Current status of viroid taxonomy.

Viroids are the smallest autonomous infectious nucleic acids known so far. With a small circular RNA genome of about 250-400 nt, which apparently does not code for any protein, viroids replicate and move systemically in host plants. Since the discovery of the first viroid almost forty-five years ago, many different viroids have been isolated, characterized and, frequently, identified as the causal agents of plant diseases. The first viroid classification scheme was proposed in the early 1990s and adopted by the International Committee on Taxonomy of Viruses (ICTV) a few years later. Here, the current viroid taxonomy scheme and the criteria for viroid species demarcation are discussed, highlighting the main taxonomic questions currently under consideration by the ICTV Viroid Study Group. The impact of correct taxonomic annotation of viroid sequence variants is also addressed, taking into consideration the increasing application of next-generation sequencing and bioinformatics for known and previously unrecognized viroids.

[Molecular properties of potato spindle tuber viroid (PSTVd) isolates from the collection of the Russian Research Institute of Phytopathology].

The complete nucleotide sequences of more than 100 isolates of PSTVd collected from locations in the territory of Russia and the former USSR have been determined. These sequences represent 42 individual sequence variants, each containing 1-10 mutations with respect to the "intermediate" or type strain of PSTVd (GenBank Acc. No. v01465). Isolates containing 2-5 mutations were the most common, and 24 sequence variants are described here for the first time. Twenty one isolates contained a mutation found only in Russian and Ukrainian isolates of PSTVd up till now; i.e., replacement of the adenine at position 121 with cytosine (A121C). Many of these isolates contained two mutations--deletion of one of the three adenine residues occupying positions 118-120 plus replacement of the adenine at position 121 with either uracil or cytosine (A120, A121U/C). Both combinations of mutations were phenotypically neutral, i.e. symptom expression in Rutgers tomato was unaffected. Phylogenetic analysis of the sequences of different PSTVd isolates presented in work together with sequences of other naturally-occurring isolates obtained from Internet databases suggesting that known PSTVd isolates may be divided into four groups: i) a group of isolates from potato, tomato and solanaceous ornamentals where the type strain of PSTVd (PSTVd.018) may be considered to represent the ancestral sequence, ii) a group of isolates from potato, tomato and solanaceous ornamentals where PSTVd.123 play the same role as PSTVd.018 for the first group, and iii) a group of potato isolates where PSTVd.125 is a possible ancestral sequence. The fourth and most divergent group of PSTVd isolates differs significantly from these first three groups. The majority of isolates in this group originate from New Zealand and Australia and infect different solanaceous hosts (tomato, pepper, cape gooseberry, potato, and others).

Mexican papita viroid and tomato planta macho viroid belong to a single species in the genus Pospiviroid.

Tomato planta macho viroid (TPMVd) and Mexican papita viroid (MPVd) are two closely related (>90% sequence identity) members of the genus Pospiviroid. Their current status as members of separate species is based upon the reported ability of TPMVd to replicate in Gomphrena globosa and the inability of this viroid to evoke flower break in N. glutinosa. Characterization of a viroid recently isolated from diseased tomato plants grown in Mexico (identical to GenBank accession GQ131573) casts doubt on this earlier report and indicates that these viroids should be classified as members of a single species. Giving priority to the older name, we propose including both of these viroids in the current species Tomato planta macho viroid.

Russian isolates of potato spindle tuber viroid.

Forty PSTVd isolates collected from five regions of Russia (North-western, Central, Volga region, Northern Caucasus and the Far East) were sequenced during 2006-2008. All isolates lacked the adenine residue present at position 123 of the type strain; i.e., PSTVd-intermediate (GenBank V01465). Nineteen Russian isolates also contained an adenosine --> cytosine substitution at position 120. Twenty two additional unique changes were also observed in one or more of the isolates sequenced.

Identification of phytoplasma species associated with potato diseases in Russia.

Four out of six known potato diseases attributed to phytoplasma infection were previously reported to occur in Russia based on a combination of biological properties such as symptomatology and/or vector relationships and electron microscopy of infected phloem tissue. In 2007, the first molecular identification of potato diseases causing symptoms including purple top, round leaves, stunting, bud proliferation and formation of aerial tubers was carried out using PCR methods. A nested PCR using primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasma in infected potato samples. PCR products were digested singly with several restriction enzymes. Comparison of RFLP profiles with published profiles was used for identification of the putative phytoplasma detected. The majority of 49 PCR positive potato samples showed RFLP profiles of 16S rDNA sequences very similar or identical to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A, and only two showed RFLP profiles similar to those of aster yellow phytoplasma strains ('Candidatus Phytoplasma asteris') belonging to aster yellows phytoplasma group (16SrI), subgroup 16SrI-A and 16SrI-B. The results demonstrated that stolbur phytoplasma is prevalent in several potato growing regions of Russia.

Molecular Detection and Identification of Group 16SrI and 16SrXII Phytoplasmas Associated with Diseased Potatoes in Russia.

Phytoplasmal diseases have long been suspected to occur in several potato-growing regions in Russia on the basis of symptoms and the presence of insect vectors. Symptoms resembling stolbur are most prevalent, but round leaf disease, potato witches'-broom, and potato purple top wilt also occur (1). The phytoplasma etiologies of these diseases have never been verified by molecular means. During the summer of 2006, 33 potato plants exhibiting various symptoms including purple top, round leaves, and stolbur-like symptoms characterized by purple top, stunting, bud proliferation, and formation of aerial tubers were randomly collected from the Volga River Region, Central Region, and Northern Caucasian Region in Russia. DNA extracts were prepared from 1.0 g of petioles and leaf mid veins according to a modified procedure with the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) as previously described (2). A nested PCR with primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasmas in infected potato samples (4). Potato plants maintained in the greenhouse were used as healthy controls. A negative control devoid of DNA templates was included in all PCR assays. One microliter of diluted (1:30) PCR product from the first amplification was used as the template in the nested PCR. Eight of 33 potato samples tested positive in the first PCR. Twelve of 33 potato samples tested positive in nested PCR. Nine of the 12 potato samples that tested positive for phytoplasma exhibited stolbur-like symptoms; the other three samples exhibited round leaves, stunting, or proliferation. The remaining symptomatic samples that exhibited nonspecific purple or yellow discoloration of terminal leaves, without other specific stolbur-like symptoms, may be infected by other pathogens. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products (R16F2n/R16R2n amplicons, 1.2 kb) was performed. PCR products (6 µl) were digested singly with the restriction enzymes AluI, HaeIII, HhaI, HpaII, KpnI, MseI, RsaI, and Tsp509I. Comparison of RFLP profiles with published profiles (3) was used for identification of the putative phytoplasmas detected. Among the 12 PCR positive potato samples, 10 showed very similar or identical RFLP profiles to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A and closely related strains, and two showed RFLP profiles similar to those of aster yellows phytoplasma group (16SrI). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession Nos. EU344884-EU344890 and EU333396-EU333400) confirmed the results of the RFLP analyses and also indicated that the two samples showing 16SrI profiles were simultaneously infected with two phytoplasma strains belonging to subgroups 16SrI-A and 16SrI-B. To our knowledge, this is the first confirmation by molecular procedures that stolbur phytoplasma (16SrXII-A) is prevalent in several potato-growing regions and is the first report of 16SrI-A and 16SrI-B phytoplasmas associated with potatoes in Russia. References: (1) D. Z. Bogoutdinov. Potato Phytoplasmas and Methods of Their Study. Samara State Agricultural University, Samara, 2000. (2) M. J. Green et al. Plant Dis. 83:482, 1999. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) I.-M. Lee et al. Plant Dis. 90:989, 2006.

Recovery of Four Novel Potato spindle tuber viroid Sequence Variants from Russian Seed Potatoes.

First described in the early 1930s, the limited distribution of potato "gothic" disease made it of little economic significance in European Russia until the early 1970s when meristem-tip culture was widely adopted throughout the former USSR to increase production of virus-free seed potatoes. Shortly thereafter, the yield and quality of Russian seed potatoes began a dramatic decline. Symptoms of potato "gothic" resemble those of Potato spindle tuber viroid (PSTVd) (3), and initial suspicions that in vitro plantlets and seed potatoes might be viroid-infected were later proved correct when Kastalyeva et al. (2) showed that approximately 50 to 70% of in vitro plantlets and tubers collected from different regions of Russia as well as the in vitro germplasm collection maintained by the All-Russian Potato Research Institute (ARPRI) were infected with PSTVd. Measures have since been taken to reduce the incidence of PSTVd infection, and numerous PSTVd isolates were collected from territories of the former USSR; however, none of these isolates have been characterized at the molecular level. Overlapping reverse transcription (RT)-PCR products (1) were generated from four PSTVd isolates maintained in field-grown tubers at the VNIIF using two pairs of primers; PSTVd180F (5'-TCACCCTTCCTTTCTTCGGGTGTC-3') + PSTVd179R (5'-AAACCCTGTTTCGGCGGGAATTAC-3') and PSTVd112F (5'-ACT GGCAAAAAAGGACGGTGGGGA-3') + PSTVd359R (5'-AGGAACC AACTGCGGTTCCAAGGG-3'). Automated sequence analysis of the resulting uncloned PCR products revealed the presence of four previously unknown PSTVd variants (GenBank Accession Nos. EF044302-EF044305). All four tubers were also infected with Potato virus M and Potato virus Y and one tuber also contained Potato virus S. ELISA tests for Potato leaf roll virus were negative. Each isolate appeared to contain only a single 358-359 nt variant differing from PSTVd-intermediate strain (GenBank Accession No. V01465) at 2-5 positions. The three closely related variants originating from Leningradskaya Province (Northwest Russia) contain two to three changes in the variable domain and central conserved region and induced intermediate symptoms in Rutgers tomato. The fourth variant originating from Samarskaya Province (Volga River Region) contains additional changes in the pathogenicity domain and induced mild symptoms. Minor differences among the Leningradskaya variants may represent sequence drift during extended (9 to 11 year) tuber passage. The presence of additional sequence changes in the variant from Samarskaya is consistent with independent origin and/or prolonged separation. Additional studies with a wider range of Russian isolates of PSTVd are currently underway to develop diagnostic methods suitable for future large-scale screening programs. References: (1) Y. Hu et al. Virology 219:45, 1997. (2) T. B. Kastalyeva et al. Vestn. RASKHN 3:22, 1992. (3) Y. A. Leontyeva. Potato spindle tuber ('gothic') as one of the most important diseases in the Volga region. (In Russian.) Ph.D. thesis. Agricultural University of Leningrad, Pushkin, 1971.

Identification of a novel structural interaction in Columnea latent viroid.

Pairwise sequence comparisons suggest that Columnea latent viroid (CLVd) may have originated from a recombination event involving Potato spindle tuber viroid (PSTVd) and Hop stunt viroid (HSVd). To examine the role of specific structural features in determining the host range of CLVd, we constructed a series of interspecific chimeras by replacing increasing portions of its terminal left and pathogenicity domains with the corresponding portions of PSTVd. Exchanges involving the left side of the pathogenicity domain led to lower rates of progeny accumulation in tomato, but one of the resulting chimeras was still able to replicate in cucumber. Exchanges involving the right side of the pathogenicity domain severely inhibited replication in tomato and appeared to abolish replication in cucumber. To identify potential interactions between nucleotides comprising the right side of the pathogenicity domain and other portions of CLVd, melting behaviors of circularized CLVd and PSTVd RNA transcripts were compared using a combination of temperature gradient gel electrophoresis and structural calculations. These analyses revealed an unexpected complementarity between the upper portion of the pathogenicity and terminal right domains of CLVd that facilitates breakdown of the rod-like native structure and formation of secondary hairpin II. Unlike secondary hairpin II, CLVd hairpin IV appears likely to act within the context of the genomic RNA.

Identification of neutral mutants surrounding two naturally occurring variants of Potato spindle tuber viroid.

Single point mutations in the pathogenicity domain of Potato spindle tuber viroid (PSTVd) can have a dramatic effect on disease expression, and only three substitutions are required for the spontaneous conversion of the type strain PSTVd-Intermediate to the rapidly replicating, highly pathogenic variant RG1 (Gruner et al., Virology 209, 60-69, 1995). To identify available evolutionary pathways linking these two variants, we mutagenized five positions in an infectious cDNA copy of PSTVd-Intermediate and screened the resulting mixture of 768 sequences for neutral or near-neutral mutants. Numerical simulations based on the bioassay data indicate that the 23 variants recovered represent >80 % of all such sequences. RG1 was the only naturally occurring variant recovered, and the overall pattern of sequence changes observed indicates that PSTVd-Int occupies a comparatively steep peak within the fitness landscape.

Possible involvement of the phloem lectin in long-distance viroid movement.

Incubation with cucumber phloem exudate in vitro results in a dramatic decrease in the electrophoretic mobility of Hop stunt viroid. UV cross-linking and a combination of size exclusion and ion exchange chromatography indicate that this phenomenon reflects a previously unsuspected ability of phloem protein 2, a dimeric lectin and the most abundant component of phloem exudate, to interact with RNA. In light of its demonstrated ability to move from cell to cell via plasmodesmata as well as long distances in the phloem, our results suggest that phloem protein 2 may facilitate the systemic movement of viroids and, possibly, other RNAs in vivo.

A Rep recognition sequence is necessary but not sufficient for nicking of DNA by adeno-associated virus type-2 Rep proteins.

The strand-specific, site-specific endonuclease (nicking) activity of the Rep68 and Rep78 (Rep68/78) proteins of adeno-associated virus type 2 (AAV) is involved in AAV replication, and appears to be involved in AAV site-specific integration. Rep68/78 cuts within the inverted terminal repeats (ITRs) of the AAV genome and in the AAV preferred integration locus on human chromosome 19 (AAVS1). The known endonuclease cut sites are 11-16 bases away from the primary binding sites, known as Rep recognition sequences (RRSs). A linear, double-stranded segment of DNA, containing an RRS and a cut site, has previously been shown to function as a substrate for the Rep68/78 endonuclease activity. We show here that mutation of the Rep recognition sequence, within such a DNA segment derived from the AAV ITRs, eliminates the ability of this substrate to be cleaved detectably by Rep78. Rep78 nicks the RRS-containing site from AAVS1 about half as well as the linear ITR sequence. Eighteen other RRS-containing sequences found in the human genome, but outside AAVS1, are not cleaved by Rep78. These results may help to explain the specificity of AAV integration.

Both point mutation and RNA recombination contribute to the sequence diversity of citrus viroid III.

Field-grown citrus trees often harbor complex mixtures of 4-5 different viroid species, and the presence of citrus viroid III (CVd-III) has been shown to reduce the rate of tree growth without inducing disease. To more fully define the structure of its quasi-species, we have examined nine citrus viroid complexes for the presence of previously undescribed sequence variants of CVd-III. Analysis of 86 full-length cDNAs generated from these nine viroid complexes by RT-PCR revealed the presence of 20 new CVd-III variants. Chain lengths ranged from 293-297 nucleotides, and sequence changes were confined largely to the lower portions of the central conserved region and variable domain. The previously described variants CVd-IIIa (297 nt) and CVd-IIIb (294 nt) were clearly predominant, but phylogenetic analysis indicated that certain isolates may contain representatives of two additional fitness peaks. At least one group of CVd-III variants appears to have arisen as a result of RNA recombination. Populations recovered from diseased/declining trees were the most diverse, but even dwarfing isolates originating from old line Shamouti trees showed considerable variability.

Mutational analysis of adeno-associated virus type 2 Rep68 protein endonuclease activity on partially single-stranded substrates.

The endonuclease activity of the Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) cuts at the terminal resolution site (trs) within the hairpin structure formed by the AAV inverted terminal repeats. Recent studies suggest that a DNA unwinding function of Rep68/78 may be required for endonuclease activity. We demonstrate that several mutant proteins which are endonuclease negative on a fully duplex hairpin substrate are endonuclease positive on a partially single-stranded hairpin substrate. Truncation analysis revealed that the endonuclease function is contained within the first 200 amino acids of Rep68/78. This endonucleolytic cleavage is believed to involve the covalent attachment of Rep68/78 to the trs via a phosphate-tyrosine linkage. A previous report (S. L. Walker, R. S. Wonderling, and R. A. Owens, J. Virol. 71:2722-2730, 1997) suggested that tyrosine 152 was part of the active site. We individually mutated each tyrosine within the first 200 amino acids of the Rep68 moiety of a maltose binding protein-Rep68/78 fusion protein to phenylalanine. Only mutation of tyrosine 156 resulted in a protein incapable of covalent attachment to a partially single-stranded hairpin substrate, suggesting that tyrosine 156 is part of the endonuclease active site.

Factors affecting the terminal resolution site endonuclease, helicase, and ATPase activities of adeno-associated virus type 2 Rep proteins.

The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Delta) produced in Escherichia coli cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Delta is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Delta helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Delta helicase activity and found that it appears to move in a 3' to 5' direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Delta or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Delta endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.

Breast growth and the urinary excretion of lactose during human pregnancy and early lactation: endocrine relationships.

Breast volume and morphology of eight subjects were measured before conception and at intervals throughout pregnancy until 1 month of lactation. Breast volume before conception ranged from 293 to 964 ml. At the end of pregnancy the volume of breast tissue had increased by 145+/-19 ml (mean+/-S.E.M., n = 13 breasts, range 12-227 ml) with a further increase to 211+/-16 ml (n = 12 breasts, range 129-320 ml) by 1 month of lactation. Urinary excretion of lactose increased at 22 weeks of pregnancy, signalling the capacity of the breast to synthesize lactose at this time. During pregnancy, both the change in breast volume and the change in cross-sectional area of the areola were related to the concentration of human placental lactogen in the plasma. The growth of the nipple and the rate of excretion of lactose were related to the concentration of prolactin in the plasma. During the first 3 days after birth, the rate of excretion of lactose was related to the rate of excretion of progesterone. There was no relationship between the growth of the breast during pregnancy and the amount of milk produced at 1 month of lactation.

Breast volume and milk production during extended lactation in women.

Quantitative measurements were made of relative breast volume and milk production from 1 month of lactation until 3 months after weaning, and the storage capacity of the breasts was calculated. The increase in breast tissue volume from before conception until 1 month of lactation was maintained for the first 6 months of lactation (means+/-S.E.M.) (190.3+/-13.1 ml, number of breasts, nb = 46). During this period of exclusive breast-feeding, 24 h milk production from each breast remained relatively constant (453.6+/-201 g, nb = 48), and storage capacity was 209.9+/-11.0 ml (nb = 46). After 6 months, breast volume, milk production and storage capacity all decreased. There was a relationship between 24 h milk production and the storage capacity of the breasts, and these both appeared to be responding to infant demand for milk. At 15 months of lactation, the 24 h milk production of each breast was substantial (208.0+/-56.7 g, nb = 6), even though the breasts had returned to preconception size. This was associated with an apparent increased efficiency of the breast (milk production per unit breast tissue) after 6 months, which may have been due to redistribution of tissues within the breast. The possible causes of the decrease in breast volume are discussed.

Analysis of the effects of charge cluster mutations in adeno-associated virus Rep68 protein in vitro.

The Rep78 and Rep68 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which are required for viral replication, regulation of AAV promoters, and preferential integration of the AAV genome into a region of human chromosome 19. These proteins bind the hairpin structures formed by the AAV inverted terminal repeat (ITR) origins of replication, make site- and strand-specific endonuclease cuts within the AAV ITRs, and display nucleoside triphosphate-dependent helicase activities. Additionally, several mutant Rep proteins display negative dominance in helicase and/or endonuclease assays when they are mixed with wild-type Rep78 or Rep68, suggesting that multimerization may be required for the helicase and endonuclease functions. Using overlap extension PCR mutagenesis, we introduced mutations within clusters of charged residues throughout the Rep68 moiety of a maltose binding protein-Rep68 fusion protein (MBP-Rep68Delta) expressed in Escherichia coli cells. Several mutations disrupted the endonuclease and helicase activities; however, only one amino-terminal-charge cluster mutant protein (D40A-D42A-D44A) completely lost AAV hairpin DNA binding activity. Charge cluster mutations within two other regions abolished both endonuclease and helicase activities. One region contains a predicted alpha-helical structure (amino acids 371 to 393), and the other contains a putative 3,4 heptad repeat (coiled-coil) structure (amino acids 441 to 483). The defects displayed by these mutant proteins correlated with a weaker association with wild-type Rep68 protein, as measured in coimmunoprecipitation assays. These experiments suggest that these regions of the Rep molecule are involved in Rep oligomerization events critical for both helicase and endonuclease activities.

Precisely full length, circularizable, complementary RNA: an infectious form of potato spindle tuber viroid.

The replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. For potato spindle tuber (PSTVd) and related viroids, the possible role of a circular (-)strand RNA as a template for synthesis of (+)strand progeny is unclear. Infected plants appear to contain only multimeric linear (-)strand RNAs, and attempts to initiate infection with multimeric (-)PSTVd RNAs generally have failed. To examine critically the infectivity of monomeric (-)strand viroid RNAs, we have developed a ribozyme-based expression system for the production of precisely full length (-)strand RNAs whose termini are capable of undergoing facile circularization in vitro. Mechanical inoculation of tomato seedlings with electrophoretically purified (-)PSTVd RNA led to a small fraction of plants becoming infected whereas parallel assays with an analogous tomato planta macho viroid (-)RNA resulted in a much larger fraction of infected plants. Ribozyme-mediated production of (-)PSTVd RNA in transgenic plants led to the appearance of monomeric circular (-)PSTVd RNA and large amounts of (+)PSTVd progeny. No monomeric circular (-)PSTVd RNA could be detected in naturally infected plants by using either ribonuclease protection or electrophoresis under partially denaturing conditions. Although not a component of the normal replicative pathway, precisely full length (-)PSTVd RNA appears to contain all of the structural and regulatory elements necessary for initiation of viroid replication.

The Rep68 protein of adeno-associated virus type 2 increases RNA levels from the human cytomegalovirus major immediate early promoter.

The Rep68 and Rep78 proteins of adeno-associated virus type-2 (AAV) are multifunctional DNA binding proteins which are involved in the positive and negative regulation of AAV genes, as well as various cellular and heterologous viral genes. In this study we report that Rep68 enhances expression from the major immediate early promoter (MIEP) of human cytomegalovirus (HCMV). This Rep-mediated enhancement of RNA levels is abrogated by the introduction of a Rep recognition sequence (RRS) at either position -18 or -244 in the HCMV-MIEP. However, a mutant RRS (mRRS), which is not bound by Rep68 is unable to negate the effect of Rep68. Sequence analysis and electrophoretic mobility shift assays showed no Rep68 binding sites within the wild-type HCMV-MIEP. Rep68 may therefore be enhancing expression from the HCMV-MIEP by interacting with other regulatory proteins that have an effect on the expression from this promoter or by altering the expression of a cellular gene whose product influences the HCMV-MIEP. Our results may also help to explain the previous observation that coinfection with AAV enhances the cytopathic effect of HCMV.

Mutational analysis of the adeno-associated virus type 2 Rep68 protein helicase motifs.

The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are required for viral replication. These proteins are encoded by unspliced and spliced transcripts, respectively, from the p5 promoter of AAV and therefore have overlapping amino acid sequences. The Rep78 and Rep68 proteins share a variety of activities including endonuclease, helicase, and ATPase activities and the ability to bind AAV hairpin DNA. The part of the amino acid sequence which is identical in Rep78 and Rep68 contains consensus helicase motifs that are conserved among the parvovirus replication proteins. In the present study, we mutated highly conserved amino acids within these helicase motifs. The mutant proteins were synthesized as maltose binding protein-Rep68 fusions in Escherichia coli cells and affinity purified on amylose resin. The fusion proteins were assayed in vitro, and their activities were directly compared to those of the fusion protein MBP-Rep68 delta, which contains most of the amino acid sequences common to Rep78 and Rep68 and was demonstrated previously to have all of the in vitro activities of wild-type Rep78 and Rep68. Our analysis showed that almost all mutations in the putative helicase motifs severely reduced or abolished helicase activity in vitro. Most mutants also had ATPase activity less than one-eighth of the wild-type levels and lacked endonuclease activity.