Family Polyomaviridae - Viruses And Human Disease

(Fig. 7.25). Starting in 1971, the military used a vaccine to

and evolutionary history of polyomaviruses and papillo-

prevent the disease. This vaccine consisted of live nonat-

maviruses are distinct and they are now classified into two

tenuated virus that was encapsulated in coated capsules and

distinct families.

taken orally. Infection of the enteric tract is asymptomatic

To date, 14 different polyomaviruses have been described

for these viruses under these conditions and results in a

and others probably exist. These viruses infect warm-blooded

good antibody response, presumably including mucosal

vertebrates. Mammalian viruses include viruses infecting

antibodies to protect the respiratory tract. Use of this vac-

humans, monkeys, cattle, rabbits, rats, mice, hamsters, while

cine reduced adenovirus-specific disease rates in trainees

bird viruses infecting parakeets, geese, and other birds are

by 95 to 99% and reduced the total respiratory disease rates

known. The majority of infections of their native host by the

by 50 to 60%, illustrating the importance of Ad4 and Ad7

mammalian viruses are asymptomatic and the viruses estab-

as causative agents of severe respiratory disease. The vac-

lish a lifelong infection. Many can cause tumors in nonnative

cine is no longer available, however. It was made by a sin-

hosts, however, and may be associated with tumors in their

gle manufacturer whose facilities for making the vaccine

native hosts, and have been intensively studied as models for

became outdated and needed upgrading. The army was

tumor induction. In contrast, the bird viruses cause severe

unwilling or unable to find a mechanism to help with the

disease with a high mortality rate in their native host. A par-

cost of the upgrade, and since the military was the sole pur-

tial listing of the members of the family Polyomaviridae is

chaser of the vaccine, production was discontinued in 1995.

given in Table 7.12. The two best studied viruses are mouse

The last of the vaccine was exhausted in 1999 and epidem-

polyomavirus (MPyV), so called because it causes many

ics of severe adenovirus respiratory disease in recruits are

(poly) different kinds of tumors (omas) in mice, and sim-

once again a serious problem.

ian virus 40 (SV40), which infects monkeys. These viruses

Adenoviral epidemics such as have occurred in mili-

share about 60% nucleic acid sequence identity. SV40 was

tary recruits are rare in the civilian sector but do occur. As

first recognized as a contaminant in monkey kidney cultures

one example, there was an epidemic of Ad11 in 1997 in a

used for the production of poliovirus vaccine. Two polyoma-

South Dakota job-training facility. Like a military camp,

viruses for which humans are the natural host are known, BK

this facility had young adults housed in crowded condi-

virus and JC virus. SV40 also circulates in humans, possibly

tions with regular multiple introductions of new suscep-

as a result of its introduction with the contaminated polio-

tible persons. Illness with severe morbidity also occurs

virus vaccine. These three viruses have received increasing

in pediatric populations, school environments, and health

attention as disease agents.

care settings.

The structures of the two polyomaviruses SV40 and

Adenoviruses are being developed as vectors to immu-

MPyV have been solved to atomic resolution (Fig. 2.10).

nize people against other viruses and for gene therapy. Two

They are icosahedral viruses 45 nm in diameter with pseudo-

approaches are being used. One is to use the military experi-

T=7 symmetry (Figs. 2.1 and 2.5). The capsid consists of

ence with the Ad4 and Ad7 oral vaccines to develop vaccines

72 pentamers of structural protein L1, and each pentamer

that could be delivered safely using this route. A second

contains one molecule of either structural protein L2 or L3.

approach is to disable the virus by deleting some of the early

The polyomavirus genome is a circular dsDNA molecule

genes. Such an attenuated virus has been used in clinical tri-

that is 5 kb in size. In the virus it is complexed with cellular

als for treatment of cystic fibrosis. In these trials, virus that

histones to form a supercoiled minichromosome.

expresses the gene that is defective in such patients is deliv-

ered directly to the lungs. Trials that use adenoviruses in an

The Early Genes

attempt to treat other diseases are also ongoing. The use of

adenovirus for gene therapy has not been very successful to

Transcription maps of SV40 and MPyV are shown in

date, and the death of a patient in a gene therapy trial from

Fig. 7.26, in which the circular genomes are represented as

adenovirus led to retrenchment in this area. This topic is dis-

linear structures for ease of presentation. The genomes are

cussed in more detail in Chapter 11.

divided into two domains, an early domain that is transcribed

from one DNA strand and a late domain transcribed from

the other strand. An origin (Ori) is present at the junction

FAMILY POLYOMAVIRIDAE

of the two domains. Ori serves as an origin of replication for

DNA replication and a transcription start site for transcription

In the past the polyomaviruses and the papillomaviruses

by RNA polymerase II. It also contains enhancer ele-

were considered to be two subfamilies within the family

ments.

Papovaviridae. The name papova came from papilloma

Following infection, the viral DNA is transported to the

virus/polyoma virus/simian vacuolating virus (= SV40),

nucleus, whether as part of a virion or subviral particle or

three characteristic members of the enlarged family.

as free DNA is not clear, and early mRNA is transcribed by

However, the mode of replication, genome organization,

cellular RNA polymerase. The MPyV or SV40 promoters

TABLE 7.12 Polyomaviridae a

Virus name

Usual

Genus/members

abbreviation

host(s)

Transmission

Disease

Polyomavirus

PMLb-like disease in rhesus monkeys and

Simian virus 40

SV-40

Monkeys

immunocompromised macaques; may cause

Reactivation of persistent

childhood brain tumors in humans

infection, virus shedding

in urine, contact, aerosols,

Murine polyoma

MPyV

Mice

Tumors when inoculated into newborn mice, virus

sexual transmission

persists in the kidney

Bovine polyomavirus

BPyV

Cattle

Common infection, persists in the kidney

BK polyomavirus

BKPyV

Humans

Transmission as above plus

Common infection of early childhood; virus persists;

tissue transplantation in humans

tumors in immuncompromised humans

Common infection of late childhood; causes PMLb in

JC polyomavirus

JCPyV

Humans

immunocompromised hosts

Budgerigar fledgling

BFPyV

Parakeets

Fatal illness in fledgling parakeets

polyomavirus

Plus other viruses infecting monkeys, hamsters, and rabbits

Most polyomaviruses are worldwide in distribution.

PML, progressive multifocal leukoencephalopathy.

are strong promoters, active in many cells. Because of this,

Binding of large T antigen to the Ori regulates its own

the SV40 promoter has been used to drive expression of

production. Binding is also required for DNA replication.

foreign genes in many different expression systems. Early

Binding of SV40 large T antigen to Rb and p53, as well

mRNA transcribed from the viral genome terminates about

as to p107 and p300, is illustrated in Fig. 7.22. Binding to these

halfway around the genome and is differentially spliced to

tumor-suppressor proteins results in continued cell cycling

yield several mRNAs (Fig. 7.26). The translation products of

and transformation of the cell. Binding to Rb and its rela-

these mRNAs are called T antigens (tumor antigens) because

tives p107 and p300 releases transcription factors required

their expression in the absence of productive viral infection

for cell cycling, of which the best studied and perhaps most

leads to cell transformation and the formation of tumors in

important are members of the E2F family. p53 is a transcrip-

animals. The two major SV40 proteins are called the small

tion factor whose activity can prevent DNA synthesis, cause

t antigen and the large T antigen, whereas the three major

G2 and G1 growth arrest, and induce apoptosis. As described

MPyV antigens are called small t, middle T, and large T anti-

earlier, cell cycling induces a state suitable for viral DNA

gens. In addition, a 17k T antigen has been reported in SV40

replication. Expression of large T in the absence of other

and a short-lived tiny t antigen has been reported for MPyV.

SV40 proteins is sufficient to transform a cell, although such

The large T antigens (size: about 700 amino acids) are

a transformed cell does not express the full complement of

multifunctional proteins that interact with viral promoters

transformed attributes unless small t is also expressed.

and several cellular proteins. A schematic representation of

The activities in MPyV virus T antigens are distributed

the large T antigen of SV40 that illustrates the locations of

somewhat differently. Polyoma large T antigen binds Rb

many of its activities within the protein sequence is shown

but does not bind p53. Binding to Rb induces cellular DNA

in Fig. 7.27. These activities are differentially regulated by

synthesis. Cell transformation is a function of middle T anti-

phosphorylation of serine and threonine residues, as shown.

gen. Middle T antigen binds to src, a known proto-oncogene

The large T antigen possesses a nuclear localization signal

located at the cell surface, and activates its kinase activity

that causes it to be transported to the nucleus, a domain that

(Fig. 7.28). The phosphorylated middle T antigen interacts

binds to sequence elements within Ori, phosphorylation

with other cellular factors and induces cell transformation,

sites in two widely separated regions that serve to regulate

and it has been shown that expression of middle T antigen

its activities, domains that interact with DNA polymerase α-

alone is sufficient to transform a cell.

primase, a domain that controls the host range of the virus, a

The oncoproteins of these small DNA viruses are excel-

domain that has DNA helicase activity, and domains that bind

lent examples of how a viral protein may be multifunctional

cellular proteins that play regulatory roles in cell cycling,

and mediate a variety of functions important for the outcome

among others.

of infection.

A. SV40 (5243 bp)

Ori

VP3

VP2

Late Transcripts

VP1

DNA

NTR

Small t

Regulatory

Early Transcripts

Region

Large T-antigen

Scale (bp)

1000

2000

3000

4000

5000

B. Mouse Polyoma (5297 bp)

Ori

Large T-antigen

Middle

T antigen

Early Transcripts

Enhancer

Small t

NTR

DNA

VP1

Late Transcripts

VP2

VP3

0 Scale (bp)

5000

400

3000

2000

1000

P Early gene promoter

Ori

ORF 1

ORF 4

Initation codon

Origin of replication

P Late gene promoter

Termination codon

ORF 2

ORF 5

NTR (regulatory

region or enhancer)

FIGURE 7.26 Comparisons of the genome organizations of two polyomaviruses: SV40 and mouse polyoma. Both are

circular genomes which have been linearized at the origin of replication (Ori) for ease of presentation. The genome of

mouse polyoma virus is shown here in the opposite sense (right to left) to that of SV40. Ag is the agnoprotein. Redrawn

after Brady and Salzman (1986).

DNA Replication

The Late Genes

Large T antigen binds to specific sites within the Ori

Large T antigen also regulates the transcription of late

to promote replication of the viral genome. Binding first

mRNAs, which are transcribed from the opposite strand as the

unwinds the DNA. Then T antigen associates with repli-

early mRNAs (Fig. 7.26). Differential splicing leads to two

cation protein A followed by DNA polymerase α-primase

mRNAs in SV40 and three mRNAs in polyoma. One mRNA

to form an initiation complex. Association with primase is

is translated into VP1, the major virion structural protein. In

species specific. As a result, SV40 productively infects only

SV40 the second mRNA is translated into both VP2 and VP3,

monkey cells and mouse polyomavirus infects only mouse

whereas in polyoma virus VP2 and VP3 are translated from

cells. After initiation by primase, DNA polymerase takes

different mRNAs. VP3 is a truncated form of VP2, consisting

over and replication proceeds. DNA synthesis is bidirec-

of the C-terminal 60% or so of VP2. Both are minor com-

tional and when the replication forks meet about halfway

ponents of the virion. VP2 is myristylated and may serve an

round the molecule, the daughter genomes separate, aided

entry function. The three structural proteins are transported to

by topoisomerase II (Fig. 1.9A).

the nucleus and assembly of virions takes place there.

Large T Protein

SST

COOH

120 124

677

679

123

100

200

300

400

500

600

700

Amino acids

ATP

Polα

Ori binding

binding

Functional

ATPase

domains of

p53 binding, Polα binding

large T

DNA helicase activity

Phosphorylation

by unknown kinases

ATP

Polα

binding

Ori binding

Activities of

ATPase

phosphorylated

p53 binding, Polα binding

large T

DNA helicase activity

Dephosphorylation by PP2A

ATP

Polα

binding

Ori binding

Activities of

ATPase

dephosphorylated

p53 binding, Polα binding

large T

DNA helicase activity

Initiation of viral DNA Synthesis

Activity enhanced

Host range helper function

Nuclear localization signal

Activity reduced

Zn finger

Phosphate group

domain

Activity unchanged

FIGURE 7.27 Functional domains of SV40 large T antigen. The top panel illustrates the location of various functional

domains and of the serine and threonine residues that are phosphorylated. The second panel shows the functions of fully

phosphorylated large T antigen. The bottom panel shows the activities of large T singly phosphorylated on threonine 124,

which can bind to the origin of replication to initiate DNA synthesis. In the middle and lower panels, blue domains are

unchanged in activity, gray domains are reduced in activity, and red functions are strongly increased. Adapted from Berg

and Singer (1997), Figures 1.23 and 1.24 and Fields et al. (1996), Figure 6 on p. 2011.

Cell Membrane

P S

c-src protein

P S

SH2

Middle T antigen

P13K protein

Association of middle T with c-src and

activation of c-src tyrosine kinase

Cell Membrane

P S

AT P

Middle T antigen

SH2

P13K protein

Phosphorylation of middle T by c-src on Y

Cell Membrane

P S

SH2

Middle T antigen

P13K protein

Transformation

FIGURE 7.28 Interactions of polyoma middle T antigen with membrane proteins to initiate cellular transformation.

Phosphatidylinositol-3-kinase (Pl3K) and c-src are localized in the plasma membrane. The src kinase phosphorylates

middle T on a tyrosine residue (Y). This generates binding sites for a variety of other cellular signaling proteins such as

P13K. The resultant complex closely resembles an activated growth factor receptor bound to signal transduction proteins.

Serine residues (S) on middle T that are phosphorylated by other protein kinases are also indicated. Drawn from data in

DiMaio et al. (1998).

VP1, when expressed alone, assembles into virus-like

Polyomavirus Infection of Humans

particles. If VP2 and VP3 are coexpressed with VP1, they

The two well-known human polyomaviruses are BK

assemble into virus-like particles whose composition is the

virus and JC virus. These viruses were first isolated in

same as that of the virion.

1971, JC from the brain of a patient with progressive

Release of virions is an active process. Membrane vesicles

multifocal leukoencephalopathy (PML) and BK from the

form and transport virions to the cell surface, where they are

urine of an immunosuppressed renal transplant patient.

released. Infection by polyomaviruses is lytic. Expression of

They were named after the initials of the patients from

the late genes and assembly of progeny virions results in the

whom they were isolated. These two viruses share 75%

death of the cell.

TABLE 7.13 Virus-Coded Proteins of Primate Polyomaviruses

Number of amino acids

% Amino acid identity

Protein

SV40

JC/BK

JC/SV40

BK/SV40

Function

Late proteins

VP1

356

362

364

77.9

76.4

82.4

Major capsid protein, attaches to cellular receptors,

hemagglutination, HI and NT epitopes

VP2

344

351

352

78.8

73.4

78.5

Minor capsid protein

VP3

225

232

234

74.5

67.2

73.6

Minor capsid protein

Agnoprotein

59.1

45.0

53.2

Facilitates capsid assembly

Early proteins

Large T

688

695

708

86.6

72.0

73.9

Initiation of replication, stimulates host DNA

synthesis; modulates transcription,

transformation

Small T

172

174

79.6

67.8

69.5

Necessary for efficient viral DNA replication

Source: Adapted from Fields et al. (1996), p. 2030 and additional data from Walker and Frisque (1986).

nucleotide sequence identity. A comparison of the proteins

kidneys, in B lymphocytes, and, for JC virus, perhaps in the

encoded by JC, BK, and SV40 viruses is given in Table

brain. Reactivation may be brought about by immunosup-

7.13. Shown are the number of amino acids in each protein

pression or by factors such as pregnancy or diabetes, and

and the amino acid identity between any two of these

results in the excretion of virus in the urine. Reactivation

viruses. This table makes obvious the close relationship

of JC virus resulting from immune suppression is serious

among these viruses.

because it can replicate in oligodendrocytes, which produce

Most humans in the United States are infected with BK

myelin in the brain, and the death of these cells results in

virus before the age of 10 (Fig. 7.29). Infection with JC

PML. PML is a rare, subacute, demyelinating disease of the

virus usually occurs somewhat later, but by the age of 14 the

central nervous system that has a worldwide distribution. It

majority of the population has been infected. Primary infec-

is an infrequent complication of a wide variety of conditions,

tion with BK virus has been associated with mild respiratory

including Hodgkin's disease, chronic diseases such as tuber-

disease or cystitis (bladder infection) in young children, but

culosis, primary acquired immunodeficiency diseases such

most infections with either BK or JC virus are not associ-

as AIDS, or immunosuppression following organ transplant.

ated with illness. The viruses establish a latent infection that

The frequency of PML, once considered a rare disease, has

persists indefinitely, and the virus may reactivate after many

increased with the AIDS epidemic and PML is now recog-

years. The latent infection appears to be established in the

nized as one of the AIDS-defining illnesses--it occurs in

about 5% of all AIDS patients. PML may be more likely to

occur when immunosuppression is due to infection by HIV

because HIV-1 transactivates the JC late promoter. The dis-

100

BK virus

ease progresses rapidly and can lead to mental deterioration

and death within 36 months after onset.

Reactivation of BK virus can cause kidney disease. Such

reactivation as a result of immune suppression accompany-

JC virus

ing kidney transplantation is a leading cause of allograft

failure.

Polyomaviruses cause tumors in laboratory animals and

many attempts to associate BK or JC virus with human

cancer have been made. Association of these viruses with

many human malignancies has been reported, but proof that

>50

they cause these malignancies is still lacking. Both viruses

persistently infect most humans, and the presence of one

Age in years

of these viruses in association with a tumor cell does not

FIGURE 7.29 Prevalence of antibodies to BK and JC viruses in humans

prove causality, as the tumor cell could provide a suitable

in the United States as a function of age. From Fields et al. (1996), p. 2039.

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