Site-specific integration by adeno-associated virus.

Cellular sequences flanking integrated copies of the adeno-associated virus (AAV) genome were isolated from a latently infected clonal human cell line and used to probe genomic blots derived from an additional 21 independently derived clones of human cells latently infected with AAV. In genomic blots of uninfected human cell lines and of primary human tissue, each flanking-sequence probe hybridized to unique bands, but in 15 of the 22 latently infected clones the flanking sequences hybridized not only to the original fragments but also to a total of 36 additional species. AAV probes also hybridized to 22 of these new bands, representing 11 of the 15 positive clones, but never to the fragment characteristic of uninfected cell DNA. From these data we conclude that the AAV genome preferentially integrates into a specific region of the cellular genome. We have determined that the integration site is unique to chromosome 19 by somatic cell hybrid mapping, and this sequence has been isolated from uninfected human DNA.

Latent infection of KB cells with adeno-associated virus type 2.

Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV-specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular-weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo.

Cloning of infectious adeno-associated virus genomes in bacterial plasmids.

We describe the construction of two Escherichia coli hybrid plasmids, each of which contains the entire 4.7-kb DNA genome of the human parvovirus, adeno-associated virus (AAV) type 2. Because the AAV genome was inserted into the plasmid DNA using BglII linkers the entire virus genome can be recovered by in vitro cleavage of the purified recombinant plasmid. Transfection of these recombinant DNAs into an adenovirus-transformed human cell line in the presence of helper adenovirus resulted in efficient rescue and replication of the AAV genome and production of fully infectious virus particles. These AAV-plasmid recombinant DNA molecules should be useful both for site-specific mutagenesis of the viral genome and to study the potential of AAV as a eukaryotic vector.

Properties of an adenovirus type 2 mutant, Ad2dl807, having a deletion near the right-hand genome terminus: failure to help AAV replication.

We have analyzed the ability of an adenovirus type 2 mutant, Ad2dl807, to support replication of adeno-associated virus (AAV). This mutant has a deletion extending from early region 3 through the late fiber gene and into early region 4. Since AAV growth does not require Ad early region 3 or the fiber gene, the Ad2dl807 mutant allows analysis of the function of Ad early region 4 in AAV growth. As determined by assay of AAV DNA, RNA, and protein synthesis as well as the number of cells producing AAV and the yield of infectious AAV particles, growth of AAV with the dl807 mutant is decreased 10- to 20-fold. The residual AAV growth is probably due to the presence of a low level of contaminating viable Ad virions in the dl807 preparations. These observations indicate that an Ad early region 4 function is required at a very early stage of AAV DNA replication to allow amplification of duplex replicating (RF) DNA. Thus growth of AAV offers an independent probe for adenovirus early region 4 functions.

Effect of deletions in adenovirus early region 1 genes upon replication of adeno-associated virus.

The growth of adeno-associated virus (AAV) is dependent upon helper functions provided by adenovirus. We investigated the role of adenovirus early gene region 1 in the AAV helper function by using six adenovirus type 5 (Ad5) host range mutants having deletions in early region 1. These mutants do not grow in human KB cells but are complemented by and grow in a line of adenovirus-transformed human embryonic kidney cells (293 cells); 293 cells contain and express the Ad5 early region 1 genes. Mutants having extensive deletions of adenovirus early region 1a (dl312) or regions 1a and 1b (dl313) helped AAV as efficiently as wild-type adenovirus in 293 cells, but neither mutant helped in KB cells. No AAV DNA, RNA, or protein synthesis was detected in KB cells in the presence of the mutant adenoviruses. Quantitative blotting experiments showed that at 20 h after infection with AAV and either dl312 or dl313 there was less than one AAV genome per cell. In KB cells infected with AAV alone, the unreplicated AAV genomes were detected readily. Apparently, infection with adenovirus mutant dl312 or dl313 results in degradation of most of the infecting AAV genomes. We suggest that at least an adenovirus region 1b product (and perhaps a region 1a product also) is required for AAV DNA replication. This putative region 1b function appears to protect AAV DNA from degradation by an adenovirus-induced DNase. We also tested additional Ad5 mutants (dl311, dl314, sub315, and sub316). All of these mutants were inefficient helpers, and they showed varying degrees of multiplicity leakiness. dl312 and dl313 complemented each other for the AAV helper function, and each was complemented by Ad5ts125 at the nonpermissive temperature. The defect in region 1 mutants for AAV helper function acts at a different stage of the AAV growth cycle than the defect in the region 2 mutant ts125.

Adeno-associated virus RNA transcription in vivo.

We have studied RNA transcripts of the defective parvovirus, adeno-associated virus (AAV) present in poly(A)rich and poly-(A)free fractions of nuclear and cytoplasmic RNA prepared from cells infected together with a helper adenovirus. Cytoplasmic poly-(A)rich RNA contains three overlapping spliced AAV RNAs having sizes of 3.9 X 10(3), 3.3 X 10(3) and 2.3 X 10(3) bases respectively. The nuclear precursors of these RNAs appear to be the coterminal unspliced poly(A)-rich RNAs containing 4.2 X 10(3), 3.6 X 10(3) and 2.6 X 10(3) bases respectively. These unspliced RNAs were also found in the cytoplasm. The nuclear poly(A)-free RNA contained a heterogenous population of AAV RNAs that were generally smaller than 2.3 X 10(3) bases. In addition, the Hirt pellet fraction of the nuclear RNA contained two discrete AAV poly(A)-free RNAs having sizes of 2.5 X 10(3) and 2.8 X 10(3) bases. The 4.2 X 10(3) and 3.6 X 10(3)-base unspliced RNAs are more abundant than the coterminal 3.9 X 10(3) and 3.3 X 10(3)-base spliced RNAs whereas the 2.3 X 10(3)-base spliced RNA is much more abundant than the 2.6 X 10(3)-base unspliced RNA. Thus, the cytoplasmic abundance of the AAV spliced RNAs appears to be controlled in part by the post-transcriptional events of splicing or message stability. We also analysed the effects of AAV defective-interfering genomes upon AAV transcription. These studies showed that when synthesis of standard AAV genomes was inhibited more than 10-fold by defective-interfering genomes there was no significant effect on the types or amounts of AAV RNA transcripts which accumulated. These observations indicate that interference by defective-interfering genomes occurs mostly at the level of DNA replication rather than transcription.

Eukaryotic translational control: adeno-associated virus protein synthesis is affected by a mutation in the adenovirus DNA-binding protein.

Growth of adeno-associated virus (AAV) requires expression of certain adenovirus (Ad) genes in the same cell. AAV particles contain three proteins, VP1 (Mr 85,700), VP2 (Mr 72,000), and VP3 (Mr 61,500). These proteins have overlapping peptide maps. We recently reported that AAV RNAs make up a 3'-coterminal family of overlapping molecules. We report here that the most abundant AAV mRNA, a 2.3-kilobase spliced RNA, codes for all three proteins--VP1, VP2, and VP3--when translated in an in vitro reticulocyte lysate. This shows that the AAV capsid proteins are coded by the genome sequence between map positions 48.0 and 96.0 (1 map unit is 1% of the genome or 47 base pairs). When AAV was grown in human KB cells with the Ad temperature-sensitive mutant Ad5ts125 at the nonpermissive temperature (40 degrees C), the accumulation in vivo of AAV capsid proteins VP1, VP2, and VP3 was decreased to less than 1/50th. However, normal amounts of the 2.3-kilobase mRNA were accumulated, and this RNA could be efficiently translated in an in vitro reticulocyte lysate system to yield VP1, VP2, and VP3. These experiments suggest that in infected cells control is exerted upon the AAV 2.3-kilobase mRNA at the translational level and that this control can be influenced by mutations in Ad. These Ad mutations map in the region 2 early gene for the Ad DNA-binding protein. The temperature-sensitive system that we have studied may be useful for analysis of translational control of a eukaryotic mRNA.

Adenovirus helper function for growth of adeno-associated virus: effect of temperature-sensitive mutations in adenovirus early gene region 2.

Adeno-associated virus (AAV) grows efficiently only in cells that are also infected with an adenovirus (Ad). We employed Ad mutants to determine which genes may be required for the AAV helper function. Two mutants of Ad type 5 (Ad5), Ad5ts125 and Ad5ts107, with temperature-sensitive lesions in the E72 DNA-binding protein coded by the Ad early region 2, were deficient for AAV helper functions at the nonpermissive temperature (40 degrees C). In contrast, Ad5ts149, with a temperature-sensitive lesion in the Ad early region 5, was an efficient helper of AAV at the nonpermissive temperature. In KB cells, with the Ad5ts125 or Ad5ts107 mutant as the helper, the accumulation of AAV capsid proteins and AAV particles was decreased by about two logs, whereas AAV DNA synthesis was decreased only severalfold. Cytoplasmic, polyadenylic acid-containing AAV RNA is composed of a set of overlapping, spliced RNAs having different 5' start points. With the ts125 helper at 40 degrees C there was a decreased accumulation of some but not all of these AAV RNAs. The Ad5 E72 protein may have an effect on transcription or more likely posttranscriptional processing of AAV RNA. These observations suggest additional pleiotropic effects of the multifunctional E72 protein and suggest further similarities in the actions of E72 and the simian virus 40 T-antigen.

Spliced adenovirus-associated virus RNA.

We describe the structure of cytoplasmic RNA species transcribed from the DNA of adenovirus-associated virus, a defective parvovirus. The RNA was hybridized with minus strand template DNA and visualized in the electron microscope. Alternatively, the DNA.RNA duplex molecules were digested with nuclease S1 or Escherichia coli exonuclease VII and analyzed by agarose gel electrophoresis. A set of RNA species was observed with 5' terminal at map positions 5, 13, 19, or 39 and a 3' terminus and poly(A) tail at position 96 (one map unit is equivalent to 1% of genome length). Most of these RNAs are spliced and lack sequences approximately between positions 40 and 49. Some RNA preparations also contained unspliced molecules with 5' and 3' terminal at positions similar to those in the spliced RNA.

Orogenital contact and the isolation of Neisseria gonorrhoeae, Mycoplasma hominis, and Ureaplasma urealyticum from the pharynx.

Men and women who came to clinics in Boston underwent pharyngeal examinations, and pharyngeal specimens were obtained for cultures for Neisseria gonorrhoeae, Mycoplasma hominis, and Ureaplasma urealyticum. Fifty-one (4.9%) of 1,037 participants had gonococcal pharyngeal infection. M. hominis and U. urealyticum were recovered from the pharynges of 149 (14.3%) and 154 (14.8%) of 1,044 participants, respectively. The history of ever having performed fellatio was associated with pharyngeal infection with N. gonorrhoeae (P less than 0.02), M. hominis (P less than 0.05), and U. urealyticum (P less than 0.006). A history of fellatio was also associated with a history of a recent sore throat. There was, however, no association between pharyngeal infection with N. gonorrhoeae, M. hominis, or U. urealyticum and a recent sore throat. Cunnilingus was not associated with symptoms or signs or pharyngitis or with the isolation of gonococci or genital mycoplasmas from the pharynx. The pharyngitis associated with fellatio remains a microbiologic enigma.

Temperature-sensitive mutants of measles virus produced from persistently infected HeLa cells.

A persistent infection with the Edmonston strain of measles virus was established in HeLa cells in the absence of measles virus antibody (HeLaPI cells). By hemadsorption or immunofluoresnce virtually 100 per cent of the cells possessed measles virus components. HeLaPI cells produced no interferon and were not resistant to superinfection with Newcastle disease virus. HeLaPI cells contained both smooth (15--18 nm) and rought (20--35 nm) nucleocapsids as detected by electron microscopy. The virus produced from the HeLaPI cells (MVPI) varied in titer between 1.5 X 10(2) and 5.5 X10(4) PFU/ml, had a smaller plque size and was more heat resistant than wild-type measles virus. MVPI was also found to be temperature-sensitive. The temperature-sensitivity of MVPI was determined by the efficiency of plaquing at 33 degrees and 39 degrees C in Vero cell monolayers. When HeLaPI cells were incubated at 33 degrees C, there was a 50-fold increase in virus production as well as a slight increase in the percentage of cells forming infectious centers compared to HeLaPI cells grown at 37 degrees C. MVPI readily established a persistent infection in HeLa cells which also rleased temperature-sensitive virus.