major roles in cell cycle regulation. It is required for mitotic exit, but must be deactivated for the G(1)/S phase transition to occur. APC consists of at least 12 subunits with the catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. APC11 facilitates ubiquitin chain formation by recruiting ubiquitin-charged conjugating enzymes through its RING-H2 domain. We report that a small number of poxviruses encode RING-H2 proteins with sequence similarities to APC11. We show that a representative of these viral proteins mimics APC11 in its interactions with APC, but unlike APC11, the viral protein fails to promote ubiquitin chain formation. This absence of ubiquitin ligase activity is linked to a distinctive sequence variation within its RING-H2 domain. Expression of the viral protein led to cell cycle deregulation and the accumulation of APC substrates in a manner consistent with impaired APC function. Our data characterize this protein as a regulator of APC activity, and consequently, we have called it PACR (poxvirus APC/cyclosome regulator). Deletion of the PACR gene substantially reduced viral replication. Here, we report a viral mimic of an APC component and reveal an intriguing mechanism by which viruses can manipulate cell cycle progression and, thereby, promote their own replication.

Progress on Poxvirus E3 Ubiquitin Ligases and Adaptor Proteins.

Poxviruses have evolved a variety of innate immunity evasion mechanisms, some of which involve poxvirus-encoded E3 ubiquitin ligases and adaptor proteins. Based on their functional domains and ubiquitin transfer mechanisms, these poxvirus-encoded E3 ubiquitin ligases and adaptor proteins can be divided into five categories: PRANC, ANK/BC, BBK, P28/RING, and MARCH proteins. Although the substrates of many poxvirus E3 ubiquitin ligases remain to be discovered, most of the identified substrates are components of the innate immune system. In this review, we discuss the current research progress on poxvirus-encoded E3 ubiquitin ligases and adaptor proteins to provide mechanistic insights into the interplay between these viruses and their hosts.

Poxvirus DNA primase.

Poxviruses are large enveloped viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. At least six virus-encoded proteins are required for synthesis and processing of the double-stranded DNA genome of vaccinia virus, the prototype member of the family. One of these proteins, D5, is an NTPase that contains an N-terminal archaeoeukaryotic primase domain and a C-terminal superfamily III helicase domain. Here we report that individual conserved aspartic acid residues in the predicted primase active site were required for in vivo complementation of infectious virus formation as well as genome and plasmid replication. Furthermore, purified recombinant D5 protein synthesized oligoribonucleotides in vitro. Incorporation of label from [alpha-(32)P]CTP or [alpha-(32)P]UTP into a RNase-sensitive and DNase-resistant product was demonstrated by using single-stranded circular bacteriophage DNA templates and depended on ATP or GTP and a divalent cation. Mutagenesis studies showed that the primase and NTPase activities of the recombinant D5 protein could be independently inactivated. Highly conserved orthologs of D5 are present in all poxviruses that have been sequenced, and more diverged orthologs are found in members of all other families of nucleocytoplasmic large DNA viruses. These viral primases may have roles in initiation of DNA replication or lagging-strand synthesis and represent potential therapeutic targets.

The circle is broken: telomere resolution in linear replicons.

Linear DNA molecules with covalently closed hairpin ends (telomeres) exist in a wide variety of organisms. Telomere resolution, a DNA breakage and reunion reaction in which replicated telomeres are processed into hairpin ends, is now known to be a common theme in poxviruses, Borrelia burgdorferi and Escherichia coli phage N15. Candidate proteins that may perform this reaction have recently been identified in poxviruses. Moreover, the first purification and definitive identification of a telomere resolvase has been reported for phage N15. This protein is the prototype for a new class of DNA enzyme that performs a unique reaction. Advances in the study of telomere resolution in poxviruses, B. burgdorferi and E. coli phage N15 are discussed.

Poxvirus uracil-DNA glycosylase-An unusual member of the family I uracil-DNA glycosylases.

Uracil-DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil-DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non-enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus-specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA-binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. The adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three-dimensional structure of D4. An overview of the current state of the knowledge on the structure-function relationship of D4 is provided here.

Viral thymidine kinases and their relatives.

Thymidine kinases were described for cellular life long before it was shown that they could also be encoded by viruses, but the viral thymidine kinase genes were the first to be sequenced. These enzymes have been extraordinarily useful to the researcher, serving first to help label DNA, then to get thymidine analogs incorporated into DNA for therapeutic and other purposes and more recently to move genes from one genome to another. Knowledge of the nucleotide and amino acid sequences of these enzymes has allowed some deductions about their possible three-dimensional structure, as well as the location on the polypeptide of various functions; it has also allowed their classification into two main groups: the herpesviral thymidine/eukaryotic deoxycytidine kinases and the poxviral and cellular thymidine kinases; the relationships of the mitochondrial enzyme are still not clear.

A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues.

Phosphatidylcholine-specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols. Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein. Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid-specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family. This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N-terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp. putative protein with a C-terminal domain likely to possess a DNA-binding function. Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi-lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event. It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two-step ping pong mechanism involving an enzyme-substrate intermediate.

A rapid method for identifying the thymidine kinase genes of avipoxviruses.

The thymidine kinase (TK) genes of poxviruses can be rapidly located without using TK- mutants or having to restriction map and clone the viral genomes. Identification of the TK gene is based on in situ gel hybridization with an end-labelled degenerate oligonucleotide probe, representing a consensus sequence near the 3' end of the gene. Restriction fragments of the viral DNAs are electrophoresed in agarose gels and annealed with the probe. Using this method, the TK genes of fowl pox (FPV) and quail pox (QPV) viruses were initially localized to HindIII fragments of approximately 3.8 and 6.7 kb, respectively. After inserting these fragments into pUC 19, recombinant plasmids containing the TK genes were screened by a modified in situ gel annealing procedure. Restriction mapping of the two cloned fragments and subsequent hybridization analysis more precisely placed at least the 3' portion of the FPV and QPV TK genes within a 1.4 kb ClaI-XbaI and 1.7 kb ClaI-PstI fragment, respectively. The site of the FPV TK gene was verified by comparison to the mapped position of the similar gene in an Australian FPV. The location of the QPV TK gene was confirmed by hybridization with the FPV TK gene, despite the apparent divergency of these two genes.

Methods for analysis of poxvirus DNA replication.

Cytoplasmic replication of poxviruses dictates the encoding of most, if not all, of the trans-acting factors required for faithful genome duplication. Several of these proteins have been identified through genetic and biochemical evaluation, including the catalytic DNA polymerase (E9), an essential and stoichiometric component of the processive polymerase (A20), a single-strand DNA-binding protein (I3), a type I topoisomerase (H6), the uracil DNA glycosylase (D4), a nucleic acid-independent nucleoside triphosphatase (D5), a serine/threonine protein kinase (B1), and a Holliday Junction resolvase (A22). All of these factors work in concert to faithfully duplicate the viral genome. Although a replication origin has not been defined for the poxviruses, cis-acting sequences found within the telomeric 200 bp have been implicated as necessary and sufficient for minichromosome replication. Replication occurs within cytoplasmic foci from approx 3 to 12 h postinfection. This chapter includes several methodologies to assay and quantitate replication in vivo, visualize replication foci microscopically, and test the integrity of central replication enzymes in vitro.

Identification and sequencing of the Choristoneura biennis entomopoxvirus DNA polymerase gene.

A degenerate oligonucleotide probe corresponding to a highly conserved amino acid sequence in several DNA polymerases was used to locate the DNA polymerase gene in the Choristoneura biennis entomopoxvirus. Southern blot analysis of the entomopoxvirus genome using the degenerate oligonucleotide probe showed specific interaction between the probe and an eight kilobasepair EcoRI fragment from the entomopoxvirus genome. Sequencing this EcoRI fragment revealed an open reading frame 2892 nucleotides in length, capable of encoding a protein about 115 kilodaltons. Homology search of this open reading frame against other proteins indicated a high degree of homology in four distinct regions with DNA polymerases from other organisms. The highest degree of homology (24.9% at the amino acid level) was found between the vaccinia DNA polymerase gene and the entomopoxvirus open reading frame.

Degradation of host microRNAs by poxvirus poly(A) polymerase reveals terminal RNA methylation as a protective antiviral mechanism.

The life cycle of several viruses involves host or virally encoded small noncoding RNAs, which play important roles in posttranscriptional regulation. Small noncoding RNAs include microRNAs (miRNAs), which modulate the transcriptome, and small interfering RNAs (siRNAs), which are involved in pathogen defense in plants, worms, and insects. We show that insect and mammalian poxviruses induce the degradation of host miRNAs. The virally encoded poly(A) polymerase, which polyadenylates viral transcripts, also mediates 3' polyadenylation of host miRNAs, resulting in their degradation by the host machinery. In contrast, siRNAs, which are protected by 2'O-methylation (2'OMe), were not targeted by poxviruses. These findings suggest that poxviruses may degrade host miRNAs to promote replication and that virus-mediated small RNA degradation likely contributed to 2'OMe evolution.