Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus.

The infection of epithelia] swine testicle and intestinal porcine epithelial (IPEC-1) cell lines by adenovirus type 5 (Ad5) has been studied in vitro by using an Ad5-luciferase recombinant containing the firefly luciferase gene as a reporter. Porcine cell lines supported Ad5 replication, showing virus titers, kinetics of virus production, and luciferase expression levels similar to those obtained in human 293 cells, which constitutively express the 5'-end 11% of the Ad5 genome. The tropism of Ad5-based vectors in swine and its ability to induce an efficient immune response against heterologous antigens expressed by foreign genes inserted in these vectors has been determined. Ad5 vectors replicate and express heterologous antigens in porcine lungs and mediastinal and mesenteric lymph nodes. Significant levels of heterologous antigen expression were also demonstrated in the small intestine (jejunum and ileum), but Ad5 replication in this organ was very poor, suggesting that Ad vectors undergo an abortive replication in the porcine small intestine. The tissues infected by Ad5 were dependent on the inoculation route. The oronasal route appeared to be best for inoculation of bronchus-associated lymphoid tissue infection, while the intraperitoneal route was best for gut-associated lymphoid tissue infection. Epithelial cells of bronchioles, macrophages, type II pneumocytes, and follicular dendritic cells were identified as targets for Ad5, while epithelial cells of the intestine were not infected by Ad5. Viruses with a deletion from 79.5 to 84.8 map units in the E3 region, with or without heterologous inserted genes, replicated to lower levels in porcine tissues than did wild-type Ad5. It was also shown that an Ad5 recombinant expressing the four antigenic sites (A, B, C, and D) of transmissible gastroenteritis coronavirus (TGEV) spike protein induced in swine immune responses which neutralized TGEV infectivity. In addition, porcine serum from Ad-TGEV-immune animals provide passive protection when mixed with fully virulent TGEV and orally administered to highly susceptible newborn piglets. These results taken together indicate that swine may be a good animal model for human Ad5 lung infection to aid in the evaluation of candidate adenovirus vaccines and that Ad5 may be suitable as a recombinant viral vaccine or for other applications in swine.     

Postinternalization inhibition of adenovirus gene expression and infectious virus production in human T-cell lines

Detection of adenovirus DNA in human tonsillar T cells in the absence of active virus replication suggests that T cells may be a site of latency or of attenuated virus replication in persistently infected individuals. The lytic replication cycle of Ad5 in permissive epithelial cells (A549) was compared to the behavior of Ad5 in four human T-cell lines, Jurkat, HuT78, CEM, and KE37. All four T-cell lines expressed the integrin coreceptors for Ad2 and Ad5, but only Jurkat and HuT78 express detectable surface levels of the coxsackie adenovirus receptor (CAR). Jurkat and HuT78 cells supported full lytic replication of Ad5, albeit at a level approximately 10% of that of A549, while CAR-transduced CEM and KE37 cells (CEM-CARhi and KE37-CARhi, respectively) produced no detectable virus following infection. All four T-cell lines bind and internalize fluorescently labeled virus. In A549, Jurkat, and HuT78 cells, viral proteins were detected in 95% of cells. In contrast, only a small subpopulation of CEM-CARhi and KE37-CARhi cells contained detectable viral proteins. Interestingly, Jurkat and HuT78 cells synthesize four to six times more copies of viral DNA per cell than did A549 cells, indicating that these cells produce infectious virions with much lower efficiency than A549. Similarly, CEM-CARhi and KE37-CARhi cells, which produce no detectable infectious virus, synthesize three times more viral genomes per cell than A549. The observed blocks to adenovirus gene expression and replication in all four human T-cell lines may contribute to the maintenance of naturally occurring persistent adenovirus infections in human T cells.     

A new type of adenovirus vector that utilizes homologous recombination to achieve tumor-specific replication

We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.IR vectors). Here, we used this system to achieve tumor-specific expression of adenoviral wild-type E1A in order to enhance viral DNA replication and spread within tumor metastases. In vitro DNA replication and cytotoxicity studies demonstrated that the mechanism of E1A-enhanced replication of Ad.IR-E1A vectors is efficiently and specifically activated in tumor cells, but not in nontransformed human cells. Systemic application of the Ad.IR-E1A vector into animals with liver metastases achieved transgene expression exclusively in tumors. The number of transgene-expressing tumor cells within metastases increased over time, indicating viral spread. Furthermore, the Ad.IR-E1A vector demonstrated antitumor efficacy in subcutaneous and metastatic models. These new Ad.IR-E1A vectors combine elements that allow for tumor-specific transgene expression, efficient viral replication, and spread in liver metastases after systemic vector application.     

Novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease

We have previously shown that replication of foot-and-mouth disease virus (FMDV) is highly sensitive to alpha/beta interferon (IFN-alpha/beta). In the present study, we constructed recombinant, replication-defective human adenovirus type 5 vectors containing either porcine IFN-alpha or IFN-beta (Ad5-pIFNalpha or Ad5-pIFNbeta). We demonstrated that cells infected with these viruses express high levels of biologically active IFN. Swine inoculated with 10(9) PFU of a control Ad5 virus lacking the IFN gene and challenged 24 h later with FMDV developed typical signs of foot-and-mouth disease (FMD), including fever, vesicular lesions, and viremia. In contrast, swine inoculated with 10(9) PFU of Ad5-pIFNalpha were completely protected when challenged 24 h later with FMDV. These animals showed no clinical signs of FMD and no viremia and did not develop antibodies against viral nonstructural proteins, suggesting that complete protection from infection was achieved.     

Efficient adenoviral-mediated gene delivery into porcine mesenchymal stem cells

Mesenchymal stem cell (MSC) mediated gene therapy research has been conducted predominantly on rodents. Appropriate large animal models may provide additional safety and efficacy information prior to human clinical trials. The objectives of this study were: (a) to optimize adenoviral transduction efficiency of porcine bone marrow MSCs using a commercial polyamine-based transfection reagent (GeneJammer, Stratagene, La Jolla, CA), and (b) to determine whether transduced MSCs retain the ability to differentiate into mesodermal lineages. Porcine MSCs (pMSCs) were infected under varying conditions, with replication-defective adenoviral vectors carrying the GFP gene and GFP expression analyzed. Transduced cells were induced to differentiate in vitro into adipogenic, chondrogenic, and osteogenic lineages. We observed a 5.5-fold increase in the percentage of GFP-expressing pMSCs when adenovirus type 5 carrying the adenovirus type 35 fiber (Ad5F35eGFP) was used in conjunction with GeneJammer. Transduction of pMSCs at 10.3-13.8 MOI (1,500-2,000 vp/cell) in the presence of Gene Jammer yielded the highest percentage of GFP-expressing cells ( approximately 90%) without affecting cell viability. A similar positive effect was detected when pMSCs were infected with an Ad5eGFP vector. Presence of fetal bovine serum (FBS) during adenoviral transduction enhanced vector-encoded transgene expression in both GeneJammer-treated and control groups. pMSCs transduced with adenovirus vector in the presence of GeneJammer underwent lipogenic, chondrogenic, and osteogenic differentiation. Addition of GeneJammer during adenoviral infection of pMSCs can revert the poor transduction efficiency of pMSCs while retaining their pluripotent differentiation capacity. GeneJammer-enhanced transduction will facilitate the use of adenoviral vectors in MSC-mediated gene therapy models and therapies.     

Tumor-specific, replication-competent adenovirus vectors overexpressing the adenovirus death protein

We have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting the adp gene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutant dl1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G(0) to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus, dl1101/1107, or wild-type Ad. They are also more efficient than dl1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1(-) E3(-) replication-defective vector expressing beta-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10(-4) PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected with dl1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.     

经NGR肽段遗传修饰的携带三种报告基因的腺病毒载体的构建及其实验研究

NGR(Asn-Gly-Arg)是通过噬菌体展示技术筛选出来的能够和肿瘤新生血管特异结合的三肽模体,可以将多种药物分子和病毒载体靶向运输到肿瘤或者进行血管再生的组织中。为此构建了腺病毒衣壳蛋白knob的HI环(HI-loop)经NGR肽段修饰的并同时表达三种报告基因的腺病毒载体Ad5/E1-mCherry/E3-luciferase-2A-eGFP/knob-NGR。体内、外实验研究表明,该病毒载体可成功表达三种报告基因;经NGR肽段修饰的腺病毒载体对人乳腺癌细胞系MDA-MB-231的感染效率高于未经修饰的对照腺病毒Ad5CMVeGFP。该载体的成功构建为进一步研究经NGR肽段修饰的腺病毒在肿瘤动物模型体内的靶向性及经NGR肽段修饰的并携带治疗基因的实验治疗研究奠定了基础。     

Species dependence of the major late promoter in adenovirus type 12 DNA.

In hamster cells human adenovirus type 12 (Ad12) is deficient in DNA replication and late gene expression whereas adenovirus type 2 (Ad2) can replicate. Functions located in the E1 region of the Ad2 or adenovirus type 5 (Ad5) genome can complement the deficiencies of the Ad12 genome in hamster cells, but, infectious viral particles are not produced. We have now investigated the activity of the major late promoter of Ad2 and of Ad12 DNA in human and hamster cells. This promoter governs the expression of most of the late viral functions. We have inserted the major late promoter (MLP) of Ad2 or of Ad12 DNA in front of the chloramphenicol acetyl transferase gene in the pSVO-CAT construct. Upon transfection into uninfected human and hamster cells, the pAd12MLP-CAT construct shows no significant activity; the pAd2MLP-CAT construct exhibits low activity. In Ad12-infected human cells, both constructs are active. These findings support the notion that other viral factors are required for MLP activity of Ad2 or Ad12 DNA in permissive human cells. In Ad2-infected hamster cells, both the pAd2MLP-CAT and the pAd12MLP-CAT constructs are active. Apparently, the Ad12 MLP can be activated by Ad2 functions, as already demonstrated for the entire Ad12 genome in double-infected cells or in Ad2- or Ad5-transformed cells superinfected with Ad12. In Ad12-infected hamster cells, however, the MLP of Ad12 DNA is inactive but that of Ad2 DNA shows activity. Thus the MLP of Ad12 DNA somehow differentiates between cellular auxiliary functions of different species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fiber-knob modifications enhance adenoviral tropism and gene transfer in malignant glioma

BACKGROUND: Malignant gliomas remain refractory to Ad5-mediated gene therapy due to deficiency of the coxsackie adenovirus receptor on tumor cells. The purpose of this study was to evaluate whether changes in adenoviral tropism can enhance gene transfer in the context of malignant glioma. METHODS: We have identified several receptors that are over-expressed on tumor cells and created a series of pseudotyped Ad5 vectors that recognize these receptors: Ad5-RGD which binds alpha(v)beta3/alpha(v)beta5 integrins; Ad5/3 which contains adenovirus serotype 3 knob and binds to CD46; Ad5-Sigma which incorporates the reovirus sigma knob and binds to junctional adhesion molecule-1; and Ad5-pk7 which contains the polylysine motif and binds heparan sulfate proteoglycans. We also investigated the Ad5-CAV1 vector, which contains the knob of canine adenovirus type 1, a virus previously shown to infect glioma via an unknown mechanism. In this study, we compared these modified vectors for their ability to promote the expression of luciferase transgene both in vitro and in vivo. RESULTS: Our results indicate that all five modified vectors attained higher mean luciferase activity vs. control. Among them, Ad5-CAV1 and Ad5-pk7 attained the highest transduction efficiency independent of different tumor lines or infection time. Ad5-Sigma and Ad5-pk7 also demonstrated the least nonspecific infection in normal human astrocytes. Most importantly, Ad5-pk7 achieved 1000-fold increased transgene expression in human glioma xenografts in vivo. CONCLUSIONS: These results indicate that modifications of adenoviral tropism can enhance gene transfer in tumors that are poorly susceptible to adenoviral vectors and warrant further development of Ad5-pk7 for glioma gene therapy.     

Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.     

Cutting edge: restoration of the ability to generate CTL in mice immune to adenovirus by delivery of virus in a collagen-based matrix

Viruses are commonly used for the delivery of genes coding for tumor-associated Ags to elicit tumor-specific immune responses. The success of viral vectors has been limited in preclinical and clinical trials in part because of antiviral immunity. We investigated the ability of a collagen-based matrix (Gelfoam; Pharmacia and Upjohn, Kalamazoo, MI) to improve CTL activation by recombinant adenovirus. The data show that coinjection of Gelfoam with type 5 adenovirus recombinant for prostate-specific Ag (Ad5-PSA) enhanced CTL activation. Ad5-PSA priming in Gelfoam also abrogated the inhibitory effects of adenoviral immunity on CTL activation in mice naive to PSA but immune to adenovirus. Finally, Gelfoam enhanced immunization in a self-Ag model using type 5 adenovirus recombinant for membrane-bound OVA (Ad5-mOVA) in rat insulin promoter (RIP)-mOVA-transgenic mice. Thus, Gelfoam enhances CTL activation by recombinant viral vectors in a setting where preformed Ab to the virus is present and also in a tolerant self-Ag model.     

Transduction and oncolytic profile of a potent replication-competent adenovirus 11p vector (RCAd11pGFP) in colon carcinoma cells

Replication-competent adenovirus type 5 (Ad5) vectors promise to be more efficient gene delivery vehicles than their replication-deficient counterparts, and chimeric Ad5 vectors that are capable of targeting CD46 are more effective than Ad5 vectors with native fibers. Although several strategies have been used to improve gene transduction and oncolysis, either by modifying their tropism or enhancing their replication capacity, some tumor cells are still relatively refractory to infection by chimeric Ad5. The oncolytic effects of the vectors are apparent in certain tumors but not in others. Here, we report the biological and oncolytic profiles of a replication-competent adenovirus 11p vector (RCAd11pGFP) in colon carcinoma cells. CD46 was abundantly expressed in all cells studied; however, the transduction efficiency of RCAd11pGFP varied. RCAd11pGFP efficiently transduced HT-29, HCT-8, and LS174T cells, but it transduced T84 cells, derived from a colon cancer metastasis in the lung, less efficiently. Interestingly, RCAd11p replicated more rapidly in the T84 cells than in HCT-8 and LS174T cells and as rapidly as in HT-29 cells. Cell toxicity and proliferation assays indicated that RCAd11pGFP had the highest cell-killing activities in HT29 and T84 cells, the latter of which also expressed the highest levels of glycoproteins of the carcinoma embryonic antigen (CEA) family. In vivo experiments showed significant growth inhibition of T84 and HT-29 tumors in xenograft mice treated with either RCAd11pGFP or Ad11pwt compared to untreated controls. Thus, RCAd11pGFP has a potent cytotoxic effect on colon carcinoma cells.     

A Novel and Simple Method for Rapid Generation of Recombinant Porcine Adenoviral Vectors for Transgene Expression

Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.     

腺病毒载体衣壳蛋白质的遗传修饰

腺病毒载体是最早用于基因治疗研究的病毒载体之一,也是目前肿瘤基因治疗中最为常见的病毒载体之一,其主要通过靶细胞表面的天然柯萨奇腺病毒受体(coxsackie and adenovirus receptor,CAR)感染宿主细胞。由于大多数肿瘤细胞表面该受体表达水平较低,降低了腺病毒载体对靶细胞的感染效率,从而制约了腺病毒载体在肿瘤基因治疗中的应用。因此,如何提高腺病毒载体对靶细胞的感染效率是腺病毒载体应用于肿瘤基因治疗的关键。目前对腺病毒载体衣壳蛋白质(capsid protein)的遗传修饰是提高其对宿主细胞感染效率的主要途径。本文将对这一领域的主要研究进展作一综述,为该方面的研究提供有用的信息。     

Recombinant Interferon-γ Lentivirus Co-Infection Inhibits Adenovirus Replication Ex Vivo

Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P = 0.005–0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P = 0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.     

Adenovirus early region 1A modulation of interferon antiviral activity.

Human adenovirus type 5 (Ad5) is a DNA virus which replicates as efficiently in human A549 cells treated with human interferon-alpha 2 (IFN) as in untreated cells. Vesicular stomatitis virus (VSV), on the other hand, is a negative-strand RNA virus which is very sensitive to the effects of IFN treatment in A549 cells. The IFN-mediated inhibition of VSV replication was not observed in cells coinfected with Ad5. Abrogation of IFN-mediated antiviral activity was maximal when Ad5 infection preceded VSV infection by at least 36 h, but did not require adenovirus DNA synthesis for manifestation. Coinfection experiments with VSV and deletion variants of adenovirus demonstrated that neither virus-associated RNA synthesis nor expression of adenovirus early regions E1B, E2A, E3, or E4 are required for abrogation of IFN-mediated inhibition of VSV replication. However, expression of early region E1A was essential, suggesting that E1A products can modulate, either directly or indirectly, IFN activity in adenovirus-infected cells.     

Repeated administration of adenoviral vectors in lungs of human CD4 transgenic mice treated with a nondepleting CD4 antibody.

The central role of CD4+ T cells in regulation of adenovirus vector-mediated immune responses has been documented previously in murine models. We analyzed the effects of a nondepleting mAb to human CD4 (CD4 mAb; Clenoliximab) on immune functions following intratracheal administration of adenoviral vectors in murine CD4-deficient mice (muCD4KO) expressing a human CD4 transgene (HuCD4 mice). Treatment of HuCD4 mice with Clenoliximab inhibited both cell-mediated and humoral immune responses to adenoviral Ags. Chronic treatment of HuCD4 mice with Clenoliximab permitted successful readministration of adenoviral vectors at least four times. The ability to readminister these vectors is associated with marked suppression of neutralizing Ab responses to viral capsid proteins. Clenoliximab also inhibited CTL and prolonged expression of the transgene. T or B cell responses to adenovirus did not emerge after the effects of a short course of Clenoliximab diminished. These data illustrate the potential utility of a nondepleting CD4 Ab in facilitating gene therapy using adenoviral vectors.     

The adenovirus type 12 early-region 1B 58,000-Mr gene product is required for viral DNA synthesis and for initiation of cell transformation.

An E1B 58K mutant of adenovirus type 12 (Ad12), dl207, was constructed by the deletion of 852 base pairs in the E1B 58K coding region. The mutant could grow efficiently in 293E1 cells but not in HeLa, KB, or human embryo kidney (HEK) cells. Viral DNA replication of dl207 was not detected in HeLa and KB cells and was seldom detected in HEK cells. Analysis of viral DNA synthesis in vitro showed that the Ad12-DNA-protein complex replicated by using the nuclear extract from Ad12 wild-type (WT)-infected HeLa cells but not by using the nuclear extract from dl207-infected cells. In dl207-infected HeLa and KB cells, early mRNAs were detected, but late mRNAs were not detected. The mutant induced fewer transformed foci than the WT in rat 3Y1 cells. Cells transformed by dl207 could grow efficiently in fluid medium, form colonies in soft agar culture, and induce tumors in rats transplanted with the transformed cells at the same efficiency as WT-transformed cells. Tumors were induced in hamsters injected with WT virions but were not induced in hamsters injected with dl207 virions. The results indicate that the E1B 58K protein is required both for viral DNA replication in productive infection and for initiation of cell transformation, but not for maintenance of the transformed phenotype.