Cloning of Herpesvirus saimiri DNA fragments representing the entire L-region of the genome

Purified particles of Herpesvirus saimiri, a potent tumor-eliciting virus of primates, contain genomic DNA molecules (145-170 kb) consisting of a unique L-DNA region (112 kb) which is flanked by variable stretches of repetitive sequences (H-DNA). Restriction fragments representing the entire L-DNA of H. saimiri strain No. 11 were cloned in plasmid and bacteriophage vectors. The internal fragments of L-DNA generated by the enzymes EcoRI and KpnI were inserted into plasmid pACYC184, cosmid pJC81, or bacteriophage lambda derivative Charon 4A. The terminal parts of L-DNA, including the junctions between repetitive DNA and unique sequences, were cloned between the cleavage sites for KpnI and SmaI in the plasmid vector pWD7, which was constructed for this purpose. Molecular cloning allowed us to confirm and modify, in part, the existing cleavage maps of H. saimiri DNA. It provides a basis for future studies on virus replication and oncogenic transformation.     

Selectable recombinant herpesvirus saimiri is capable of persisting in a human T-cell line.

Strategies were developed to insert the neo resistance marker into the junction between L DNA and the right terminal repetitive H DNA of herpesvirus saimiri. Recombinant viruses were selectable in permissive epithelioid cultures. The human T-cell line Jurkat could be infected persistently with the Neor virus; the cells contained episomal viral DNA in high copy number. This selectable vector should be generally applicable for gene expression in human T cells.     

Construction of fowlpox virus vectors with intergenic insertions: expression of the beta-galactosidase gene and the measles virus fusion gene.

A DNA fragment from fowlpox virus cloned on a plasmid vector was modified to contain foreign DNA inserts within an intergenic region. In a first step, a 32-base-pair intergenic region from the fowlpox virus genome corresponding to the position of the thymidine kinase locus in the vaccinia virus genome was enlarged to 55 base pairs by site-directed mutagenesis. A unique restriction endonuclease site introduced upstream of the intergenic region was then used to insert various foreign DNA fragments. The lacZ gene encoding beta-galactosidase and the measles virus gene encoding the fusion protein were positioned downstream of two vaccinia virus p7.5 promoter elements in either a direct repeat or inverted repeat orientation. Foreign DNA inserts contained within the fowlpox virus sequence were transferred to the viral genome by homologous recombination occurring in cells infected with a fowlpox virus temperature-sensitive mutant and transfected with both wild-type viral DNA and plasmid DNA. Recombinant viruses were selected for the expression of beta-galactosidase activity by screening for blue plaques in the presence of a chromogenic substrate. Stable recombinants expressing both the lacZ gene and the unselected measles gene were obtained when the p7.5 promoter was present as an inverted repeat. However, when the p7.5 promoter was in the direct repeat orientation, viral recombinants which initially expressed both gene inserts readily deleted the lacZ gene flanked by the promoter repeat. The methods described enable precise insertion and deletion of foreign genes in the fowlpox virus genome and could be applied to other intergenic regions of the same virus as well as other poxviruses.     

Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene.

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.     

Second-site homologous recombination in Epstein-Barr virus: insertion of type 1 EBNA 3 genes in place of type 2 has no effect on in vitro infection.

This study was undertaken to develop a general strategy for the introduction of mutations into specific sites in the Epstein-Barr virus (EBV) genome. Previous approaches were limited by the need for physical linkage of the transfected EBV DNA fragment to a positive selection marker. In our experiments, a positive selection marker was introduced into one site in the EBV genome and a distant, nonlinked, marker was introduced into another site. Each marker was on a large EBV DNA fragment and was inserted into the genome by transfection into cells carrying a resident EBV genome. The resident EBV genome was simultaneously induced to replicate by using a cotransfected expression plasmid for the EBV immediate-early transactivator, Z (J. Countryman, H. Jenson, R. Seibl, H. Wolf, and G. Miller, J. Virol. 61:3672-3679, 1987; G. Miller, M. Rabson, and L. Heston, J. Virol. 50:174-182, 1984). Eleven percent of the resultant EBV genomes which incorporated the positive selection marker also incorporated the nonlinked marker. Both markers uniformly targeted the homologous EBV genome site. In this way novel EBV recombinants were constructed in which the EBV type 1 EBNA 3A, EBV type 1 EBNA 3A and 3B, or EBV type 1 EBNA 3A, 3B, and 3C genes were introduced into a largely type 2 EBV genome, replacing the corresponding type 2 gene(s). No difference was observed in primary B-lymphocyte growth transformation, in latent EBV gene expression, or in spontaneous lytic EBV gene expression. These new recombinants should be useful for ongoing analyses of the type specificity of the immune response.     

Transfection of a plasmid with a retrovirus promoter: distribution of sequences in the genome of induced tumors

The structure of the integration site of plasmid with LTR of Rous sarcoma virus (pLTR1,5) in the genome of nude mice tumors, induced as a result of N1H3T3 cells' implantation, cotransfected by pLTR1,5 with the DNA of malignant human glioma cells, carrying amplified c-Ha-ras genome, has been studied. The restriction map of the investigated region of the cell genome was obtained. Molecular cloning of the integrated plasmid and adjacent cell sequences has been carried out. It was shown that the exogenic vector in the DNA of the tumor cells is included in BamHI structure of the repeat of mice genome.     

Transient host range selection for genetic engineering of modified vaccinia virus Ankara

Recombinant vaccinia viruses are extremely valuable tools for research in molecular biology and immunology. The extension of vaccinia vector technology to replication-deficient and safety-tested virus strains such as modified vaccinia virus Ankara (MVA) have made this versatile eukaryotic expression system even more attractive for basic and clinical research. Here, we report on easily obtaining recombinant MVA using stringent growth selection on rabbit kidney RK-13 cells. We describe the construction and use of new MVA vector plasmids that carry an expression cassette of the vaccinia virus host range gene, K1L, as a transient selectable marker. These plasmids allow either stable insertion of additional recombinant genes into the MVA genome or precisely targeted mutagenesis of MVA genomic sequences. Repetitive DNA sequences flanking the K1L gene were designed to remove the marker gene from the viral genome by homologous recombination under nonselective growth conditions. The convenience of this new selection technique is demonstrated by isolating MVA recombinants that produce green fluorescent protein and by generating MVA deletion mutants.     

The URNA genes of herpesvirus saimiri (strain C488) are dispensable for transformation of human T cells in vitro

The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.     

Development of a cloning system in Mycoplasma pulmonis

A system suitable for recombinant DNA manipulation in mycoplasmas was developed using the cloned antibiotic-resistance genes of Tn4001 and Tn916. An integrative plasmid containing one of the resistance markers was inserted into the genome of Mycoplasma pulmonis to form a recipient strain. This was accomplished by transformation and homologous recombination between chromosomal DNA sequences cloned onto the integrative plasmid. A second vector, the cloning vector, containing the same plasmid replicon and alternate resistance marker, carried cloned foreign DNA. When transformed into mycoplasmal recipients, homologous recombination between plasmid sequences resulted in integration of the cloning vector and foreign DNA. A Brucella abortus gene coding for a 31-kDa protein and the P1 structural gene and operon from Mycoplasma pneumoniae were introduced to examine the feasibility of developing mycoplasma as cloning hosts. Recombinant plasmids as large as 20 kb were inserted into M. pulmonis, and the integrated foreign DNA was stably maintained. The maximum size of clonable DNA was not determined, but plasmids larger than 22 kb have not been transformed into mycoplasmas using polyethylene glycol. Also the size of genome (800-1200 kb) may affect the stability of larger inserts of foreign DNA. This system is applicable to any mycoplasma capable of transformation, homologous recombination and expression of these resistance markers. Because of their lack of a cell wall, mycoplasmas may be useful cloning hosts for membrane or excreted protein genes from other sources.     

Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells.

The objective of these experiments was to develop strategies for creation and identification of recombinant mutant Epstein-Barr viruses (EBV). EBV recombinant molecular genetics has been limited to mutations within a short DNA segment deleted from a nontransforming EBV and an underlying strategy which relies on growth transformation of primary B lymphocytes for identification of recombinants. Thus, mutations outside the deletion or mutations which affect transformation cannot be easily recovered. In these experiments we investigated whether a toxic drug resistance gene, guanine phosphoribosyltransferase or hygromycin phosphotransferase, driven by the simian virus 40 promoter can be recombined into the EBV genome and can function to identify B-lymphoma cells infected with recombinant virus. Two different strategies were used to recombine the drug resistance marker into the EBV genome. Both utilized transfection of partially permissive, EBV-infected B95-8 cells and positive selection for cells which had incorporated a functional drug resistance gene. In the first series of experiments, B95-8 clones were screened for transfected DNA that had recombined into the EBV genome. In the second series of experiments, the transfected drug resistance marker was linked to the plasmid and lytic EBV origins so that it was maintained as an episome and could recombine with the B95-8 EBV genome during virus replication. The recombinant EBV from either experiment could be recovered by infection and toxic drug selection of EBV-negative B-lymphoma cells. The EBV genome in these B-lymphoma cells is frequently an episome. Virus genes associated with latent infection of primary B lymphocytes are expressed. Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells. Moreover, the EBV-infected B-lymphoma cells are often partially permissive for early replicative cycle gene expression and virus replication can be induced, in contrast to previously reported in vitro infected B-lymphoma cells. These studies demonstrate that dominant selectable markers can be inserted into the EBV genome, are active in the context of the EBV genome, and can be used to recover recombinant EBV in B-lymphoma cells. This system should be particularly useful for recovering EBV genomes with mutations in essential transforming genes.     

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.     

Development of improved Sindbis virus-based DNA expression vector

We have constructed an improved DNA expression vector based on the Sindbis virus. Several DNA-based Sindbis virus vectors were constructed to investigate the efficiency of transgene expression. These vectors, when transfected into mammalian cells, have been used to express heterologous genes. A recombinant genome of Sindbis plasmid DNA, in which the structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes, was placed under the control of a simian virus (SV 40) promoter with a hepatitis delta virus (HDV) antigenomic ribozyme and a polyadenylation signal. Transfection of mammalian cells with this Sindbis-based plasmid vector, pSin-SV40-HDV-SV40pA, resulted in transient high-level expression of the beta-galactosidase reporter gene. The expression level of beta-galactosidase from pSin-SV40-HDV-SV40pA was more than 16-fold higher than that of pSin-Lux originally reported by Herweijer et al. In vivo expression was also detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable after day 14. Furthermore, we demonstrate that the transfection of cells with this Sindbis virus vector results in apoptotic death on glioma cells. We have demonstrated a high-level expression of the exogenous beta-galactosidase gene from the pSin-SV40-HDV-SV40pA construct using a Sindbis replication system.     

A novel host-vector system for direct selection of recombinant baculoviruses (bacmids) in Escherichia coli

Site-specific transposition in Escherichia coli was used to introduce foreign genes into the Autographica californica nuclear polyhedrosis baculovirus genome. Using a temperature-sensitive donor plasmid and an E. coli host strain with an occupied Tn7 attachment site it was possible to select directly for ‘bacmid’ recombinants at 44°C. A blue to white color screen provided further confirmation of insertion at the correct site in the baculovirus genome. After cloning the gene of interest into a donor plasmid, a single transformation and plating on selective medium resulted in homogeneous baculovirus DNA which could immediately be transfected into insect cells. The utility of the host-vector system for expression in insect cells was illustrated using three heterologous genes encoding β-glucuronidase, human N-myristoyl transferase and murine preproguanylin. Using this approach, bacmid recombinants could be produced at a frequency of 10⁵ per pg input DNA. This system should not only greatly enhance the ability to obtain recombinant viruses for heterologous protein production, but should also be useful for protein engineering applications and expression cloning in insect cells.     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.     

Human adenovirus cloning vectors based on infectious bacterial plasmids

By making use of the fact that human adenovirus DNA circularizes in infected cells, and that circular forms of the viral genome are infectious, we have developed an improved adenovirus-based cloning system. A deletion mutant of adenovirus type 5 (Ad5) with deletions in early regions 1 (E1) and 3 (E3) was converted to a bacterial plasmid which can regenerate infectious virus following transfection into human 293 cells. A single XbaI recognition site in the deleted E3 region serves as a site for the insertion of foreign DNA. We have used this system to clone a number of genes into the Ad5 genome and describe the insertion of the neomycin/G418 resistance marker into Ad5 as an example.     

Recovery of biologically active spleen focus-forming virus from molecularly cloned spleen focus-forming virus-pBR322 circular DNA by cotransfection with infectious type C retroviral DNA.

The genome of the Lilly-Steeves strain of spleen focus-forming virus (SFFV) was molecularly cloned in the plasmid vector pBR322. Infectious SFFV could be recovered by releasing the SFFV DNA from the vector, transfecting the released DNA onto NIH 3T3 cells, and rescuing the SFFV either by superinfection with helper virus or by cotransfection with molecularly cloned infectious helper viral DNA. By using transfections with SFFV DNA still attached to the plasmid vector, infectious SFFV activity could also be recovered with either method of rescue. Studies performed with these latter types of transfections indicated that only a portion of the SFFV genome was required for biological activity. Since gp52, a marker protein for SFFV, could be detected in all cultures from which adequate titers of biologically active SFFV were recovered, the results are consistent with the hypothesis that gp52 is necessary for SFFV-induced erythroblastosis and polycythemia.     

鸡马立克氏病病毒814株细菌人工染色体的构建

为构建全基因组鸡马立克氏病病毒814株感染性细菌人工染色体(bacterial artificial chromosome, BAC), 首先通过构建表达Eco-gpt(xanthine-guanine phosphoribosyl transferase, XGPRT, gpt)的哺乳动物细胞基因转移遗传选择标记(1.3 kb)和带有细菌人工染色体的基本功能基因序列的鸡马立克氏病病毒重组病毒转移载体pUAB-gpt-BAC11, 将重组病毒转移载体与鸡马立克氏病病毒细胞总DNA共转染鸡胚成纤维细胞, 在选择培养基中经过8轮加压筛选, 获得并纯化重组病毒; 将重组病毒细胞总DNA电转化大肠杆菌, 筛选共获得38个BAC分子克隆化病毒, 提取BAC-DNA转染鸡胚成纤维细胞以拯救重组病毒。结果表明, MDV-BAC2 DNA再次启动病毒感染, 拯救了重组鸡马立克氏病病毒。成功构建了鸡马立克氏病病毒814株基因组全长感染性细菌人工染色体, 为方便利用现代RED/ET基因重组系统对病毒进行反向遗传操作提供了技术平台; 同时为研究鸡马立克氏病病毒的基因功能和开发新型马立克氏病疫苗奠定了基础。     

Generation and effective enrichment of selectable human cytomegalovirus mutants using site-directed insertion of the neo gene

Studies on the biology and function of human cytomegalovirus (HCMV) genes have been hampered by the limited number of viral mutants available for genetic analyses. We have developed a simple procedure to generate and enrich for HCMV recombinants. By inserting the bacterial neo gene, encoding neomycin/kanamycin phosphotransferase, into the large HCMV DNA genome using homologous recombination, selectable mutants of this complex herpesvirus were isolated for the first time. The synthesis of Neo from the viral genome was used to effectively enrich for recombinant viruses (re-viruses) in permissive culture cells grown in the presence of Geneticin (G418). A quick assay for Neo activity in infected cells, based on phosphorylation of kanamycin (Km), was used to easily identify viral recombinants in the process of screening and isolation. This procedure, not used previously to identify re-viruses, proved to be very useful for screening of large numbers of HCMV recombinants. Analysis of re-virus by Southern blotting revealed that the insertion of the marker gene had resulted in the expected deletion of the open reading frames, TRL 13/14 and UL 1–5, of HCMV. Re-virus was stable and showed no differences in growth kinetics as compared to wild-type (wt) virus. The insertion of a selectable marker gene into the HCMV genome and identification of viral recombinants by the Km phosphorylation assay, as presented here, provides the rationale for effective generation, enrichment and stable propagation of HCMV mutants.