Holliday junction resolving enzymes of archaeal viruses SIRV1 and SIRV2

In the final stages of genetic recombination, Holliday junction resolving enzymes transform the four-way DNA intermediate into two duplex DNA molecules by introducing pairs of staggered nicks flanking the junction. This fundamental process is apparently common to cells from all three domains of life. Two cellular resolving enzymes from extremely thermophilic representatives of both kingdoms of the domain Archaea, the euryarchaeon Pyrococcus furiosus and the crenarchaeon Sulfolobus solfataricus, have been described recently. Here we report for the first time the isolation, purification and characterization of Holliday junction cleaving enzymes (Hjc) from two archaeal viruses. Both viruses, SIRV1 and SIRV2, infect Sulfolobus islandicus. Their Hjcs both consist of 121 amino acid residues (aa) differing only by 18 aa. Both proteins bind selectively to synthetic Holliday-structure analogues with an apparent dissociation constant of 25 nM. In the presence of Mg(2+) the enzymes produce identical cleavage patterns near the junction. While S. islandicus shows optimal growth at about 80 degrees C, the nucleolytic activities of recombinant SIRV2 Hjc was highest between 45 degrees C and 70 degrees C. Based on their specificity for four-way DNA structures the enzymes may play a general role in genetic recombination, DNA repair and the resolution of replicative intermediates.     

Sulfolobus tengchongensis spindle-shaped virus STSV1: virus-host interactions and genomic features

A virus infecting the hyperthermophilic archaeon Sulfolobus tengchongensis has been isolated from a field sample from Tengchong, China, and characterized. The virus, denoted STSV1 (Sulfolobus tengchongensis spindle-shaped virus 1), has the morphology of a spindle (230 by 107 nm) with a tail of variable length (68 nm on average) at one end and is the largest of the known spindle-shaped viruses. After infecting its host, the virus multiplied rapidly to high titers (>10(10) PFU/ml). Replication of the virus retarded host growth but did not cause lysis of the host cells. STSV1 did not integrate into the host chromosome and existed in a carrier state. The STSV1 DNA was modified in an unusual fashion, presumably by virally encoded modification systems. STSV1 harbors a double-stranded DNA genome of 75,294 bp, which shares no significant sequence similarity to those of fuselloviruses. The viral genome contains a total of 74 open reading frames (ORFs), among which 14 have a putative function. Five ORFs encode viral structural proteins, including a putative coat protein of high abundance. The products of the other nine ORFs are probably involved in polysaccharide biosynthesis, nucleotide metabolism, and DNA modification. The viral genome divides into two nearly equal halves of opposite gene orientation. This observation as well as a GC-skew analysis point to the presence of a putative viral origin of replication in the 1.4-kb intergenic region between ORF1 and ORF74. Both morphological and genomic features identify STSV1 as a novel virus infecting the genus Sulfolobus.     

A novel terminal resolution-like site in the adeno-associated virus type 2 genome.

The adeno-associated virus 2 (AAV) contains a single-stranded DNA genome of which the terminal 145 nucleotides are palindromic and form T-shaped hairpin structures. These inverted terminal repeats (ITRs) play an important role in AAV DNA replication and resolution, since each of the ITRs contains a terminal resolution site (trs) that is the target site for the AAV rep gene products (Rep). However, the Rep proteins also interact with the AAV DNA sequences that lie outside the ITRs, and the ITRs also play a crucial role in excision of the proviral genome from latently infected cells or from recombinant AAV plasmids. To distinguish between Rep-mediated excision of the viral genome during rescue from recombinant AAV plasmids and the Rep-mediated resolution of the ITRs during AAV DNA replication, we constructed recombinant AAV genomes that lacked either the left or the right ITR sequence and one of the Rep-binding sites (RBSs). No rescue and replication of the AAV genome occurred from these plasmids following transfection into adenovirus type 2-infected human KB cells, as expected. However, excision and abundant replication of the vector sequences was clearly detected from the plasmid that lacked the AAV left ITR, suggesting the existence of an additional putative excision site in the left end of the AAV genome. This site was precisely mapped to one of the AAV promoters at map unit 5 (AAV p5) that also contains an RBS. Furthermore, deletion of this RBS abolished the rescue and replication of the vector sequences. These studies suggest that the Rep-mediated cleavage at the RBS during viral DNA replication may, in part, account for the generation of the AAV defective interfering particles.     

Theoretical study about the variability of the genome of foot-and-mouth disease viruses.

1. The possibilities of change in amino acids of a protein are discussed in terms of a point mutation. 2. Whereas Met and Trp are forced to change due to a point mutation, other amino acids (Ala, Arg, Gly, Leu, Pro and Val) have a probability of 1/3 to survive in the sequence. 3. On basis of these considerations, the genome from 5 strains (CSP, C3Ind, O1K, A10 and A12) of the foot-and-mouth disease virus was studied. 4. A hypothetical genealogic tree for these strains is suggested, where CSP and C3Ind are close and also A10 and A12. O1K is closer to A10 and A12 than to CSP or C3Ind.     

Structural variability in the genome of the Thermoproteus tenax virus TTV1

Summary Six variants of the TTV1 genome, including the primary isolate, have been characterized. DNA sequence comparison of wild-type virus (WT) and one of the variants (VT3) showed that differences are due to insertions and deletions that were confined to contiguous portions of two distinctClaI fragments. Seven similar short DNA sequences (30–102 bp) were involved in the variation. The deletions and insertions of these short DNA sequences occurred in every case adjacent to the 8 by consensus sequence 5-ACXCCTAC-3 which formed the 5 flank of the segments involved.     

Structure of the hepatitis B virus genome.

The extent and position of the single-stranded gap in DNA molecules from Dane particles isolated from two donors of the adw serotype were determined by molecular hybridization and electron microscopic methods. The results showed that in each preparation more than 99% of the circular molecules are of uniform length and contain both single- and double-stranded regions. They confirmed that one end of the short strand is fixed with respect to the single EcoRI site within the molecule and to the nick in the long strand, but they also showed that although the position of the other end is variable, there is a preferred minimum length of about 650 to 700 nucleotides for the single-stranded region.     

Structure of the Abelson murine leukemia virus genome.

Virions produced from cells transformed by A-MuLV contain a 30S, 5.6 kb RNA that can be translated in a cell-free system to form the characteristic A-MuLV protein. This RNA was mapped by heteroduplex methods using DNA probes from M-MuLV, the presumed parent of A-MuLV. The overall organization of the RNA was determined by using full-length M-MuLV reverse transcribed DNA and visualizing the heteroduplexes in the electron microscope. This showed that A-MuLV and M-MuLV have homologous sequences at both ends of their RNAs but that the central portion of the A-MuLV genome is not homologous to sequences in M-MuLV RNA. A precise measure of the lengths of the shared regions was obtained by using S1 nuclease to digest hybrids between 32P-labeled M-MuLV DNA and A-MuLV RNA; the resulting fragments were analyzed for their length by electrophoresis. The regions of homology were shown to be 1320 nucleotides long at the 5' end and 730 nucleotides long at the 3' end. Thus approximately 6200 nucleotides of the approximately 8300 in M-MuLV RNA were deleted when the A-MuLV genome was formed, but an insert of 3600 nucleotides, presumably derived from the normal murine genome, was inserted in place of the deleted region.     

From small hosts come big viruses: the complete genome of a second Ostreococcus tauri virus, OtV-1

Ostreococcus tauri virus (OtV-1) is a large double-stranded DNA virus and a prospective member of the family Phycodnaviridae , genus Prasinovirus . OtV-1 infects the unicellular marine green alga O. tauri , the smallest known free-living eukaryote. Here we present the 191 761 base pair genome sequence of OtV-1, which has 232 putative protein-encoding and 4 tRNA-encoding genes. Approximately 31% of the viral gene products exhibit a similarity to proteins of known functions in public databases. These include a variety of unexpected genes, for example, a PhoH-like protein, a N -myristoyltransferase, a 3-dehydroquinate synthase, a number of glycosyltransferases and methyltransferases, a prolyl 4-hydroxylase, 6-phosphofructokinase and a total of 8 capsid proteins. A total of 11 predicted genes share homology with genes found in the Ostreococcus host genome. In addition, an intein was identified in the DNA polymerase gene of OtV-1. This is the first report of an intein in the genome of a virus that infects O. tauri. Fifteen core genes common to nuclear-cytoplasmic large dsDNA virus (NCLDV) genomes were identified in the OtV-1 genome. This new sequence data may help to redefine the classification of the core genes of these viruses and shed new light on their evolutionary history.     

Structural variability of the herpes simplex virus 1 genome in vitro and in vivo

Herpes simplex virus 1 (HSV-1) is a human pathogen that leads to recurrent facial-oral lesions. Its 152-kb genome is organized in two covalently linked segments, each composed of a unique sequence flanked by inverted repeats. Replication of the HSV-1 genome produces concatemeric molecules in which homologous recombination events occur between the inverted repeats. This mechanism leads to four genome isomers (termed P, IS, IL, and ILS) that differ in the relative orientations of their unique fragments. Molecular combing analysis was performed on DNA extracted from viral particles and BSR, Vero, COS-7, and Neuro-2a cells infected with either strain SC16 or KOS of HSV-1, as well as from tissues of experimentally infected mice. Using fluorescence hybridization, isomers were repeatedly detected and distinguished and were accompanied by a large proportion of noncanonical forms (40%). In both cell and viral-particle extracts, the distributions of the four isomers were statistically equivalent, except for strain KOS grown in Vero and Neuro-2a cells, in which P and IS isomers were significantly overrepresented. In infected cell extracts, concatemeric molecules as long as 10 genome equivalents were detected, among which, strikingly, the isomer distributions were equivalent, suggesting that any such imbalance may occur during encapsidation. In vivo, for strain KOS-infected trigeminal ganglia, an unbalanced distribution distinct from the one in vitro was observed, along with a considerable proportion of noncanonical assortment.     

Structure of D-63 from sulfolobus spindle-shaped virus 1: surface properties of the dimeric four-helix bundle suggest an adaptor protein function

Sulfolobus spindle-shaped virus 1 (SSV1) and its fusellovirus homologues can be found in many acidic (pH or=70 degrees C) around the world. SSV1 contains a 15.5-kb double-stranded DNA genome that encodes 34 proteins with greater than 50 amino acids. A site-specific integrase and a DnaA-like protein have been previously identified by sequence homology, and three structural proteins have been isolated from purified virus and identified by N-terminal sequencing (VP1, VP2, and VP3). The functions of the remaining 29 proteins are currently unknown. To assign functions to these proteins, we have initiated biochemical and structural studies on the SSV1 proteome. Here we report the structure of SSV1 D-63. The structure reveals a helix-turn-helix motif that dimerizes to form an antiparallel four-helix bundle. Mapping residues conserved among three fusellovirus isolates onto the structure shows that one face of the rod-shaped molecule is highly conserved. This conserved surface spans the dimer axis and thus exhibits 2-fold symmetry. Two smaller conserved patches, also related by 2-fold symmetry, are found on the opposite face of the molecule. All of these conserved surfaces are devoid of clefts or pockets typically used to bind small molecules, suggesting that D-63 may function as an adaptor protein in macromolecular assembly.     

Structure similarity, difference and variability in the filamentous viruses fd, If1, IKe, Pf1 and Xf. Investigation by laser Raman spectroscopy

The filamentous bacteriophages fd, If1, IKe, Pf1, Xf and Pf3 in aqueous solutions of low, moderate and high ionic strength have been investigated as a function of temperature by laser Raman difference spectroscopy. By analogy with Raman spectra of model compounds and viruses of known structure, the data reveal the following structural features: the predominant secondary structure of the coat protein subunit in each virus is the alpha-helix, but the amount of alpha-helix differs from one virus to another, ranging from an estimated high of 100% in Pf1 to a low of approximately 50% in Xf. The molecular environment and intermolecular interactions of tyrosine, tryptophan and phenylalanine residues differ among the different viruses, as do the conformations of aliphatic amino acid side-chains. The foregoing features of coat protein structure are highly sensitive to changes in Na+ concentration, temperature or both. The backbones of A-DNA and B-DNA structures do not occur in any of the viruses, and unusual DNA structures are indicated for all six viruses. The alpha-helical protein subunits of Pf1, like those of Pf3 and Xf, can undergo reversible transitions to beta-sheet structures while retaining their association with DNA; yet fd, IKe and If1 do not undergo such transitions. Raman intensity changes with ionic strength or temperature suggest that transgauche rotations of aliphatic amino acid side-chains and stacking of aromatic side-chains are important structural variables in each virus.     

Phase separation of a plant virus movement protein and cellular factors support virus-host interactions

Both cellular and viral proteins can undergo phase separation and form membraneless compartments that concentrate biomolecules. The p26 movement protein from single-stranded, positive-sense Pea enation mosaic virus 2 (PEMV2) separates into a dense phase in nucleoli where p26 and related orthologues must interact with fibrillarin (Fib2) as a pre-requisite for systemic virus movement. Using in vitro assays, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 genomic RNAs formed droplets that may provide the basis for self-assembly in planta. Mutating basic p26 residues (R/K-G) blocked droplet formation and partitioning into Fib2 droplets or the nucleolus and prevented systemic movement of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana. Mutating acidic residues (D/E-G) reduced droplet formation in vitro, increased nucleolar retention 6.5-fold, and prevented systemic movement of TMV, thus demonstrating that p26 requires electrostatic interactions for droplet formation and charged residues are critical for nucleolar trafficking and virus movement. p26 readily partitioned into stress granules (SGs), which are membraneless compartments that assemble by clustering of the RNA binding protein G3BP following stress. G3BP is upregulated during PEMV2 infection and over-expression of G3BP restricted PEMV2 RNA accumulation >20-fold. Deletion of the NTF2 domain that is required for G3BP condensation restored PEMV2 RNA accumulation >4-fold, demonstrating that phase separation enhances G3BP antiviral activity. These results indicate that p26 partitions into membraneless compartments with either proviral (Fib2) or antiviral (G3BP) factors.     

Structure and variability of recently inserted Alu family members.

The HS subfamily of Alu sequences is comprised of a group of nearly identical members. Individual subfamily members share 97.7% nucleotide identity with each other and 98.9% nucleotide identity with the HS consensus sequence. Individual subfamily members are on the average 2.8 million years old, and were probably derived from a single source 'master' gene sometime after the human/great ape divergence. The recent Alu family member insertions provide a better image of the structure of Alu retroposons before they have had the opportunity to change significantly. All of the HS subfamily members are flanked by perfect direct repeats as a result of insertion at staggered nicks. The 'master' gene from which the HS subfamily members were derived had an oligo-dA rich tail at least 40 bases long. The 'master' gene is very rich in CpG dinucleotides, but nucleotide substitutions within subfamily members accumulated in a random manner typical for Alu sequence with CpG substitutions occurring 9.2 fold faster than non-CpG substitutions.