Intranasal inoculation of type 5 adenovirus (AdS) produced pneumonia in mice even though the virus did not replicate. To induce the pneumonia, however, a large viral infectious dose was required-i.e., 1010 plaque-forming units.Four strains of inbred mouse were studied (C57BL/6N, C57BL/1OScN, CBA/N, and C3H/N): all showed similar inflammatory responses, although the greatest infiltration occurred in the C57BL/6N mice. The pathological response to AdS infection resembled that previously described in cotton rats: it consisted of overlapping early and late phases, and the infiltration contained primarily lymphocytes and monocytes/macrophages with a scattering of polymorphonuclear leukocytes. The prominent early phase and the presence of polymorphonuclear leukocytes suggested that induction of cytokines may play an important role in the pathogenesis ofthis pneumonia. Assays showed the appearance of tumor necrosis factor a (TNF-a), interleukin 1 (IL-1), and IL-6 in the infected mouse lungs concomitant with the developing early-phase infiltration. Only IL-6 was found in the peripheral blood. IL-6 reached maximum titers 6-24 hr after infection, whereas maximum levels of TNF-a and IL-1 were attained 2-3 days after infection. Specific RNAs for each of these cytokines were demonstrated in the infected lungs. To test the hypothesis that a cytotoxic T-cell response was responsible for the second phase, which primarily consisted of a perivascular and peribronchial infiltration of lymphocytes, AdS was used to infect C57BL/lOScN Nu/Nu and parent mice. The nude mice showed a normal early-phase response, but essentially no peribronchial and only minimal perivascular infiltrations occurred.Intranasal inoculation of type 5 adenovirus (Ad5) into the cotton rat Sigmodon hispidus initiates the development of a pneumonia that closely resembles that produced in humans (1, 2). This model was then used to investigate the viral genes required to produce the pathogenesis of the disease, which for these studies is termed "molecular pathogenesis." The results obtained, using mutants that contained defects in a 19-kDa glycoprotein, implied that the inflammatory response resulted from both the. production of cytokines and the infiltration of cytotoxic T cells (3). Thus, it was hypothesized that the first phase of the pneumonia, which consists of a lymphocyte and monocyte/macrophage intraalveolar and interstitial infiltration as well as a scattering of polymorphonuclear leukocytes (PMNs), resulted from a local elaboration of cytokines. It was further hypothesized that the second phase, which is composed of a lymphocytic perivascular and bronchiolar infiltration, was due to a virus-specific cytotoxic T-cell response.It was not readily possible to test these hypotheses in cotton rats since reagents are not available either to assay their cytokines or to identify the species of lymphocytes present in the pneumonic infiltration. It appeared possible to overcome these experimental barriers, however, when we demonstrated that H5ts125 (4), a co...

The cotton rat Sigmodon hispidus has provided an animal model of adenovirus pneumonia that permits investigation of the viral gene products required to produce the disease and the molecular mechanisms effecting the damage. This study was One utility of the modern techniques of virology and molecular biology should be to reveal the basic molecular mechanisms by which viruses such as adenoviruses produce disease. The discovery that intranasal inoculation of type 5 adenovirus (AdS) into cotton rats results in development of a pneumonia that pathologically simulates that produced in humans (1) provided the opportunity to determine the viral gene functions required to produce the pneumonia and the molecular mechanisms by which the virus induced the disease. Extensive studies have been carried out on the replication of adenovirus in the lungs of the Sigmodon hispidus species of cotton rats and on the relationship of viral multiplication to the development of pneumonia (2, 3). Productive viral replication was only detected in the epithelial cells of the bronchi and bronchioles of the lung and the nasal mucosa. Onset of viral multiplication, which reached maximum titers 2-4 days after infection (depending upon the size of the inoculum), was soon followed by progressively increasing peribronchial, perivascular, and alveolar septal infiltration of lymphocytes and monocyte/macrophages and finally by lymphocytic infiltration of the basal bronchiolar wall into the epithelium; scattered polymorphonuclear leukocytes (PMN) were also present (2, 3). The maximum pathology was attained 5-7 days after infection, which was also dependent upon the viral inoculum. The use of conditionally lethal, temperature-sensitive mutants [e.g., H5ts125 (4)] unable to replicate their DNA at the cotton rat's normal body temperature, about 39.2°C, led to unexpected results: the cellular inflammatory response was the same as in wild-type (Wt) virus-infected lungs, although quantitatively not as extensive. These data indicate that only early gene products appear necessary to induce the inflammatory response to viral infection. It was further demonstrated that the E1B 58-kDa protein, which is required to shut off host protein synthesis during adenovirus productive infection (5, 6), is also essential for producing maximum viral pneumonia (3).Early region 3 (E3) has been termed a "nonessential" region since naturally occurring mutants or hybrid viruses in which almost the entire region is deleted still replicate like Wt virus in cultured cells (7). It seemed unlikely that almost 10% of the genome would have survived in evolution if its encoded genes did not play a significant role in the virus's life cycle. Therefore, extensive studies were done to determine whether the E3 region might play a critical role in pathogenesis. It is the objective of this communication to report the results of that investigation. Data will be presented to show that gene products of the E3 region do in fact play a strategic role in viral pathogenesis. However, their ...

Late in adenovirus infection, large amounts of viral mRNA accumulate while cell mRNA transport and translation decrease. Viruses deleted in the ElB region of type 5 adenovirus do not produce the same outcome: (i) viral mRNA synthesis by the mutants is normal, delivery to the cytoplasm is 50 to 75% of normal, but steady-state levels of viral mRNA are decreased 10-fold; (ii) cell mRNA synthesis and transport continue normally in the mutant virus-infected cell; and (iii) translation of preexisting cell mRNA which is disrupted in wild-type infection remains normal in mutant-virus-infected cells. Thus ElB proteins are required for accumulation of virus mRNA and for induction of the failure of host cell mRNA transport and translation. If a single function is involved, by inference the transport and some aspect of translation of mRNAs could be linked.Late in the course of adenovirus infection, host protein synthesis is replaced almost entirely by virus protein synthesis. This phenomenon has received considerable study (2-5, 15, 27). It is now established that preexisting host mRNA is not destroyed, but is no longer translated (2, 15). Furthermore, it is now known that VA RNA is required to maintain late viral translation (27) and apparently does so by preventing the excess phosphorylation of the protein initiation factor E1F-2 (20). How this restitution of active EIF-2 specifically leads to viral mRNA translation is not understood. Without VA late in infection, however, neither viral nor host mRNA is translated (2, 27).In addition to the translation changes that occur late in infection, new cell mRNA fails to accumulate in the cytoplasm despite continued nucleus synthesis (2, 5). Whether this interruption is related to the effect on translation of cell mRNA is unknown, but both events do occur.In studying two type 5 adenovirus mutants containing deletions in the E1B region (H5d1118 and H5dl163; referred to as d1118 and d1163), it was earlier noted that DNA replication occurred normally, and, based on [3H]uridine incorporation, late viral RNA synthesis began normally, but late viral protein synthesis was decreased (3). In addition, total host protein synthesis did not appear to be deranged.We have further characterized host and viral mRNA metabolism in cells infected with these mutant viruses compared with that in the wild-type virus (sub3O9). We find further quantitative evidence that transcription of late viral sequences is normal and that delivery of late viral mRNA to the cytoplasm is near normal, but that the total late viral mRNA is decreased 10-fold. This viral phenotype is associated with a dramatic reversal of the effects of wild-type infection on host cell mRNA functions. Not only does host cell mRNA synthesis continue, but entrance into the cytoplasm is normal and host cell translation is quantitatively maintained. Thus it appears that the two major effects of late viral infection, high accumulation and near exclusive trans- H5sub309, a type 5 adenovirus with wild-type properties (13), was provided by T. Shen...

To investigate whether DNA viruses can augment gene expression of the human immunodeficiency virus (HIV), cotransfection experiments were carried out in which a recombinant plasmid containing the HIV long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene was transfected into cultured cells along with plasmids containing DNA from various distinct classes of DNA viruses. Molecular clones containing JC virus, BK virus, lymphotropic papovavirus, bovine papilloma virus, type 1 herpes simplex virus (HSV-1), and varicella-zoster virus sequences increased CAT expression directed by the HIV LTR. Trans-activation of the HIV LTR varied in different cell lines, but in each case the HIV tat gene product elicited the greatest stimulation. Primer-extension assays specific for HIV LTR mRNA revealed increased levels of steady-state RNA following transfection with HIV tat as well as with several of the DNA viruses. Virus-specific RNA expression paralleled the stimulation of CAT activity. More-than-additive effects were observed at both the RNA and protein levels when tat plus type 1 herpes simplex virus DNAs or tat plus JC virus DNAs were transfected into cells with the HIV LTR-CAT plasmid. These data suggest that coinfection of cells by HIV and some DNA viruses can stimulate the expression of HIV.

Type 5 adenovirus mutants that differentially express ElA 13S, 12S, or 9S mRNAs were constructed to study the role of their gene products in transformation. H5dl520 expresses the 243-amino-acid (AA) protein encoded in the 12S mRNA but not the 13S mRNA-encoded 289-AA protein. This mutant transformed both cloned rat embryo fibroblast (CREF) cells and baby rat kidney (BRK) cells at a frequency 40-100 times greater than did wild-type viruses. In addition, all of the foci produced were fibroblastic and grew very slowly at 320C. In contrast, H5dl21, which was mutated so that only the 54-AA protein encoded by the 9S mRNA was synthesized, did not transform either cell type. DNA transfection studies with plasmids from which these mutants were constructed demonstrated that the differences in transformation frequencies were not as marked. The plasmid pDl/D2, which directs the synthesis of the 54-AA protein only, was found to transform baby rat kidney cells at low frequency, provided the gene was linked to a fragment from the simian virus 40 genome containing the transcriptional enhancer element.The early region genes ElA and E1B, located at the lefthand end of the genome, have been shown to be essential for complete morphological transformation of rodent cells by human adenoviruses (Ad) (1-4). However, transfections of baby rat kidney (BRK) cells (5) or an established cloned rat embryo fibroblast (CREF) cell line (6) with various fragments of the Ad type 5 (AdS) or type 12 (Adl2) genome containing only the ElA sequences produce partial or incomplete morphological transformation, whereas DNA fragments containing only the E1B genes do not transform (7).The exact roles of the ElA genes in transformation are not well understood, but they do provide an establishment function that is concerned with the immortalization of primary cells (2, 8). The ElA sequences encode three different but related products. During early times of productive infection, the ElA primary transcript is differentially spliced to give 12S and 13S messages that differ in the extent of internal sequences removed by RNA splicing (9-12). The translational reading frames of both messages are identical, and, thus, the proteins encoded by each [a 289-amino-acid (AA) protein by the 13S mRNA and the 243-AA protein by the 12S mRNA] differ only by an additional 46 AA encoded by the sequences unique to the 13S mRNA. At late times of infection, a third message, 9S in size, is synthesized. The predicted polypeptide encoded by this message is 54 AA in size and has the same amino-terminal sequence before the splice as in the protein products of the 12S and 13S mRNAs (13). A wellcharacterized function of the ElA region in the productive infection of cells is to produce a product that hastens the transcription of the other early viral genes (4, [14][15][16] (3,5,18,20); some are transformation defective, whereas, others are cold sensitive for the initiation and maintenance of the transformed phenotype. From these results it was deduced that the 289-AA protein was di...

Two mutants containing large deletions in the E4 region of the adenovirus genome H5dl366 (91.9-98.3 by replication-induced recombination (11). The last 45 bp of the genome contain the necessary sequence information for replication, and any deletion of the terminal cytosine abolishes DNA synthesis (12, 13).Other viral gene products may be involved in viral DNA synthesis, and other mechanisms not yet elucidated may be required for completion of viral DNA replication. Some phenomena not accounted for in the current model include the following: circular, supercoiled, and multimeric viral DNA molecules, which were identified in electron microscopic studies (14); the pL 5'-to-3' exonuclease required in the in vitro model (9) produces defective strands that need additional processing for maturation; a large proportion of viral particles produced during infection carry partially replicated single-stranded DNA (15); and concatemers of input DNA are formed in the in vivo plasmid model of viral DNA synthesis (10)(11)(12)(13). Topoisomerase, which is required in the in vitro model, should not be needed to relieve tortional stress in the linear molecules predicted by the current model. In addition, more than the three viral genes defined by the current model are needed for viral DNA replication, since mutations in the E4 region have a significant effect on viral DNA synthesis (16)(17)(18).To gain a better understanding of the functions of the E4 genes in adenovirus DNA synthesis, the DNA replication phenotypes of E4 mutants were investigated. These experiments revealed the formation of viral DNA concatemers, which suggests that multimeric genomes are a consequence of disruption of a normal viral gene function and that the mechanism of adenovirus DNA synthesis may differ from the presently accepted model. MATERIALS AND METHODS

The ability to analyze the molecular events involved in adenovirus transformation of mammalian cells critically has been impeded by the lack of a highly transformable target cell. The majority of investigations have used heterogeneous primary or secondary rat embryo cells that are semipermissive for group C adenovirus-i.e., serotypes 1, 2, or 5 (Adl, Ad2, and AdS, respectively) (1-6). With primary rat embryo cells, a certain proportion ofthe infected population is permissive for viral replication and produces infectious virus, whereas a small fraction of infected cells integrate viral DNA and become morphologically transformed (usually <10-5 transformants per infected cell) (3, 6). The frequency of Ad5 transformation of rat embryo cells can be enhanced 7-to 8-fold by treating the cells with chemical carcinogens prior to viral infection and by growing infected cells in the presence of the tumor promoting agent phorbol 12-myristate 13-acetate (6-8). However, even with these modifications, the frequency ofadenovirus transformation is still too low to allow accurate appraisal ofthe crucial virus-cell interactions required for stable transformation. In addition, because the majority of transformation assays have used genetically diverse animals as sources of embryo cultures, the quantitation and reproducibility oftransformation in different assays and laboratories have been major problems.By systematically screening >100 cloned populations derived from five established rat embryo cell lines, a specific clone of Fischer rat embryo cells (CREF cells) was isolated. The cells derived from this clone are transformed by Ad5 at least 150-fold more frequently than primary or secondary rat embryo cells. This report describes the CREF transformation system and the phenotypic properties of CREF clones transformed with wildtype Ad5 and the temperature-sensitive mutant H5ts125. Since previous studies have shown that wild-types Ad5 and Ad2 transformed rat embryo cells usually contain only part of the viral genome, including approximately the left hand 15% (9-11), whereas H5tsl25-transformed rat embryo cells transformed and cultured at 36 or 39.5°C usually contain the complete viral genome (10, 11), the patterns of viral DNA integration in wildtype-and H5tsl25-transformed CREF clones have also been determined. MATERIALS AND METHODSCell Cultures. A series of five early passage and established rat embryo cell cultures isolated from 10-to 14-day gestation Sprague-Dawley and Fischer rats were cloned and analyzed for stability at confluence, anchorage dependence, and transformability by adenovirus (6,12). The cell cultures studied included NRK, a normal rat kidney cell line, provided by David K. Howard; 3Y1, an established Fischer rat embryo cell line supplied by Nada Ledinko; SDRE-1 and SDRE-2, two independently isolated early passage Sprague-Dawley rat embryo fibroblast cell lines established in this laboratory as described (13); and an early passage F2408 Fischer rat embryo fibroblast cell line provided by Claudio Basilico. The CREF ...

Eight temperature-sensitive (ts) mutants that replicate normally at 32 C but poorly, if at all, at 39.5 C have been isolated from mutagenized stocks of a wildtype strain of type 5 adenovirus. Three mutagens were employed: nitrous acid, hydroxylamine, and nitrosoguanidine. Ts mutants were isolated from mutagenized viral stocks with frequencies between 0.01 and 0.1. All eight mutants had reversion frequencies of 10-5 or less. Complementation experiments in doubly infected cultures at the nonpermissive temperature separated the mutants into three nonoverlapping complementation groups. Complementation yields ranged from a 2.3to a 3,000fold increase over the sums of the yields from the two singly infected controls. Genetic recombination was also demonstrated; approximate recombination frequencies ranged from 0.1 to 15%. Preliminary biochemical and immunological characterization of the mutants indicated that: (i) the single mutant in complementation group I did not replicate its deoxyribonucleic acid (DNA) or synthesize late proteins at the nonpermissive temperature but did inhibit host DNA synthesis to 25 % of an uninfected control; (ii) the four group II mutants replicated viral DNA, shut off host DNA synthesis, synthesized penton base and fiber, but did not synthesize immunologically detectable hexon; the three mutants in complementation group III synthesized viral DNA, shut off host DNA synthesis, and made immunologically reactive capsid proteins (hexon, penton base, and fiber).