Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors.

We describe replication competent retroviruses capable of expressing heterologous genes during multiple rounds of infection. An internal ribosome entry site (IRES) from encephalomyocarditis virus was inserted in the U3 region of Akv- and SL3-3-murine leukemia viruses (MLV) to direct translation of neo or the enhanced green fluorescence protein gene (EGFP). Akv-MLV's with IRES-neo and IRES-EGFP cassettes replicated with titers of about 10(6) infectious units/ml while SL3-3-MLV with IRES-neo gave about 10(3)-fold lower titers. Interestingly, RNA analysis showed a drastic reduction in the amount of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds and these revertants only slowly became dominant in the virus population.     

The Influence of Primary and Secondary DNA Structure in Deletion and Duplication between Direct Repeats in Escherichia Coli

We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events.     

Packaging capacity and stability of human adenovirus type 5 vectors.

Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus type 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb combined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size ranged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors during serial passage in tissue culture revealed that when the size exceeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb insert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the insert after only a few passages. In contrast, vectors with inserts resulting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, viruses with genomes only slightly above 105% of that of the wt were unstable and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relatively tight constraint on the amount of DNA which can be packaged into virions and that exceeding the limit results in a sharply decreased rate of virus growth. The resultant strong selection for variants which have undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population.     

Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance.

A series of replication-competent Moloney murine leukemia virus vectors was constructed in which each vector contained a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat. Two of the resulting viruses, MLV (murine leukemia virus) DHFR*-5 and MLV DHFR*-7, were able to stably transfer methotrexate resistance to infected fibroblast cells upon multiple rounds of virus replication and in the absence of drug selection. Cell lines producing recombinant virus with high titers were established, which indicated that the insert did not grossly interfere with viral replication functions. These vectors should be useful for introducing and expressing foreign genes in vivo in tissues and whole animals in which virus spread is needed for efficient infection.     

Characterization of unintegrated retroviral DNA with long terminal repeat-associated cell-derived inserts.

We have used a replication-competent shuttle vector based on the genome of Rous sarcoma virus to characterize genomic rearrangements that occur during retrovirus replication. The strategy involved cloning circular DNA that was generated during an acute infection. While analyzing a class of retroviral DNA clones that are greater than full length, we found several clones which had acquired nonviral inserts in positions adjacent to the long terminal repeats (LTRs). There appear to be two distinct mechanisms leading to the incorporation of cellular sequences into these clones. Three of the molecules contain a cell-derived insert at the circle junction site between two LTR units. Two of these molecules appear to be the results of abortive integration attempts, because of which, in each case, one of the LTRs is missing 2 bases at its junction with the cell-derived insert. In the third clone, pNO220, the cellular sequences are flanked by an inappropriately placed copy of the tRNA primer-binding site on one side and a partial copy of the U3 sequence as part of the LTR on the other side. A fourth molecule we characterized, pMD96, has a single LTR with a U5-bounded deletion of viral sequences spanning gag and pol, with cell-derived sequences inserted at the site of the deletion; its origin may be related mechanistically to pNO220. Sequence analysis indicates that all of the cellular inserts were derived from the cell line used for the acute infection rather than from sequences carried into the cell as part of the virus particle. Northern (RNA) analysis of cellular RNA demonstrated that the cell-derived sequences of two clones, pNO220 and pMD96, were expressed as polyadenylated RNA in uninfected cells. One mechanism for the joining of viral and cellular sequences suggested by the structures of pNO220 and pMD96 is recombination occurring during viral DNA synthesis, with cellular RNA serving as the template for the acquisition of cellular sequences.     

Deletion mutagenesis independent of recombination in bacteriophage T7.

Deletion between directly repeated DNA sequences in bacteriophage T7-infected Escherichia coli was examined. The phage ligase gene was interrupted by insertion of synthetic DNA designed so that the inserts were bracketed by 10-bp direct repeats. Deletion between the direct repeats eliminated the insert and restored the ability of the phage to make its own ligase. The deletion frequency of inserts of 85 bp or less was of the order of 10(-6) deletions per replication. The deletion frequency dropped sharply in the range between 85 and 94 bp and then decreased at a much lower rate over the range from 94 to 900 bp. To see whether a deletion was predominantly caused by intermolecular recombination between the leftmost direct repeat on one chromosome and the rightmost direct repeat on a distinct chromosome, genetic markers were introduced to the left and right of the insert in the ligase gene. Short deletions of 29 bp and longer deletions of approximately 350 bp were examined in this way. Phage which underwent deletion between the direct repeats had the same frequency of recombination between the left and right flanking markers as was found in controls in which no deletion events took place. These data argue against intermolecular recombination between direct repeats as a major factor in deletion in T7-infected E. coli.     

Human immunodeficiency virus type 1 genetic recombination is more frequent than that of Moloney murine leukemia virus despite similar template switching rates

Retroviral recombinants result from template switching between copackaged viral genomes. Here, marker reassortment between coexpressed vectors was measured during single replication cycles, and human immunodeficiency virus type 1 (HIV-1) recombination was observed six- to sevenfold more frequently than murine leukemia virus (MLV) recombination. Template switching was also assayed by using transduction-type vectors in which donor and acceptor template regions were joined covalently. In this situation, where RNA copackaging could not vary, MLV and HIV-1 template switching rates were indistinguishable. These findings argue that MLV's lower intermolecular recombination frequency does not reflect enzymological differences. Instead, these data suggest that recombination rates differ because coexpressed MLV RNAs are less accessible to the recombination machinery than are coexpressed HIV RNAs. This hypothesis provides a plausible explanation for why most gammaretrovirus recombinants, although relatively rare, display evidence of multiple nonselected crossovers. By implying that recombinogenic template switching occurs roughly four times on average during the synthesis of every MLV or HIV-1 DNA, these results suggest that virtually all products of retroviral replication are biochemical recombinants.     

Evolutionarily selected replication origins: functional aspects and structural organization.

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.     

DNA replication and chromatin structure of simian virus 40 insertion mutants.

Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure.     

Equimolar generation of the four possible arrangements of adjacent L components in herpes simplex virus type 1 replicative intermediates.

Herpes simplex virus type 1 (HSV-1) replication generates high-molecular-weight intermediates containing branched DNA and concatemers carrying adjacent genomes with inverted L components. We have studied replicative intermediates generated by (i) wild-type HSV-1; (ii) 5dl1.2, an ICP27 null mutant which fails to synthesize normal amounts of DNA and late proteins; (iii) RBMu3, a mutant containing a deletion in the inverted repeats which fails to generate genomic isomers; and (iv) amplicon plasmids and vectors which contain no inverted sequences. Replication intermediates were analyzed by pulsed-field gel electrophoresis, after restriction enzyme digestion of infected-cell DNA, followed by blot hybridization. DNA fragments were statistically quantified after phosphorimaging. We observed that (i) the four possible configurations of L components of two adjacent genomes in the concatemers are present at equimolar amounts at any time during virus replication, (ii) ICP27 is not required for inversions or for branched DNA to occur, and (iii) replication intermediates of both RBMu3 mutant and amplicon plasmids or vectors do contain branched structures, although the concatemers they generate contain no inversions. These data indicate that inversions are generated by a mechanism intrinsically linked to virus DNA replication, most likely homologous recombination between inverted repeats. Branched structures are detected in all replicating molecules, including those that do not invert, suggesting that they are constitutively linked to virus DNA synthesis. Our results are consistent with the notion that the four HSV-1 genomic isomers are generated by alternative cleavage frames of replication concatemers containing equimolar amounts of L-component inversions.     

Retroviral Recombination Rates Do Not Increase Linearly with Marker Distance and Are Limited by the Size of the Recombining Subpopulation

Recombination occurs at high frequencies in all examined retroviruses. The previously determined homologous recombination rate in one retroviral replication cycle is 4% for markers 1.0 kb apart in spleen necrosis virus (SNV). This has often been used to suggest that approximately 30 to 40% of the replication-competent viruses with 7- to 10-kb genomes undergo recombination. These estimates were based on the untested assumption that a linear relationship exists between recombination rates and marker distances. To delineate this relationship, we constructed three sets of murine leukemia virus (MLV)-based vectors containing the neomycin phosphotransferase gene (neo) and the hygromycin phosphotransferase B gene (hygro). Each set contained one vector with a functional neo and an inactivated hygro and one vector with a functional hygro and an inactivated neo. The two inactivating mutations in the three sets of vectors were separated by 1.0, 1.9, and 7.1 kb. Recombination rates after one round of replication were 4.7, 7.4, and 8.2% with markers 1.0, 1.9, and 7.1 kb apart, respectively. Thus, the rate of homologous recombination with 1.0 kb of marker distance is similar in MLV and SNV. The recombination rate increases when the marker distance increases from 1.0 to 1.9 kb; however, the recombination rates with marker distances of 1.9 and 7.1 kb are not significantly different. These data refute the previous assumption that recombination is proportional to marker distance and define the maximum recombining population in retroviruses.     

Replication of the cauliflower mosaic virus: role and stability of the cloned delta 3 discontinuity sequence

A fragment of cauliflower mosaic virus (CaMV) DNA, containing delta 3, one of the three discontinuity sequences, was cloned in various ways into CaMV DNA deleted for the delta 3 sequence. The series of constructions was monitored for the appearance of the typical single-strand (ss) discontinuity after hybrid CaMV replication in plants. The delta 3 discontinuity was observed only if the orientation of inserted DNA sequence was the same as in the wild-type virus. Long polylinker sequences used for insertion of the fragment into cloned viral DNA, affected the stability of the insert in progeny viral DNA in plants by acting as recombination targets.     

Amino acid insertions near Gag cleavage sites restore the otherwise compromised replication of human immunodeficiency virus type 1 variants resistant to protease inhibitors

A variety of amino acid substitutions in the protease and Gag proteins have been reported to contribute to the development of human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors. In the present study, full-length molecular infectious HIV-1 clones were generated by using HIV-1 variants isolated from heavily drug-experienced and therapy-failed AIDS patients. Of six full-length infectious clones generated, four were found to have unique insertions (TGNS, SQVN, AQQA, SRPE, APP, and/or PTAPPA) near the p17/p24 and p1/p6 Gag cleavage sites, in addition to the known resistance-related multiple amino acid substitutions within the protease. The addition of such Gag inserts mostly compromised the replication of wild-type HIV-1, whereas the primary multidrug-resistant HIV infectious clones containing inserts replicated significantly better than those modified to lack the inserts. Western blot analyses revealed that the processing of Gag proteins by wild-type protease was impaired by the presence of the inserts, whereas that by mutant protease was substantially improved. The present study represents the first report clearly demonstrating that the inserts seen in the proximity of the Gag cleavage sites in highly multi-PI resistant HIV-1 variants restore the otherwise compromised enzymatic activity of mutant protease, enabling the multi-PI-resistant HIV-1 variants to remain replication competent.     

Differential DNA secondary structure-mediated deletion mutation in the leading and lagging strands.

The frequencies of deletion of short sequences (mutation inserts) inserted into the chloramphenicol acetyl-transferase (CAT) gene were measured for pBR325 and pBR523, in which the orientation of the CAT gene was reversed, in Escherichia coli. Reversal of the CAT gene changes the relationship between the transcribed strand and the leading and lagging strands of the DNA replication fork in pBR325-based plasmids. Deletion of these mutation inserts may be mediated by slipped misalignment during DNA replication. Symmetrical sequences, in which the same potential DNA structural misalignment can form in both the leading and lagging strands, exhibited an approximately twofold difference in the deletion frequencies upon reversal of the CAT gene. Sequences that contained an inverted repeat that was asymmetric with respect to flanking direct repeats were designed. With asymmetric mutation inserts, different misaligned structural intermediates could form in the leading and lagging strands, depending on the orientation of the insert and/or of the CAT gene. When slippage could be stabilized by a hairpin in the lagging strand, thereby forming a three-way junction, deletion occurred by up to 50-fold more frequently than when this structure formed in the leading strand. These results support the model that slipped misalignment involving DNA secondary structure occurs preferentially in the lagging strand during DNA replication.     

Herpes simplex virus amplicon: effect of size on replication of constructed defective genomes containing eucaryotic DNA sequences.

Previous studies (R. R. Spaete and N. Frenkel, Cell 30:295-304, 1982) have documented the potential use of defective virus vectors (amplicons) derived from herpes simplex virus for the efficient introduction of foreign DNA sequences into eucaryotic cells. Specifically, cotransfection of cells with helper virus DNA and cloned amplicons (8 to 10 kilobases [kb]) containing bacterial plasmid DNA sequences linked to a set of herpes simplex virus cis-acting propagation signals (a replication origin and a cleavage-packaging signal) resulted in the generation of virus stocks containing packaged defective genomes that consisted of uniform head-to-tail reiterations of the chimeric seed amplicon sequences. The chimeric defective genomes could be stably propagated in virus stocks and could thus be used to efficiently infect cells. We now report on additional studies designed to propagate relatively large sets of eucaryotic DNA sequences within chimeric packaged defective genomes. These studies have utilized a 12-kb chicken DNA sequence encoding the chicken ovalbumin gene and cloned by Lai et al. (Proc. Natl. Acad. Sci. U.S.A. 77:244-248, 1980) in the plasmid pOV12. Virus stocks derived from cells cotransfected with helper virus DNA and chimeric amplicons (overall size of 19.8 kb, of which 12 kb corresponded to the chicken DNA) contained defective genomes composed of reiterations of the 19.8-kb seed amplicon sequences. However, in addition to the authentically sized repeat units, defective genomes in the derivative virus stocks contained smaller repeat units representing deleted versions of the seed 19.8-kb amplicons. The recombinational events leading to the formation of deleted repeats did not appear to occur at unique sites, as shown by comparative analyses of multiple, independently generated virus series propagated from separate transfections. In contast, seed amplicons ranging in size from 11 to 15 kb and containing subsets of the 12-kb chicken DNA sequences replicated efficiently and could be stably propagated in virus stocks. The results of these studies suggest the existence of size restrictions (up to 15 kb) on the efficient replication of seed herpes simplex virus amplicons.