General Concepts
Clinical Manifestations
Adenoviruses cause acute respiratory disease (usually), pneumonia (occasionally),acute follicular conjunctivitis, epidemic keratoconjunctivitis, cystitis, andgastroenteritis (occasionally). In infants, pharyngitis andpharyngeal-conjunctival fever are common.
Structure
The icosahedral capsid (70 to 100 nm) is made up of 252 capsomeres: 240 hexonsforming the faces and 12 pentons at the vertices. Each penton bears a slenderfiber. The double-stranded linear DNA is associated with two major core proteinsand carries a 55-kDa protein covalently attached to its 5′ end.
Classification and Antigenic Types
More than 100 antigenic types of adenoviruses have been identified that infectmammals (mastadenoviruses) and birds (aviadenoviruses); 47 human adenovirustypes are classified, 5 more candidate types are presently studied.
Multiplication
Infection may be productive, abortive, or latent. In productive infections, theviral genome is transcribed in the nucleus, mRNA is translated in the cytoplasm,and virions self-assemble in the nucleus. In latent infections and intransformed and tumor cells, viral DNA is integrated into the host genome.Virus-host DNA recombinants are also found in productive infections.
Pathogenesis
Infection is usually transmitted in droplets of respiratory or ocular secretions.Persistent infection occurs in the tonsils. Some adenovirus types are oncogenicin newborn rodents and can transform cells. A few transformed human cell linesexist. Human oncogenesis has not been found but may nevertheless occur (e.g., bya “hit-and-run” mechanism).
Host Defenses
Most adolescents and adults have circulating neutralizing antibodies; immunity iswidespread. Cytotoxic T lymphocytes destroy adenovirus-infected cells.
Epidemiology
Infection is common in children. Epidemics do not occur in the generalpopulation, but outbreaks of acute respiratory disease occur in militaryrecruits. Serious complications are very rare.
Diagnosis
Adenovirus infection is suggested clinically by fever, upper respiratory tractinfections, and conjunctivitis; the diagnosis is confirmed by a rise in antibodytiters and by virus isolation.
Control
There is no treatment. Whole-virus vaccines are not used because of the potentialrisk of oncogenesis. Other vaccines, including recombinant vaccines, are underdevelopment, but adenoviruses do not represent a serious health hazard.
Vector in Gene Therapy
Adenoviral genomes have been developed into vectors in experimental therapy sinceadenoviruses readily infect human and other mammalian cells. Vector genomescarry deletions in the E1 and E3 regions; the gaps in the genome are used totake up foreign genes, e.g., the gene for the cystic fibrosis transmembraneconductance regulator (CFTR). Deletions in E1 minimize the potential of thesevector genomes to elicit an infection cycle in human cells. The first clinicalapplications in patients suffering from the genetic disease cystic fibrosis havebeen reported but problems with adenovirus toxicity remain.
Introduction
The adenoviruses are common pathogens of humans and animals. Moreover, severalstrains have been the subject of intensive research and are used as tools inmammalian molecular biology. More than 100 serologically distinct types ofadenovirus have been identified, including 49 types that infect humans. The familyAdenoviridae is divided into two Genera, the mammalian adenoviruses(mastadenoviruses) and the avian adenoviruses (aviadenoviruses). The adenovirusesare named after the human adenoids, from which they were first isolated.
Several adenoviruses can cause respiratory and conjunctival diseases. In addition, afew types of human adenoviruses induce undifferentiated sarcomas in newborn hamstersand other rodents and can transform certain rodent and human cell cultures. There iscurrently no evidence that adenoviruses are oncogenic in humans, but the possibilityremains of interest.
Clinical Manifestations
The main target for human adenoviruses is the respiratory tract. Various adenovirusescan also cause acute follicular conjunctivitis, epidemic keratoconjunctivitis, and,less frequently, cystitis and gastroenteritis (Fig.67-1).In infants, the most common clinical manifestations of adenovirusinfections are acute febrile pharyngitis and pharyngeal-conjunctival fever. Inmilitary recruits, acute respiratory disease is the predominant form of adenovirusdisease, with adenovirus pneumonia as a not infrequent complication. Except foroutbreaks in military groups and occasionally among children, adenovirus infectionsdo not occur epidemically. The virus is probably transmitted via droplets ofrespiratory or ocular secretions.
Structure
The adenovirus particle consists of an icosahedral protein shell surrounding aprotein core that contains the linear, double-stranded DNA genome (Fig. 67-2). The shell, which is 70 to 100 nmin diameter, is made up of 252 structural capsomeres. The 12 vertices of theicosahedron are occupied by units called pentons, each of which has a slenderprojection called a fiber. The 240 capsomeres that make up the 20 faces and theedges of the isocahedron are called hexons because they form hexagonal arrays. Theshell also contains some additional, minor polypeptide elements. The core particleis made up of two major proteins (polypeptide V and polypeptide VII) and a minorarginine-rich protein (μ). A 55 kDa protein is covalently attached to the5′ ends of the DNA.
Figure 67-2
Structural model of the adenovirus virion. The Roman numerals refer to the standard designations of the viralstructural proteins according to their decreasing molecular masses. FPstands for fracture plane in freeze etching. (From Brown DT, Westphal (more...)
Figure 67-3 shows the genetic map of aprototype adenovirus, adenovirus type 2 (Ad2). The genome is divided into earlyfunctions (E1A, E1B, E2A, E2B, E3, and E4 regions), which are expressed first duringviral replication, and late functions (L1 to L5 regions), which are usuallyexpressed after the early functions and after the beginning of viral DNAreplication. The late genes encode the viral structural proteins. In the case ofAd2, DNA replication begins 6 to 8 hours after infection of cultured human cells.The VA segment of the genome codes for small RNAs (VAI and VAII RNAs) about 160nucleotides long, which are not translated but regulate the translation of viralmRNAs. The VA RNAs are transcribed by eukaryotic RNA polymerase III. The genome alsocodes for a tripartite RNA leader sequence that is spliced onto all the late viralmRNAs. In 1977, RNA splicing was discovered in adenovirus-infected cells. Bothstrands of the double-stranded DNA code for specific viral functions (Fig. 67-3). The termini of the DNA moleculecarry inverted repeat sequences so that denatured single strands can form circularDNA molecules.
Figure 67-3
Genetic map of adenovirus type 2. The coding capacities of individual genome segments are indicated by thesizes of polypeptides (K represents 1,000 Dalton) or by the designationsof the virion structural proteins (Roman numerals; see Fig. 2). The double-stranded (more...)
Classification and Antigenic Types
At present, 47 types of human adenoviruses have been identified (Table 67-1) five additional candidatetypes are under investigation. The genomes of the different adenoviruses aregenetically distinct and vary somewhat in size.
Table 67-1
Human Adenovirus Types.
Multiplication
Host cells differ in permissivity for adenovirus types (Table 67-2). In permissive cells, the virus multipliesproductively and kills the host cell. Other cells are semipermissive, allowingreplication at low efficiency, whereas in still others replication is blocked andthe infection is abortive. As discussed below, in some abortive infections all orpart of the genome may be integrated into the host DNA, resulting in a latentinfection, which may lead to oncogenic transformation.
Table 67-2
Adenovirus-Host Cell Interactions.
Productive Infection
The virion enters host cells either by attaching to the cytoplasmic membrane andthen being engulfed into the cytoplasm in a membrane-bound vesicle (viropexis)or by directly penetrating the cytoplasmic membrane. The viral DNA is graduallyuncoated and enters the nucleus of the cell, most probably as a nucleoproteincomplex that still contains viral core proteins (Fig. 67-4).
Figure 67-4
Early events in the interaction of the adenovirion with a hostcell. The figure shows a model based on biochemical studies that depictsthe major steps in viral penetration and uncoating. (FromLonberg-Holm K, Philipson L: Early events of virus-cell interactionin (more...)
The viral DNA is transcribed and replicates in the nucleus of the host cell. Theviral mRNA undergoes processing in the nucleus and/or during transport throughthe nuclear membrane into the cytoplasm, where it is translated by polysomesinto viral proteins. These proteins return to the nucleus, where new virionsself-assemble. The mass of newly synthesized virus particles can assumecrystal-like arrangements. The bulk of the virions may not be easily releasedfrom the nucleus and the cell. There is evidence that extracellular adenovirustype 12 virions have a considerably higher specific infectivity thanintracellular virions. During active viral release, the newly synthesizedvirions may receive properties conferring high infectivity toward the hostcells.
The initiation of adenovirus DNA replication is atypical in that theβ-hydroxyl group of a serine residue in the precursor to the terminalprotein (pTP), an 80- to 87-kDa polypeptide, acts as a primer in DNAreplication. Viral DNA replication can proceed bidirectionally and bysingle-strand displacement from either end of the DNA duplex. Theadenovirus-encoded DNA polymerase, pTP, the adenovirus E2A protein, and severalhost proteins catalyze viral DNA replication.
Most of the adenovirus genes (Fig. 67-3)are transcribed by the host DNA-dependent RNA polymerase II in a complextranscriptional program. This program is regulated by the nucleotide sequencesand the structure of the viral promoters and by a host of cell-encodedtranscription factors that recognize specific upstream and downstream nucleotidesequence motifs in the promoters. Genes in the E1A region of the adenovirusgenome are the first to be transcribed. One protein product of this gene regionis a transactivator that is essential for the activation of all other viralgenes. This immediate-early viral function can also activate or inactivatecertain cellular genes.
The jointly controlled E2A and E2B regions code for proteins that are essentialfor viral DNA replication. Among the E3-encoded functions, one is a 25,000(19,000)-molecular-weight glycoprotein responsible for the interaction with cellmembrane-associated proteins (major histocompatibility complex). The E3region-encoded functions may be unnecessary for viral replication in cellculture, but essential for the interaction with the intact defense system of anorganism and for the modulation of host functions. The late viral L1 region canalso be transcribed early in the infection cycle, probably to a limited extent.Genes encoded in the L1 region of Ad5 DNA are essential for virion assembly.
All the late viral functions are under the control of the major late promoter(MLP) components, which are located at about 17, 20 and 27 map units on theviral genome. The gene encoding the fiber structural protein can also becontrolled by the x, y, and z leaders (Fig.67-3)
The regulation of promoter activity in all biologic systems is dominated by theinteraction of promoter sequence motifs with specific factors. These (protein)factors in turn bind to a host of further proteins, cofactors, that determinethe structure of transcription complexes. Viral promoters are conditioned to thefactors present in specific host cells. Enhancers and silencers are quantitativemodulators of promoter function. Both act independently of position andorientation and can exert their influence over relatively long distances.Enhancers strengthen promoter activity, whereas silencers have a negativeeffect, abrogating or diminishing promoter function. Enhancer and silencerelements are species specific.
The VAI and VAII RNAs (Fig. 67-3) aretranscribed by RNA polymerase III. VAI RNA is an important translationalactivator of host cell and viral messenger RNAs (mRNAs) late after infection. Itprevents activation of a protein kinase that is responsible for thephosphorylation and ensuing inhibition of the eIF-2 translation factor. Thiskinase can be induced by interferon. VAI RNA, thus, can be viewed as a viraldefense mechanism against interferon.
Abortive Infection
Virus interaction with a host cell can be blocked at many different steps, thusleading to an incomplete or abortive cycle. Depending on the permissivity of thehost cell, different types of adenovirus-host cell interactions can bedistinguished (Table 67-2). Manycultured human epithelioid cell lines are productively infected by humanadenoviruses. Rat cells are semipermissive (e.g., for Ad5), and permit viralreplication only at low efficiency. The outcome of an adenovirus infectiondepends on the animal species, cell type, and virus type involved. For example,hamster cells are abortively infected with human Ad12. The viral DNA istransported to the nucleus, where part of it is integrated into the host cellgenome. Both in productively and in abortively-infected cells, the viral DNAgravitates towards and becomes transiently associated with the host cellchromosomes as demonstrated by fluorescent in situhybridization. Most of the early viral genes are transcribed, but the late genesremain silent in the host cells. Ad12 DNA replication in hamster cells cannot bedetected with the most sensitive techniques. The major late promoter of Ad12 DNAis inactive in both uninfected and Ad12-infected hamster cells, whereas itfunctions in infected human cells. Ad2 cannot replicate in monkey cells; in thiscase, the translation of some of the late viral mRNAs is amiss. The adenovirusgenome persists, perhaps for a very long time, in cells of the human tonsils. Itis not known how adenovirus replication in this human organ is restricted.
Pathogenesis
Adenovirus disease results from localized virus multiplication at the portals ofentry (Fig. 67-1). The pathogenesis oflocalized infections is presented in Chapter45.
Integration of Adenovirus DNA into the Host Genome
Latency and persistence of, as well as oncogenicity by, DNA viruses arefrequently associated with integration of all or part of the viral genome intothe host cell DNA. Integration of adenovirus DNA has been demonstrated inabortively infected cells, in adenovirus-transformed cells, and in Ad12-inducedtumor cells. In productively infected human cells, recombination betweenadenovirus DNA and host cell DNA has also been observed. However, it is notknown whether this recombination can lead to stable integration, because in theproductive infection cycle the host cells are eventually killed. There isevidence that early in productively infected human cells Ad12 DNA becomespreferentially integrated into human chromosome 1.
Soon after infection, the viral genome may be inserted into selective sites ofthe cellular genome. The initial steps of viral malignant transformation couldinvolve insertional mutagenesis at a certain number of selective cellular sites.From the viewpoint of the geneticist, this model of viral oncogenesis is stillone of the more attractive possibilities. Moreover, after being insertedinitially at a limited number of sites and eliciting decisive mutagenic events(e.g., deletions), the viral DNA could perhaps be transposed to other loci inthe host genome or could be lost.
Recently, an interesting alternate mechanism of insertional mutagenesis inadenovirus-transformed or Ad12-induced tumor cells was discovered. Insertion ofAd12, plasmid or bacteriophage lambda DNA into established mammalian genomes canlead to extensive changes in patterns of cellular DNA methylation far away fromand on chromosomes different from those of the site of viral DNA integration.Since patterns of DNA methylation are related to expression patterns and genomeorganization, alterations in patterns of DNA methylation might affect manycellular functions whose altered expression may play a role in insertionalmutagenesis and viral oncogenesis.
Analyses of several different integration sites in transformed cell lines suggestthat transcriptionally active regions of the host cellular genome, which have acharacteristic chromatin structure, are most apt to recombine with foreign(viral) DNA. Adenovirus DNA frequently recombines with cellular DNA via itstermini, and terminal viral nucleotides are often deleted from the integratedviral DNA molecule. In general, considerable variability is observed in thestructure of the site of integration. No specific cellular DNA sequence has beenfound at the site of viral DNA insertion in established cell lines. Cellular DNAcan be deleted at the insertion site, or the cellular site can be preserved tothe last nucleotide. Ad12 DNA is frequently integrated nearly intact in the DNAof nonpermissive hamster cells. However, Ad2 DNA is usually integrated infragments in hamster cells permissive for Ad2.
The adenovirus system has also served as a model for studying the function ofsequence-specific promoter methylations in mammalian cells. Upon integration ofthe adenovirus genome into the host cell genome, a highly specific pattern ofmethylation is de novo imposed on the integrated viral genomeduring many cell generations. This de novo methylation is notprimarily dependent on nucleotide sequence. Site of integration, structure ofintegrate and genetics of the host cell are contributing factors. There isevidence from analyses in many different biologic systems that sequence-specificpromoter methylations can cause long-term gene inactivation.
Ad12-transformed hamster cells or Ad12-induced hamster tumor cells maintained inculture can eventually lose the integrated copies of viral DNA. This losssuggests that adenoviruses might cause transformation by a “hit andrun” mechanism.
Malignant Transformation and Oncogenesis
Cells from a number of rodent species and humans can be transformed in culture byadenoviruses. The frequency of malignant transformation is extremely low, andthis has prohibited quantitative studies in this system. Transformed human celllines have also been described. Some adenoviruses, such as Ad2 and Ad5, are notoncogenic in animals at all. Tumorigenic potential has been attributed to thecapacity of some adenoviruses (e.g., Ad12) to turn off the expression of genesof the major histocompatibility complex and thus to allow the transformed cellsto overcome host defenses and grow into solid tumors. Most of theadenovirus-induced tumors, tumor cell lines, and transformed cell lines carryone or several copies of the viral genome integrated into the chromosomes. Thetumor or transformed state is also associated with the differential expressionof the integrated viral genes. The early viral genes are often the predominantgenes expressed. It is thought that the E1 region of the viral genome isparticularly important in eliciting the transformed state. However, thecontinued presence of the viral genome, or of parts of it, may not be essentialfor the maintenance of the transformed state.
The so-called oncogenes represent a set of cellular genes that are involved inmany different ways in growth control. Oncogenes in adenovirus-induced tumor ortransformed cells have received surprisingly little attention. The few studieson this topic have reported occasional changes of oncogene activity,particularly for the myc gene. Moreover, E1 proteins can bindtightly to the product of the retinoblastoma (RB) or the p53 gene, which areconsidered to be anti-oncogenes. It has been suggested that the fixation of theanti-oncogene products by E1 proteins might contribute to the transformation ofcells. The interplay of several viral and cellular factors may eventually alterthe cellular growth control and weaken or overcome the host defenses in such away that an adenovirus-transformed rodent cell can grow into a solid tumor.
Since many human tumors do not contain even traces of adenovirus genes or geneproducts, the possibility that adenoviruses cause human tumors is low. New, moresensitive techniques are now available. Moreover, the “hit andrun” hypothesis has not been ruled out. Since even experimentallyinduced tumors can lose the viral genome and retain oncogenicity, this possiblemechanism of transformation of human cells is still being studied.
Persistence of Adenoviruses in Human Tonsils
Adenoviruses were first isolated from human adenoids, and the persistence ofthese viruses or their DNA in the human adenoids has been studied. It is notknown whether adenoviruses or their genomes can persist in other human organsystems. When the adenoids are removed during acute adenovirus infection, intactviral genomes are present. In contrast, when adenoid tissue obtained during asymptom-free interval or from a chronically-infected carrier is analyzed, only asmall number of cells seem to harbor the viral genome, which may not be intact.In some cases, in situ hybridization is needed to show that individual cells inthe adenoids contain the viral DNA and/or adenovirus-specific RNA. These cellsdo not produce infectious virus. It is not known to what extent adenovirusvirions continue to replicate in the adenoids throughout adult life.
Host Defenses
In adolescents and adults a high prevalence of circulating neutralizing antibodiescontributes to widespread immunity against adenovirus infections. Cytotoxic Tlymphocytes also recognize and destroy adenovirus-infected cells. Interferon isinduced by adenoviruses in vitro but fails to inhibit many adenovirus types, perhapsdue to the function of VA RNA. Nevertheless, in a few preliminary studies interferonhas been reported to be effective in the treatment of adenovirus conjunctivitis.
Epidemiology
Adenovirus infections are widely distributed in human populations. The highestsusceptibility is found among children from 6 months to 2 years of age and extendsto the group of 5 to 9 year old children. Types 2, 1, 3, 5, 7, and 6 (in that order)are most frequently isolated from adenovirus-infected children, with types 1 and 2constituting some 60 percent of all isolates. Nevertheless, adenovirus infectionsare responsible for only 2 to 5 percent of acute respiratory infections inchildren.
Adenovirus also infects military recruits in the United States, where this infectionhas been studied well, and most likely in other countries as well. Adenovirus types4, 7, and 3 cause acute respiratory diseases, including pneumonia, in thispopulation.
Adenoviruses have been isolated from severely immunocompromised patients, such asthose with acquired immune deficiency syndrome (AIDS). Many of these isolates,including the adenovirus types 42 to 47, are found in the urine of AIDSpatients.
Diagnosis
Infection with an adenovirus may be suspected on the basis of a characteristicclinical presentation e.g., respiratory disease, conjunctivitis. The diagnosis canbe confirmed by demonstrating a rise in antibody titer between acute-phase andconvalescent-phase sera or by virus detection or isolation.
Control
Since adenoviruses are excellent antigens, vaccination could be very effective.However, viral vaccines usually have not been used because adenoviruses are involvedin tumorigenesis in animals and in cell culture. Moreover, adenovirus infectionsonly rarely cause serious complications. Nevertheless, efforts are under way toproduce vaccines by recombinant DNA technology. Purified hexon or fiber preparationsinduce high levels of neutralizing antibodies, and vaccines based on these proteinshave been tested successfully.
Vector in Human Somatic Gene Therapy
Adenoviruses have been used as vector systems in approaches towards human somaticgene therapy. The early region E3 of the viral genome is not essential for viralreplication in cell culture and can be removed to yield space in the genome forthe insertion of foreign genes constructed for therapeutic purposes. Moreover,the E1 region of the adenoviral genome can be excised to incapacitate viralreplication in human tissues thus offering further space for foreign geneinsertions. Manipulated, E1-deficient adenovirions can be propagated in thehuman cell line 293 which contains in an integrated form and constitutivelyexpresses the E1 region of Ad5. Results adduced to date indicate thatmanipulated adenoviral genomes, e.g. with the test gene forβ-galactosidase under eukaryotic promoter control inserted, persist andcontinue to express this test gene in different organs of rodents for periods upto months. It is not known whether these viral genomes can integrate into thehost genome under these conditions.
In the genetic disease cystic fibrosis, mutations in the human gene for thecystic fibrosis transmenbrane conductance regulator (CFTR) cause severe symptomsin the respiratory and gastrointestinal tracts, mainly because of a drasticallyincreased viscosity of secretions. This disease can lead to death early in thelives of afflicted individuals. Recombinant adenoviruses carrying the c-DNA forthe CFTR gene have been shown to facilitate synthesis of the CFTR gene productin infected human cells. Recent clinical trials in human cystic fibrosispatients have demonstrated that CFTR gene-recombinant adenovirus infection canlead to improved pulmonary function in these patients. Rise in antibody titeragainst adenoviruses and bronchial irritations - presumably due to adenovirustoxicity - have also been reported. Adenoviruses may not be the final answer tothe solution of vector problems in human somatic gene therapy, but they may helpopen the path towards the construction of more suitable vector systems.
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