Adenovirus Gene Products Research Articles

Inactivation of p53 but not p73 by adenovirus type 5 E1B 55-kilodalton and E4 34-kilodalton oncoproteins.

The adenovirus E1B 55-kDa and E4 34-kDa oncoproteins bind and inactivate the p53 tumor suppressor gene product, resulting in cell transformation. A recently discovered cellular protein, p73, shows extensive similarities to p53 in structure and function. Here we show that the simultaneous transient expression of E1B 55-kDa and E4 34-kDa proteins is sufficient to drastically shorten the intracellular half-life of p53, leading to strongly reduced steady-state p53 levels. Concomitantly, the E1B 55-kDa and E4 34-kDa proteins act synergistically to inactivate the transcriptional activity of p53. Mutational analysis suggests that physical interactions between the E1B 55-kDa protein and p53 and between the E1B 55-kDa and E4 34-kDa proteins are both required for p53 degradation. In contrast, the ability of p53 to interact with the cellular mdm2 oncoprotein or with its cognate DNA element appears to be dispensable for its destabilization by adenovirus gene products. The adenovirus E1B 55-kDa protein did not detectably interact with p73 and failed to inhibit p73-mediated transcription; also, the E1B 55-kDa and E4 34-kDa proteins did not promote p73 degradation. When five amino acids near the amino termini were exchanged at corresponding positions between p53 and p73, this rendered p53 resistant and p73 susceptible to complex formation and inactivation by the E1B 55-kDa protein. Our results suggest that while p53 inactivation is a central step in virus-induced tumor development, efficient transformation can occur without targeting p73.

Inhibition of NF-kappaB activation in combination with bcl-2 expression allows for persistence of first-generation adenovirus vectors in the mouse liver.

NF-kappaB is a key regulator of the innate antiviral immune response, due in part to its transcriptional activation of cytokines and adhesion molecules, which, in turn, function in chemotaxis and activation of inflammatory cells. We reported earlier that viral gene expression in hepatocytes transduced with first-generation (E1-deleted) adenoviruses induced NF-kappaB activation, elevation of serum cytokines, and hepatocellular apoptosis during the first days postinfusion. These events did not occur in mice infused with an adenovirus vector deleted for E1, E2, E3, and late gene expression. In the present study, we used an adenovirus expressing an IkappaBalpha supersuppressor (Ad.IkappaBM) and bcl-2 transgenic mice to unravel the role of virus-induced NF-kappaB activation and apoptosis in the clearance of recombinant adenovirus vectors from the liver. The combined action of IkappaBM and Bcl-2 allowed for vector persistence in livers of C57BL/6 x C3H mice. In the absence of Bcl-2, IkappaBM expression in mouse livers significantly reduced NF-kappaB activation, cytokine expression, leukocyte infiltration, and the humoral immune response against the transgene product; however, this was not sufficient to prevent the decline of vector DNA in transduced cells. Infusion of Ad.IkappaBM caused extended apoptosis predominantly in periportal liver regions, indicating that NF-kappaB activation may protect transduced hepatocytes from apoptosis induced by adenovirus gene products. To confer vector persistence, bcl-2 transgene expression was required to block virus-induced apoptosis if NF-kappaB protection was inactivated by IkappaBM. Expression of gene products involved in early stages of apoptotic pathways was up-regulated in response to virus infusion in bcl-2 transgenic mice, which may represent a compensatory effect. Our study supports the idea that the suppression of innate defense mechanisms improves vector persistence.

Human Adenoviruses: Evading Detection by Cytotoxic T Lymphocytes

Human adenoviruses cause lytic and persistent respiratory, enteric, and other infections. Adenoviruses can transform primary rodent cells and certain serotypes of human adenoviruses (such as adenovirus type 12 from subgenus A) induce tumors in newborn rodents. In both cases, adenovirus gene products mediate evasion of the cellular immune system by infected and tumor cells. In viral infection, a product of the early regionE3gene, a glycoprotein termed E3-19K, binds to and retains newly synthesised major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum thus rendering infected cells resistant to lysis by cytotoxic T lymphocytes. In addition, other products of theE3region confer on infected cells resistance to tumor necrosis factor-α-mediated lysis. Not all human adenovirus serotypes encode an E3-19K protein: viruses from subgenus A (such as adenovirus 12) and F (the enteric adenoviruses 40 and 41) do not encode an E3-19K molecule. In the case of Ad12, products of the viral E1A gene repress MHC class I heavy chain gene transcription. This leads to loss of MHC class I molecules from the surface of adenovirus 12-transformed cells and contributes to their evasion from cytotoxic T lymphocytes. Considerable progress has been made toward identifying the targets for E1A-mediated repression of the class I heavy chain promoter. In addition, adenovirus 12 mediates transcriptional repression of other genes in the MHC complex involved in antigen presentation, namely the transporter associated with antigen presentation (TAP) genes and MHC-encoded proteasome components, the low molecular weight proteins termed LMPs. Overall, adenoviruses display a variety of differing mechanisms for posttranslational (E3-19K) and transcriptional (E1A) repression of MHC class I expression that operate in all human viral serotypes studied, suggesting that evasion of cytotoxic T cell lysis forms an important part of the infection and oncogenic transformation strategies adopted by human adenoviruses.

Down-regulation of CD95 (Fas/Apo-1) in the epithelia of adenovirus-infected appendices.

Adenoviral inclusions are commonly seen in appendices from infants with intussusception. They are associated with focal epithelial budding and less frequently with epithelial shedding. These morphological changes could depend on the opposing effects of adenoviral gene products on CD95-mediated apoptosis. Appendices from intussusceptions with viral inclusions (n = 4) and normal appendices (n = 10) were studied by immunochemistry with anti-adenovirus, anti-CD95 and anti-HLA-DR antibodies. Apoptosis was studied by the TUNEL method. The mucosa of normal appendices contained no adenoviral protein. CD95 was present in all epithelial cells except Paneth cells. HLA-DR was absent in epithelial cells and apoptosis was seen only in germinal centres and in a few surface epithelial cells. The epithelium of appendices from intussusceptions contained nuclear inclusions labelled with anti-adenovirus antibody, always found in the epithelial buds. The epithelial CD95 pattern was drastically altered in adenovirus-infected appendices. CD95 was absent from the budding foci. In these foci, HLA-DR was overexpressed. There was also increased epithelial apoptosis in areas remote from those lacking CD95 antigen. The appearance of epithelial budding or shedding in appendices from intussusception could be due to focal in situ differences in the expression of adenoviral genes.

High level of transgene expression in cell cultures and in the mouse by replication-incompetent adenoviruses harboring modified VAI genes.

Replication-incompetent adenoviruses are currently used in gene therapy trials. Most of the work designed to increase the expression from these vectors concerns the modification of cis sequences of the foreign transcription unit, so as to improve the transcription level or the stability of the mRNA. In this report, we show that an alternative strategy based on the coexpression of modified VAI genes can efficiently increase gene expression both in cell cultures and in animals. The VAI RNA is synthesized mainly during the late phase of the adenovirus cycle and increases the translation of late adenovirus gene products by counteracting the effect of an interferon-induced kinase, the PKR. We have constructed several modified VAI genes in which the central domain was deleted or substituted by exogenous sequences. These modified VAI genes, or the native VAI gene, were cloned into the left part of adenovirus type 5 genomes harboring their own endogenous VAI gene. One of the resulting viruses (Ad-VAr) increased 12.5- to 502-fold the expression level of reporter genes, either expressed as a constitutive cell line from an extrachromosomal DNA or introduced into cells by coinfection with another adenovirus vector. This effect was independent of the promoter, the coding sequence, and the 5' untranslated mRNA sequence and was obvious in the two non-E1-complementing cell lines tested (HeLa and Vero). Coinfection of Ad-VAr with adenoviruses expressing the luciferase gene from the major late promoter or Rous sarcoma virus (RSV) promoter by the intravenous route in mice increased by more than 33 (MLP)- to 128 (RSV)- and 4,700 (MLP)- to 30,000 (RSV)-fold the expression level of the reporter gene in the lungs and liver, respectively. The intramuscular coinoculation of Ad-VAr and Ad-MLP-gD (a recombinant adenovirus vaccine expressing gD from the pseudorabies herpes virus) led to a 10-fold decrease in the protective dose of Ad-gD in mice. Ad-VAfull, a similar adenovirus in which the native VAI gene was cloned at the left part of the genome, showed no evidence of efficacy in cell culture and in mice. These results suggest that the use of modified VAI genes expressed at the early phase of the cycle can be helpful in the design of potent adenovirus vectors.

Recombinant adeno-associated virus for muscle directed gene therapy.

Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli beta-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

A late adenovirus factor induces eIF-4E dephosphorylation and inhibition of cell protein synthesis

Adenovirus prevents host cell protein synthesis during its late phase of replication in large part by causing the underphosphorylation of translation initiation factor eIF-4E, a component of initiation factor eIF-4F (cap-binding protein complex). Late adenovirus mRNAs are preferentially translated because they possess a reduced requirement for eIF-4F. This study continues the characterization of the mechanism by which adenovirus inhibits cellular protein synthesis. First it is shown that adenovirus blocks the addition of phosphate to eIF-4E rather than enhancing its removal, establishing that the virus impairs a signalling pathway or protein kinase activity involved in eIF-4E phosphorylation. It is then shown that shutoff of cell protein synthesis and translation of late viral mRNAs are uncoupled, in that shutoff actually occurs a short time (1 to 3 h) after late adenovirus mRNAs are already undergoing translation. Finally, by using a variety of genetic mutants stalled at different stages in the viral life cycle, it was found that dephosphorylation of eIF-4E and inhibition of cell translation are not caused by early adenovirus gene products acting at late times or by events related to viral DNA replication. Instead, it is shown that inhibition of eIF-4E phosphorylation and cell translation are mediated upon activation of the viral major late transcription unit. These and other results presented indicate that the adenovirus signal which induces eIF-4E dephosphorylation and shutoff of cell protein synthesis is linked either to an activity of one or more late viral polypeptides, to double-stranded RNA produced by opposition of the early and late viral transcription units, or to both.

Adenovirus-mediated correction of the genetic defect in hepatocytes from patients with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an inherited deficiency of LDL receptors that has been an important model for liver-directed gene therapy. We are developing approaches for treating FH that are based on direct delivery of recombinant LDL receptor genes to liver in vivo. As a first step towards this goal, replication-defective recombinant adenoviruses were constructed which contained either the lacZ gene or the human LDL receptor cDNA expressed from a beta-actin promoter. Primary cultures of hepatocytes were established from two patients with homozygous FH and one nonFH patient, and subsequently exposed to recombinant adenoviruses at MOIs ranging from 0.1 to 5. Essentially all of the cells expressed high levels of the transgene without demonstrable expression of an early or late adenoviral gene product; the level of recombinant-derived LDL receptor protein in transduced FH hepatocytes exceeded the endogenous levels by at least 20-fold. These studies support the utility of recombinant adenoviruses for efficient transduction of recombinant LDL receptor genes into human FH hepatocytes without expression of viral proteins.

The EBNA2-Related Resistance Towards Alpha Interferon (IFN-α) in Burkitt’s Lymphoma Cells Effects Induction of IFN-Induced Genes but Not the Activation of Transcription Factor ISGF-3

Transfection of a plasmid encoding the Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) gene confers resistance to the antiproliferative effect of alpha interferon (IFN-alpha) in EBV-negative U968 cells (P. Aman and A. von Gabain, EMBO J. 9:147-152, 1990). We studied the expression of IFN-stimulated genes (ISGs) in two pairs of Burkitt's lymphoma cell lines, differing in the expression of the putative immortalizing gene of EBV, EBNA2. In EBNA2-expressing cells, the induction of four ISGs by IFN-alpha was strongly reduced or, in some cases, abolished. Chloramphenicol acetyltransferase reporter gene constructs containing different IFN-stimulated response elements were transfected into EBNA2-negative and EBNA2-positive cells. Induction of chloramphenicol acetyltransferase activity by IFN was impaired in EBNA2-positive cells. Also, a reporter gene construct driven by an IFN-gamma-sensitive promoter element was affected. However, as revealed by gel shift assays, EBNA2-positive and EBNA2-negative cells exhibited a nearly identical pattern of IFN-stimulated response element-binding proteins. Most important, activation of the factor ISGF-3, which previously has been shown to be required and sufficient for transcriptional activation of IFN-induced genes, was not inhibited in IFN-resistant cells expressing EBNA2. The mechanism of the EBNA2-related IFN resistance seems to be distinct both from the resistance mediated by hepatitis virus and adenovirus gene products and from the IFN resistance in Daudi cell variants. In these three cases, the transcriptional block of IFN-induced genes is due to inhibition of ISGF-3 activation and binding. Our data suggest that the EBNA2-related IFN resistance in Burkitt's lymphoma cells acts downstream of the activation of ISGF-3.

The EBNA2-related resistance towards alpha interferon (IFN-alpha) in Burkitt's lymphoma cells effects induction of IFN-induced genes but not the activation of transcription factor ISGF-3.

Transfection of a plasmid encoding the Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) gene confers resistance to the antiproliferative effect of alpha interferon (IFN-alpha) in EBV-negative U968 cells (P. Aman and A. von Gabain, EMBO J. 9:147-152, 1990). We studied the expression of IFN-stimulated genes (ISGs) in two pairs of Burkitt's lymphoma cell lines, differing in the expression of the putative immortalizing gene of EBV, EBNA2. In EBNA2-expressing cells, the induction of four ISGs by IFN-alpha was strongly reduced or, in some cases, abolished. Chloramphenicol acetyltransferase reporter gene constructs containing different IFN-stimulated response elements were transfected into EBNA2-negative and EBNA2-positive cells. Induction of chloramphenicol acetyltransferase activity by IFN was impaired in EBNA2-positive cells. Also, a reporter gene construct driven by an IFN-gamma-sensitive promoter element was affected. However, as revealed by gel shift assays, EBNA2-positive and EBNA2-negative cells exhibited a nearly identical pattern of IFN-stimulated response element-binding proteins. Most important, activation of the factor ISGF-3, which previously has been shown to be required and sufficient for transcriptional activation of IFN-induced genes, was not inhibited in IFN-resistant cells expressing EBNA2. The mechanism of the EBNA2-related IFN resistance seems to be distinct both from the resistance mediated by hepatitis virus and adenovirus gene products and from the IFN resistance in Daudi cell variants. In these three cases, the transcriptional block of IFN-induced genes is due to inhibition of ISGF-3 activation and binding. Our data suggest that the EBNA2-related IFN resistance in Burkitt's lymphoma cells acts downstream of the activation of ISGF-3.

Translation by the adenovirus tripartite leader: elements which determine independence from cap-binding protein complex

The adenovirus tripartite leader is a 200-nucleotide-long 5' noncoding region which facilitates translation of viral mRNAs at late times after infection. The tripartite leader also confers the ability to initiate translation independent of the requirement for cap-binding protein complex or eIF-4F without any requirement for adenovirus gene products. To elucidate the manner by which the tripartite leader functions, the primary determinants of leader activity were investigated in vivo by testing a series of mutations expressed from transfected plasmids. The results of these experiments indicate that the tripartite leader does not promote internal ribosome binding, at least in a manner recently described for picornavirus mRNAs. In addition, despite an unusual arrangement of sequences complementary to the 3' end of 18S rRNA in the tripartite leader, we could find no evidence for involvement in its translation activity. Instead, our results are consistent with a model in which much of the first leader is maintained in an unstructured conformation which determines the ability of the tripartite leader to facilitate translation and bypass a normal requirement for eIF-4F activity. Several possible translation models are discussed, as well as the implications for translation of late viral mRNAs.

Cis-acting elements and a trans-acting factor affecting alternative splicing of adenovirus L1 transcripts.

Expression of the L1 region of adenovirus is temporally regulated by alternative splicing to yield two major RNAs encoding the 52- to 55-kilodalton (52-55K) and IIIa polypeptides. The distal acceptor site (IIIa) is utilized only during the late phase of infection, whereas the proximal site (52-55K) is used at both early and late times. Several parameters that might affect this alternative splicing were tested by using expression vectors carrying the L1 region or mutated versions of it. In the absence of a virus-encoded or -induced factor(s), only the 52-55K acceptor was used. Decreasing the distance between the donor and the IIIa acceptor had no effect. Removal of the 52-55K acceptor induced IIIa splicing slightly, implying competition between the two acceptors. Fusion of the IIIa exon to the 52-55K intron greatly enhanced splicing of the IIIa junction, suggesting that the IIIa exon does not contain sequences that inhibit splicing. Thus, the lack of splicing to the IIIa acceptor in the absence of a virus-encoded or -induced factor(s) is probably due to the absence of a favorable sequence and/or the presence of a negative element 5' of the IIIa splice junction, or both. The presence of several adenovirus gene products, including VA RNAs, the E2A DNA-binding protein, and the products of E1A and E1B genes, did not facilitate use of the IIIa acceptor. In contrast, the simian virus 40 early proteins, probably large T antigen, induced IIIa splicing. This result, together with those of earlier studies, suggest that T antigen plays a role in modulation of alternative RNA splicing.

Cis-Acting Elements and a trans-Acting Factor Affecting Alternative Splicing of Adenovirus L1 Transcripts

Expression of the L1 region of adenovirus is temporally regulated by alternative splicing to yield two major RNAs encoding the 52- to 55-kilodalton (52-55K) and IIIa polypeptides. The distal acceptor site (IIIa) is utilized only during the late phase of infection, whereas the proximal site (52-55K) is used at both early and late times. Several parameters that might affect this alternative splicing were tested by using expression vectors carrying the L1 region or mutated versions of it. In the absence of a virus-encoded or -induced factor(s), only the 52-55K acceptor was used. Decreasing the distance between the donor and the IIIa acceptor had no effect. Removal of the 52-55K acceptor induced IIIa splicing slightly, implying competition between the two acceptors. Fusion of the IIIa exon to the 52-55K intron greatly enhanced splicing of the IIIa junction, suggesting that the IIIa exon does not contain sequences that inhibit splicing. Thus, the lack of splicing to the IIIa acceptor in the absence of a virus-encoded or -induced factor(s) is probably due to the absence of a favorable sequence and/or the presence of a negative element 5' of the IIIa splice junction, or both. The presence of several adenovirus gene products, including VA RNAs, the E2A DNA-binding protein, and the products of E1A and E1B genes, did not facilitate use of the IIIa acceptor. In contrast, the simian virus 40 early proteins, probably large T antigen, induced IIIa splicing. This result, together with those of earlier studies, suggest that T antigen plays a role in modulation of alternative RNA splicing.

Efficient synthesis of adeno-associated virus structural proteins requires both adenovirus DNA binding protein and VA I RNA

We have shown previously that replication of defective parvoviruses [adeno-associated viruses (AAV)] requires several early adenovirus (Ad) gene products [ J. E. Janik, M. M. Huston, and J. A. Rose (1981) Proc. Natl. Acad. Sci. USA 78, 1925–1929 ]. To examine their possible roles in the transcription and translation of AAV mRNA, 293-31 cells, a human embryonic kidney cell line that constitutively expresses the Ad early region IA and IB gene products, were transfected with a pBR325 plasmid (pLHi) that contains a duplex AAV2 DNA segment (0.03–0.97 map units) which encompasses the promoters and coding sequences necessary for expression of all AAV polypeptides. When cells were transfected with pLH1 alone, both spliced and unspliced AAV-specific cytoplasmic RNAs accumulated. These transcripts were capable of directing synthesis of the three AAV capsid polypeptides in vitro, whereas in vivo synthesis of AAV protein was not detected by immunofluorescence or immunoprecipitation. When cells were cotransfected with pLH1 and intact Ad DNA, the level of cytoplasmic AAV RNA was enhanced and AAV protein was synthesized in vivo. Additional experiments demonstrated that in vivo AAV protein synthesis also could be induced when pLH1 was cotransfected with plasmids that contain the Ad DNA-binding protein (pDBP) and VA I RNA (p2BalM) genes; however, a low level of in vivo AAV capsid protein was occasionally detected in cotransfections with pLHi and a plasmid that contains both VA I and VA II RNA coding sequences (p2SaIC). Cotransfection of pLH1 and pDBP or pLHi and p2SaIC showed complex alterations in the steady-state patterns of AAV cytoplasmic transcripts. In both cases, increased levels of transcripts, particularly the 2.3-kb spliced species, were detected in comparison to levels seen in cells transfected with pLH1 alone. Despite these increases, however, there was little, if any, induction of AAV protein synthesis unless both the DNA-binding protein (DBP) and VA I RNA coding sequences were present in cotransfection with pLH1. We conclude that, in 293-31 cells, the Ad VA I RNA and DBP gene products regulate AAV capsid protein synthesis at least at two levels: (i) by increasing the steady-state levels of structural protein transcripts in the cytoplasm, especially the spliced species, and (ii) by enhancing the translation of these messages.

The adenovirus tripartite leader may eliminate the requirement for cap-binding protein complex during translation initiation.

The adenovirus tripartite leader is a 200-nucleotide 5' noncoding region that is found on all late viral mRNAs. This segment is required for preferential translation of viral mRNAs at late times during infection. Most tripartite leader-containing mRNAs appear to exhibit little if any requirement for intact cap-binding protein complex, a property previously established only for uncapped poliovirus mRNAs and capped mRNAs with minimal secondary structure. The tripartite leader also permits the translation of mRNAs in poliovirus-infected cells in the apparent absence of active cap-binding protein complex and does not require any adenovirus gene products for this activity. The preferential translation of viral late mRNAs may involve this unusual property.

Requirement for either early region 1a or early region 1b adenovirus gene products in the helper effect for adeno-associated virus.

Several adenovirus early genes act together to promote growth of the helper-dependent adeno-associated virus (AAV). Data from several laboratories have implicated adenovirus early regions 1a, 1b, 2a, and 4 in the helper effect, as well as the small RNA polymerase III transcript, virus-associated RNA I. Although a subset of these must participate directly in the AAV life cycle, some may play an indirect role by influencing expression of the others. This paper is concerned particularly with the roles of early regions 1a and 1b in the helper effect. We introduced DNA fragments representing the various early regions into AAV-infected or uninfected Vero cells, by the manual microinjection procedure. After labeling the cells with [35S]methionine, we visualized immunoprecipitates of AAV or adenovirus proteins on sodium dodecyl sulfate-polyacrylamide gels. When over 200 copies of each DNA fragment per cell were injected, early regions 2a and 4 were themselves sufficient to provide the helper effect. At 100 copies per cell, however, a third gene became essential, and this could be either early region 1a or 1b. The role of early region 1a is easily explained by its known ability to stimulate transcription of the other early genes. The function of early region 1b is less clear, but it does not simply mimic the action of early region 1a. Instead, there appear to be at least two distinct regulatory pathways which can lead to expression of AAV. To investigate the sequence of regulatory interactions, we microinjected purified adenovirus mRNAs, or combinations of mRNA and DNA, into AAV-infected cells. Our results suggest that adenovirus early products enhance viral gene expression by several mechanisms which can operate independently, but whose effects may be cumulative.

Genetic expression of human adenoviruses in simian cells. Evidence for interserotypic inhibition of viral DNA synthesis.

Most simian cells are permissive for SV40 and adenovirus-SV40 hybrids but nonpermissive for human adenoviruses, and the defect has been shown to take place at the level of processing of late viral mRNAs (Klessig and Grodzicker, 1979). Viral DNA synthesis and virus progeny production were studied in simian cells infected with different adenovirus serotypes. Adenoviruses belonging to oncogenic subgroups A and B (Ad31 and Ad3) failed to replicate their DNA in CV1 cells, whereas DNA replication occurred for all the other serotypes. Co-infection of CV1 cells with SV40 and Ad3 (or Ad31) resulted in the inhibition of SV40 DNA synthesis, as well as cellular DNA synthesis. The inhibition was not related to adenovirus DNA replication, since SV40 did not complement the Ad3/Ad31 replication defective function. Similar results were obtained in coinfected BSC and MK2 simian cell lines. Inhibition of Ad2ND1 DNA synthesis and gene expression also occurred in co-infection of simian cells with nondefective Ad2ND1 hybrid and defective Ad3/Ad31. In permissive human cell lines (HeLa or KB) co-infected with Ad2 and Ad3 (or Ad31), a dominant, inhibitory effect of Ad3 (or Ad31) over Ad2 was also observed. The inhibition appeared to function stoichiometrically and not catalytically, and to involve early adenovirus gene products. In both simian and human cells a hierarchy of dominance appeared between serotypes belonging to different subgroups. The degree of inhibitory effect occurred in the following decreasing order: Ad3 and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad31 (A), Ad2 and Ad5 (C).

Classification of Human Adenoviruses by SDS-Polyacrylamide Gel Electrophoresis of Structural Polypeptides

The virion polypeptide compositions of 15 human adenovirus types, representing all members of subgroups B and C and selected members of subgroups A and D, have been analyzed by SDS-PAGE. The capsid polypeptides II, III, IIIa and IV displayed a pattern of apparent molecular weights which was characteristic for each adenovirus serotype analyzed. Adenovirus serotypes belonging to the same subgroup were also demonstrated to share an apparent molecular weight pattern of the internal structural polypeptides in the following manner: Subgroup A:V-resolved into two bands of 46--48.5 K and 51--51.5 K; VI-25.5 K; and VII-18 K. Subgroup B:V-53.5--54.5 K; VI-24 K; and VII-18 K. Subgroup C:V-48.5 K; VI-24 K; and VII-18.5 K. Subgroup D:V-50.5 K; VI-23.5 K; and VII-18 K. Adenovirus type 4: V-48 K; VI-24.5 K; and VII-18 K. The structural polypeptides represent a major portion of the adenovirus gene products. It is therefore proposed that the apparent molecular weight differences of viral polypeptides be used (i) to confirm the identification of an adenovirus type, and (ii) for subgroup classification of adenoviruses and, consequently, that adenovirus type 4 should be classified as a member of a separate subgroup designated E.