Family Adenoviridae - Viruses And Human Disease

FAMILY ADENOVIRIDAE

and HHV-8. Intriguingly, Kaposi's sarcoma is 15-fold more

common in homosexual male AIDS patients than in patients

who acquired HIV by a nonsexual route. HHV-8 is also asso-

Adenoviruses are widespread viruses of mammals and

ciated with body cavitybased lymphoma, a lymphoid tumor

birds, but a few of the known viruses are able to infect rep-

in some AIDS patients, and with multifocal Castleman's dis-

tiles or frogs. The virions are a T=25 icosahedron, 7090 nm

ease, a rare lymphoproliferative disorder. In common with a

in diameter, with fibers 9 to 77 nm in length projecting from

number of other herpesviruses, HHV-8 encodes factors that

the 12 fivefold axes of the icosahedron (Figs. 2.1 and 2.12).

stimulate cells to divide, that interfere with the host immune

Virions contain about a dozen proteins, of which 4 are present

response, and that block apoptosis. It also produces a protein

in the core. The major structural proteins are a hexon protein

in latently infected cells that tethers the viral DNA to mitotic

called II, three copies of which form a hexon, of which there

chromosomes.

are 240 in the virion, and a penton protein called III, five

copies of which form a penton base, of which there are 12 in

the virion. The genome of adenoviruses is a linear dsDNA of

Monkey B Virus

size 26 to 45 kb. A terminal protein that served as a primer

Many herpesviruses infect vertebrates other than humans,

during DNA replication is covalently attached to the 5¢ end

but only one nonhuman herpesvirus is known to be highly

of both strands.

pathogenic for humans. Cercopithecine herpesvirus 1 or

Adenoviruses are named after adenoids, a glandlike col-

monkey virus B is indigenous to Old World monkeys in the

lection of lymphoid tissue in the nasopharynx. Many human

genus Macaca. In its native host it causes a disease that is

adenoviruses establish a long-term infection in this tissue and

similar to that caused by HSV-1 in humans. A latent, lifelong

adenoviruses were first isolated from human adenoids. Four

infection is established in the monkey that seldom leads to

genera are currently recognized. The genus Mastadenovirus

serious illness. Sporadic reactivation of the virus occurs that

contains viruses that infect only mammals and the genus

results in the formation of vesicular lesions, particularly on

Aviadenovirus contains viruses that infect only birds, whereas

the tongue and cheeks. However, infection of humans or of

the genera Atadenovirus and Siadenovirus contain viruses that

a number of monkeys other than macaques results in a very

infect a wide variety of vertebrates (Table 7.10). Most viruses

serious neurological disease that has a high fatality rate. As

are species specific and in general will only undergo a com-

described earlier, HSV-1 is also neurotropic and occasion-

plete replication cycle in cells isolated from their native host.

ally causes fatal encephalitis.

Fifty-one human adenoviruses have been distinguished

B virus has usually been transmitted to humans through

on the basis of serological reactivity--an adenovirus is con-

the bite of a monkey in which infectious virus was present

sidered distinct if it resists neutralization by antisera against

in the saliva. However, transmission has also occurred by

the other known adenoviruses. All belong to the genus

other means. In at least one case transmission resulted from

Mastadenovirus and have a genome size of 3036 kb. The

contact of infectious material with the eye and two cases are

51 viruses are simply numbered in order of their isolation

thought to have resulted from exposure to aerosols. One case

and are often referred to as Ad1, Ad2, etc., or more formally

of transmission of virus from an animal worker to his wife

as HAdV-1, etc., to distinguish them from adenoviruses

has also been documented. The majority of human infec-

that infect other species. The human viruses were originally

tions have resulted in fatal neurological disease, but some

divided into six subgroups on the basis of serological cross-

infections resulted in only mild disease and the establish-

reactions in a hemagglutination-inhibition assay. In this

ment of a latent infection. Acyclovir has been used to treat

assay, the ability of an antiserum to bind to the virus and

persons infected by B virus with apparent success, but the

prevent it from agglutinating red blood cells is examined.

number of cases is small and no controlled trials of efficacy

An antiserum against one of the viruses of subgroup A,

have been conducted.

for example, inhibits hemagglutination by all members of

Recurrence of herpes infections is often associated with

that subgroup but not by members of other subgroups. This

stress. Newly captured or shipped animals are subject to a

grouping correlated with a number of other properties of

great deal of stress, so that active infection in these animals

the viruses as well, such as their ability to form tumors in

is not uncommon. Animal handlers or researchers using

rodents. These original subgroups are now considered to be

these animals are at risk for the disease and must take proper

different adenovirus species, human adenovirus A through

precautions when handling macaques. Because of the dan-

F, with grouping relying on sequence identities where pos-

gers from B virus infection, most laboratories in the United

sible. Two viruses are considered to belong to the same spe-

States use only monkeys that lack antibodies to B virus, an

cies if they differ by less than 10% in their sequence, and

indication that they are not infected. Although this greatly

by this criterion the human adenovirus species may contain

reduces the risk of handling the animals, it does not elimi-

adenoviruses of other animals.

nate all risk because occasionally monkeys that are infected

Because they replicate to high titer in cultured human cells,

do not have detectable antibodies.

several human adenoviruses have been intensively studied,

TABLE 7.10 Adenoviridae

Genus/members

Serotypes

Usual host(s)

Disease in natural host

Mastadenovirus

Human adenovirus A

Types 12,18,31

Humans

Enteritis

Human adenovirus B

Types 3,7,11,14,16,21,34,35

Humans

Enteritis; military recruits' disease (3, 7, 14, 21); type 35

causes pneumonia in elderly and immunocompromised

humans

Human adenovirus C

Types 1,2,5,6

Humans

Respiratory infection in children

Human adenovirus D

Types 8,9,10,13,15,17, 19,20,

Humans

Enteritis

22-30,32,33,36-39,42-47

Human adenovirus E

Human type 4, simian types

Humans

Enteritis, pneumonia and upper respiratory disease in

22,23,24,25

military recruits

Human adenovirus F

Humantypes 40,41, simian type 19

Humans

Infant diarrhea

Murine adenovirus A

Mice

Bovine adenovirus A, B, C

Cattle

Asymptomatic or mild respiratory disease

Ovine adenovirus A, B

Sheep

Porcine adenovirus A, B, C

Swine

Canine adenovirus

Types 1 and 2

Dogs

Hepatitis (type 1)

Respiratory disease (type 2)

Aviadenovirus

Fowl adenovirus A, B, C,

Chickens, ducks

Hepatitis, bronchitis, duck hepatitis (rare)

D, E.

Goose adenovirus

Geese

Atadenovirus

Ovine adenovirus D

Sheep

Basis of some vectors for human gene therapy

Bovine adenovirus D

Cattle

Duck adenovirus A

Ducks

Egg drop syndrome

Siadenovirus

Frog adenovirus

Amphibians

Nonpathogenic

Turkey adenovirus 3

Turkey, pheasants,

Hemorrhagic enteritis in turkeys, marble spleen

chickens

disease in pheasants, splenomegaly in chickens

In general adenoviruses are transmitted by both aerosols and fomites and by the oralfecal route.

in particular Ad2, Ad5, and Ad12. Further interest has been

produce a set of early RNAs. Later, a set of late RNAs is

generated by the fact that, although they will not undergo a

produced. A transcription map of Ad2 is shown in Fig. 7.20.

complete replication cycle in rodent cells, they will infect

Transcription of early RNAs occurs from five promoters,

and transform rodent cells in culture. Members of HAdV-A

three on the so-called R strand and two from the L strand.

will also cause tumors in rodents at a high rate and members

The R strand is transcribed rightward on the chromosome

of HAdV-B at a moderate rate. Other human adenoviruses

as conventionally drawn, and the L strand is transcribed

cause tumors in rodents at a low or undetectable rate. There

leftward. There are two other promoters for transcription of

is no evidence that adenoviruses are associated with tumors

delayed early mRNAs. Multiple splicing of these transcripts

in humans, however. Because of the extensive background of

leads to the production of about 30 mRNAs. The proteins

information about them and the fact that they usually cause

translated from these early mRNAs are required for repli-

only minor illness, attempts are being made to use them as

cation of the viral genome. The E1A and E1B gene prod-

expression vectors for gene therapy (Chapter 11).

ucts are oncogenes that stimulate the cell to enter S phase

and thereby induce an ideal environment for the replication

of the viral DNA. Their mode of action is described later.

Transcription of Adenovirus mRNAs

Proteins from the E2 region are directly involved in repli-

Adenovirus replication takes place in the nucleus. After

cating adenovirus DNA and include a DNA polymerase, an

the entry of the infecting genome into the nucleus, the infect-

ssDNA-binding protein, and a precursor to the terminal pro-

ing genome is transcribed by host RNA polymerase II to

tein, which is involved in initiation of DNA replication. E3

irion structural proteins (green units)

E1A E1B

R 3

DNA

E2B

E2A

Polymerase

72K - ssDNA binding

Map

Units

100

Promoter

Transcription Unit

IGURE 7.20

Genome organization of human adenovirus type 2. The double blue lines represent the two linear DNA

strands that make up the genome of 36 kbp. The genes have been mapped by superimposing an arbitrary scale of 100

map units. Each arrow represents a transcription unit composed of a nested set of spliced messages, transcribed in the

direction of the arrow. Black dots indicate short spliced sequences that form leaders. General functions of the various

transcription units are shown. Proteins in the E3 cluster interact with the host immune system, and E4 genes are involved

in DNA replication. The major late transcription unit includes the leaders "1," "2," and "3" and the L1, L2, L3, L4, and

L5 families of genes. Adapted from Wold and Golding (1991).

proteins modulate the host response to adenovirus infection,

Replication of the Viral DNA

and this region is nonessential in cultured cells. The func-

tions of the E3 proteins are described in Chapter 10. Region

Inverted terminal repeats that contain the origins

E4 encodes proteins involved in transcription and transport

of replication are present at the ends of the adenovirus

of viral mRNAs and in DNA replication.

genome. DNA synthesis is initiated at one of the two ends

Late genes are all transcribed beginning from a single pro-

and proceeds to the other end. There is no lagging strand

moter on the R strand, and transcription leads to an RNA prod-

synthesis and the partner of the strand being copied is

uct that is about 80% the length of the adenovirus genome.

displaced as an ssDNA (illustrated schematically in Fig.

Multiple splicing occurs to produce at least 18 different mRNAs

1.9C). The precursor of the terminal protein serves as a

that fall into five families, based on the use of five different

primer during initiation. It forms a complex in solution

polyadenylation sites. Each late mRNA has the same tripartite

with the adenovirus DNA polymerase, and it is assumed

leader, formed by splicing. The late mRNAs are translated into

that these two proteins bind to the origin of replication

the proteins required for the assembly of progeny virions.

as a complex during initiation of DNA replication. There

The multiple splicing events that occur during process-

are also binding sites within the terminal repeats for sev-

ing of adenoviral mRNA, especially the late mRNA, which

eral cellular proteins that stimulate the initiation of DNA

is made in abundance, led to the discovery of RNA splic-

synthesis. The first step in initiation is the covalent link-

ing by Phillip Sharp. Upon examination of adenoviral

age of dCMP, the first nucleotide in adenovirus DNA,

mRNADNA hybrids with an electron microscope, he

to the preterminal protein. Subsequent chain elongation

observed that regions of the genome were missing from the

requires the activity of the adenovirus DNA polymerase

mRNA transcripts. For his discovery of RNA splicing, he

and of the ssDNA binding protein, as well as of a cellular

was awarded a Nobel Prize, along with Richard Roberts, in

topoisomerase. The use of a protein primer eliminates the

1993 (Table 1.1).

need for a primase to initiate DNA synthesis with an RNA

In addition to the many genes transcribed by RNA

primer, and thus solves the problem of how to maintain

polymerase II, one or two adenovirus genes, called VA, are

the ends of the linear adenoviral DNA molecule during

transcribed by host RNA polymerase III. Short VA RNA

replication.

molecules are produced that are not translated. They func-

The products of the first round of replication are a dou-

tion to inhibit the host interferon system and the host RNAi

ble-strand progeny genome and a single-strand copy of one

system and will be described in Chapter 10, after the descrip-

of the two strands of the genome. Initiation of DNA syn-

tion of these host systems.

thesis can also occur on this ssDNA. It is proposed that a

panhandle structure is formed by the terminal repeats so that

from an mRNA that sediments at 12S) and 13S E1A (because

an origin of replication is present that is identical to that in

its mRNA sediments at 13S). Rb or retinoblastoma suscep-

the dsDNA. Copying this ssDNA renders it double stranded

tibility protein is a tumor suppressor. It was first identified

and completes the production of two copies of the dsDNA

because it is absent in patients suffering from retinoblastoma.

genome from the parental genome.

In its hypophosphorylated form, Rb binds a cellular transcrip-

tion factor called E2F and causes the cell cycle to arrest in G1.

Hyperphosphorylation of Rb causes it to dissociate from E2F.

Assembly and Release of Progeny Virions

Free E2F activates the transcription of genes that cause the

Progeny viruses are assembled in the nucleus from pre-

cell to enter S phase. The binding of Rb by E1A prevents it

assembled hexons and pentons (see Fig. 2.12). Viral DNA

from complexing with E2F, and E2F is thus free to induce the

is required for assembly. A packaging signal of about 260

cell to enter S phase. p107 and p130 are other members of the

nucleotides at the left end of the viral DNA leads to polar-

Rb family that also interact with E2F, as well as with cyclins

ized encapsidation starting from this end. During assembly,

and cyclin-dependent kinases, whose activities are disrupted

the viral protease cleaves at least four viral products, and

by binding to E1A. Binding of E1A to p300 appears to be

these cleavages are required to stabilize the particle and

an independent method of disrupting the cell cycle; p300 is

make it infectious. Release of virions from the cell is

thought to bind to DNA and activate transcription of factors

associated with the disruption of intermediate filaments.

involved in cell cycle progression.

Vimentin is cleaved early after infection by an unknown

The E1B 55-kDa protein also targets a tumor suppressor

protease, and cytokeratin K18 is cleaved late by the viral

protein called p53. p53 is another cellular protein that regu-

protease. A schematic of the relative timing of the major

lates cell cycle progression. It is the most commonly mutated

events in the adenovirus life cycle is presented in Fig. 7.21.

gene associated with human tumors. It is both a transcrip-

Virus infection is lytic and the cell eventually dies.

tional activator and repressor. It activates the transcription

of genes whose function is to arrest cell cycle progression.

E1B blocks the activity of p53, and when p53 is inactive or

Adenovirus Oncogenes

absent, cell cycle progression continues. Ad5 E1B binds p53

and sequesters it outside the nucleus, whereas Ad12 E1B

Two early genes of adenovirus, E1A and E1B, encode pro-

does not appear to bind p53 but inhibits its activity in some

teins that induce the cell to enter S phase, in which cellular

indirect way.

DNA is replicated. E1A targets a number of cell proteins that

The net result of the expression of E1A and E1B is the con-

are involved in cell cycling, forming complexes with Rb, p107,

tinued cycling of the cell. The expression of these two genes

p130, p300, and several other cellular proteins. These proteins

alone will transform cells in culture. Rat cells transformed by

are listed in Table 7.11 for two forms of E1A that result from

Ad12 (HAdV-A) will produce tumors in syngeneic newborn

differential splicing, called 12S E1A (because it is translated

104

Infectious Virus

Host DNA Synthesis

Host Protein Synthesis

103

Interference with host defenses

(antagonize IFN-α, IFN-β, TNF-α;

antagonize CTL recognition)

Virion protein synthesis (L1-L5)

102

Late transcription

Viral DNA replication

101

Early protein synthesis

Early transcription (E1A, E2A, E4)

Hours post infection

FIGURE 7.21 Relative timing of major events during the adenovirus life cycle. Example shown is for HeLa cells

infected with adenovirus at a multiplicity of infection (MOI) of 10. L1L5 are the major late transcription units mapped

in Fig. 7.20. Adapted from Fields et al. (1996), p. 2119 and Ginzberg (1984), Fig. 1 on p. 2.

TABLE 7.11 Cellular Proteins That Bind Directly

Adenovirus

to Adenovirus E1A Proteinsa

p300

Binds tob

E1A binding

Other proteins

E1B

protein

12S E1A 13S E1A

in complex

p53

E1A

p107

pRB

None known

p107

Cyclin A, cdk2/cyclin E, cdk2

p130

Cyclin A, cdk2/cyclin E, cdk2

p300

None known

TATA

TBP-associated factors

SV40

binding

proteins

p300

(TBP)

ATF-2

None known

Large-T

p53

YY1

None known

p107

E1A is the first viral transcription unit to be expressed. In the early phase

of infection, differential splicing results in two mRNAs from this gene,

which sediment at 12S and 13S respectively. The translation products of

these two mRNAs are referred to as 12S E1A and 13S E1A.

RB is the retinoblastoma susceptibility protein; cdk2 is cyclin-dependent

Papillomavirus

kinase 2; ATF-2 is a member of the ATF family of transcription factors;

YY1 is a human transcriptional repressor. These cellular proteins have

been shown to bind directly to the E1A protein in a biologically relevant

p53

way. See also Fig. 7.22.

Source: Adapted from Fields et al. (1996), p. 2122.

FIGURE 7.22 Known interactions between the oncogenic proteins

rats but cells transformed by Ad2 or Ad5 (HAdV-C) are not

(shaded with pink patterns) of an adenovirus, a polyomavirus, and a

tumorigenic. The differences in the ability to induce tumors

papillomavirus and cellular proteins that are regulators of cell cycle

appear to be in the immune responses of the host. In par-

progression. (A) Protein E1A of adenoviruses binds to the Rb family,

ticular, Ad12 appears to interfere more effectively with host

promoting entry into S phase. The 19-kD form of E1B also binds to p53,

CTL responses, as described in Chapter 10. Although some

blocking apoptosis. (B) The large T antigen of the polyomavirus, SV40,

interacts with the Rb family of proteins as well as with, p53 (see also Fig.

adenoviruses cause tumors in rats, there is no evidence that

7.28). (C) The human papillomavirus proteins E6 and E7 bind Rb and p53,

they do so in humans. The regulation of cell cycling may be

respectively, the latter promoting the destruction of p53. Adapted from Berg

different in rodents and humans, or the adenovirus proteins

and Singer (1997), Fig. 1.35 on p. 61.

may interact with rodent and human regulatory proteins in

different ways.

effector products of the interferon pathway. VA RNAs are

The proteins targeted by E1A and E1B are key regulatory

highly structured RNAs that are abundantly produced and

elements of the cell. The polyomaviruses and the papilloma-

act as dsRNA decoys. VA RNA binds to PKR, which requires

viruses target many of these same proteins in order to induce

bound dsRNA for activity, but does not activate the enzyme.

cell cycling, emphasizing their importance in the control

VA RNA also binds to several molecules in the RNA inter-

of the cell cycle. The oncogenes of these three families of

ference (RNAi) pathway, including Exportin 5 required for

viruses and their interactions with these key cellular proteins

the export of pre-microRNAs from the nucleus, and DICER

are illustrated schematically in Fig. 7.22.

required for processing of these RNAs, and VA RNA proc-

essed by DICER binds to RISC (see Chapter 10). The result

Interference with Host Defenses

is to saturate this pathway, thereby inactivating it.

Adenoviruses also inhibit the lysis of infected cells by

Adenoviruses interfere with host antiviral defenses in

CTLs. Ad12 E1A protein blocks the transcription of the

multiple ways. This topic is covered in detail in Chapter 10,

genes for class I MHC molecules, whereas the Ad2 or Ad5

so only a summary is presented here. Adenoviruses have two

E3 19-kDa protein prevents the export of class I MHC mol-

independent mechanisms to suppress the interferon system.

ecules to the cell surface. In either case, presentation of

First, E1A inhibits the transcription of interferon response

peptide antigens to CTLs is blocked.

genes by inhibiting the activity of ISGF3, a cellular tran-

Apoptosis is a cell suicide pathway by which cells die

scription factor. Second, VA RNA prevents the activation

after infection by many viruses. Adenoviruses encode

of a protein kinase called PKR, which is one of the major

proteins that delay the advent of apoptosis in order to give

to some extent Ad3 and Ad4, are the adenoviruses most

the virus more time to replicate. First, E3 region proteins

often associated with severe disease, and Ad7 accounts for

interfere with the action of TNF-α, blocking TNF-α-induced

about 20% of adenoviruses reported to the World Health

apoptosis. Second, E1B blocks, or at least delays, apoptosis

Organization.

otherwise induced by E1A.

Virus can be shed for months following primary infec-

Because adenoviruses block the antiviral defenses of the

tion, especially in stools (Fig. 7.23). Children who are shed-

host, they have the ability to persist in the infected host for

ding virus are infectious and spread is particularly efficient

considerable periods of time. Virus may be present in tonsils

between close family members. Figure 7.24 diagrams the

and adenoids and may be shed in the stools for a year or

spread of infection within four families in which older sib-

more following primary infection (Fig. 7.23).

lings were excreting adenovirus at the time of the birth of the

youngest child. In all cases the new baby was infected within

a year. Day care centers are also important in the spread of

these viruses.

Adenoviruses and Human Disease

Adenoviruses also cause respiratory disease in adults

and probably account for about 3% of such illnesses. The

The human adenoviruses replicate primarily in the upper

disease is usually mild, but Ad4 and Ad7 have caused

respiratory tract or in the gastrointestinal tract. Some rep-

epidemics of more serious respiratory illness in military

licate well in both while others express a tropism for one

recruits. Such epidemics of acute respiratory disease have

or the other. Spread of the viruses is by a respiratory route

resulted in the infection of 80% of the recruits in a unit and

or by an oralfecal route. Many infections by adenoviruses

2040% of these have required hospitalization. The stress,

appear to be asymptomatic or to result in only mild illness,

crowding, and bringing together of young men from dif-

but about 5% of acute respiratory disease in children under

ferent backgrounds and from all over the country seems

5 years old is due to adenovirus infection. Some serotypes

to potentiate the illness. Illness may be exacerbated if

can also cause gastroenteritis, but the overall importance

infection begins by deep inhalation of aerosolized virus,

of these viruses as causative agents of gastroenteritis is not

which results during the vigorous exercise that is required

resolved. Ad1, 2, and 5 are the most common viruses found

of recruits. Such epidemics result in protective immunity

in human populations, and antibodies to these viruses

because seasoned troops do not suffer recurrent epidemics

are present in about one-half of all children. Ad7, and

100

200

400

600

800

Days following initial infection

FIGURE 7.23 Adenovirus shedding by patients. The percent of 133 patients who shed virus in stools for at least the

number of days indicated after an initial adenovirus infection is plotted. Note that the last point is almost 2 years. Data

from Strauss (1984).

Family

Age

4 yr

2 yr

3 yr

17 mo

recrudescent

2 yr

10 11 12 13 14 15 16 17

Months

Male

Female

Fecal shedding

Respiratory shedding

First excretion

FIGURE 7.24

Adenovirus transmission between siblings in four families. Schematic representation of infection with

adenovirus of newborn babies in families where one or more older siblings was already infected. The newborns became

infected between 3 and 12 months after birth. It is believed that shedding of virus was continual (red lines) after the first

virus-positive stool sample from a given child. From Fox et al. (1969).

Special Training Unit (recruits)

Field Artillery (recruits)

Field Artillery Battalions

(seasoned men)

Nov

Dec

Jan

Feb

Mar

Apr

May

1944

1945

FIGURE 7.25

Admission to the base hospital for treatment of respiratory disease, presumably primarily adenovirus

related, of two recruit groups and one group of seasoned troops. From Dingle and Langmuir (1968).

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