C H A P T E R

36
Family Retroviridae

O U T L I N E

Virion Structure 289 Transcription of Retroviral mRNA 294

Genome Organization 289 Structural Proteins 295
Overview of the Retroviral Replication Cycle 292 Assembly, Release, and Maturation 295
The Process of Reverse Transcription 292 Mechanisms of Retroviral Oncogenesis 296
Integration 294

After reading this chapter, you should be able to insert or integrate their genomes into the host cell
answer the following questions: chromosome, an activity that is inherently mutagenic
(Fig. 36.2). But as we see in this chapter, the retro-
• What are the major steps in a retrovirus replication
viruses are much more than just cancer agents. About
cycle?
8% of the human genome is comprised of retroviral
• Which step in the retrovirus replication cycle is
sequences; retroviruses have shaped our genomes and
unique to this virus family?
driven our evolution. The human immunodeficiency
• What are the enzymatic activities of reverse
virus (HIV), discovered only a few decades ago, infects
transcriptase, RNaseH and integrase?
millions of people worldwide. It causes disease primar-
• What is the function of retroviral protease (PR) and
ily by damaging of a subset of T-lymphocytes that are
when is it active?
central regulators of the immune response. Modified
• Explain the difference between exogenous and
retroviruses are commonly used for gene delivery,
endogenous retroviruses.
genetic engineering, and gene therapy; they are often
• What is insertional activation and how does it lead
used as tools to alter animal genomes. Clearly there is a
to cell transformation?
lot to know about these unique viruses!
• How are retroviral oncogenes and cellular proto-
All of the viruses discussed to this point in this text-
oncogenes related?
book are transmitted via infectious particles (virions).
Retroviruses are enveloped viruses with RNA gen- However retroviruses can be transmitted either as
omes (Fig. 36.1). The name retrovirus was coined infectious particles or as heritable genes. While some
when it was discovered that the RNA genome is a viruses occasionally insert their genomes into host
template for synthesis of double-stranded (ds) DNA. DNA, this is usually a “mistake” that does not benefit
This process was counter to the prevailing dogma that the virus. In contrast, retroviruses must insert their
DNA was a template for RNA synthesis (the process genomes into host DNA, in order to complete a single
of transcription), never the reverse. An older name for replication cycle (Box 36.1).
the group of viruses we now call retroviruses was There is important, but sometimes confusing, termi-
“RNA tumor virus” because they were associated with nology used to describe retroviruses. Retroviruses
transmissible cancers in mammals and birds. transmitted as virions are called exogenous (think extra-
Retroviruses can transform cells because they must cellular) while retroviruses transmitted exclusively via

Viruses. DOI: http://dx.doi.org/10.1016/B978-0-12-803109-4.00036-2 287 © 2017 Elsevier Inc. All rights reserved.
288 36. FAMILY RETROVIRIDAE

FIGURE 36.1 Left: Retrovirus structure. Right: Micrograph showing both the HTLV-1 and the HIV. From CDC/Cynthia Goldsmith. CDC
Public Health Image Library, Image ID# 8241.

FIGURE 36.2 Retrovirus replication process showing steps of reverse transcription, integration, transcription, and translation of viral proteins.

the germ-line are called endogenous retroviruses. The HIV is an exogenous retrovirus; it is transmitted
many retroviral sequences that populate our genomes from person to person via infectious virions in
are endogenous retroviruses. Most are defective or blood and body fluids. But confusion often stems
fragmented but some do express proteins. Endogenous from the following: Exogenous retroviruses must
retroviruses are essentially cellular genes; they rarely, integrate their genomes into host DNA for replication.
if ever, are transmitted as virus particles. The presence We call the integrated versions of retroviral genomes
of large numbers of endogenous retroviruses in our proviruses. RNA transcribed from the provirus is
genomes is a reminder that we have coexisted with packaged into particles for transmission. In order
these viruses for hundreds of millions of years. for an exogenous retrovirus to become endogenous,
Retroviruses are one group of a larger family of repli- it must integrate into germ-line cells and become
cating molecules called retrotransposons. fixed in the host genome. The remainder of this

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 GENOME ORGANIZATION 289

BOX 36.1

GENERAL CHARACTERISTICS OF RETROVIRUSES

Packaged within retroviruses are two identical copies produced by cleavage of the ENV precursor. One is a trans-
of capped, polyadenylated, positive-strand RNA. Upon membrane (TM) glycoprotein with a cytoplasmic tail. The
penetration/uncoating, the RNA molecules are not second glycoprotein is the surface (SU) glycoprotein. SU is
translated, instead they are temples for synthesis of a the attachment protein and TM is the fusion protein. capsids
molecule of ds DNA that is integrated into the host are assembled from the capsid protein (CA). A basic, RNA-
chromosome. Transcription of mRNAs from the inte- binding nucleocapsid (NC) protein binds the genomic
grated DNA genome (called the provirus) is catalyzed RNA. A membrane-associated matrix (MA) protein associ-
by host RNA Pol II. Transcripts are capped and polyade- ates with the cytoplasmic tails of TM.
nylated and some are spliced. Upon penetration into the cell, the RNA genome
Retroviral genomes are 712 kb. All retroviral serves as a template for the synthesis of a molecule of ds
genomes encode three precursor polyproteins (GAG, DNA. This is accomplished by reverse transcriptase (RT)
GAG-POL, and ENV). Some retroviruses encode addi- a viral enzyme that is an RNA-dependent DNA poly-
tional proteins with a variety of regulatory and auxiliary merase. ds DNA is then inserted (integrated) into the
activities. cell genome by the viral integrase (IN) protein.
Virions are enveloped with icosahedral-shaped capsids
or cores. Their envelopes contain two glycoproteins

chapter deals almost exclusively with exogenous the HIV replication cycle. They are sometimes called
retroviruses. “accessory” proteins and they serve to counter host
cell defenses to retroviral infection. They will be
discussed in more detail in Chapter 37, Replication
and Pathogenesis of Human Immunodeficiency Virus
VIRION STRUCTURE (HIV).

Retrovirus particles are enveloped with icosahedral

(or icosahedral-like) cores (Fig. 36.1). Two glycoproteins
are associated with the viral envelope. A transmem- GENOME ORGANIZATION
brane (TM) glycoprotein is anchored into the viral enve-
lope and a surface (SU) glycoprotein is found on the Retroviral genomes are 50 capped and 30 polyadeny-
exterior of the virion. SU associates with TM via nonco- lated mRNAs. However, upon entry into the cell, the
valent interactions. Under the viral envelope, the matrix mRNA is not translated (in contrast to the replication
protein (MA) associates with the cytoplasmic tails of cycle of plus-strand RNA viruses). Instead the RNA
TM, as well as with the lipid envelope. Moving further genome serves as the template for the synthesis of a
into the virion, the capsid (or core) is assembled from molecule of ds DNA and the RNA genome is
capsid proteins (CA). Within the capsid are two identi- destroyed during this process.
cal copies of RNA (capped and polyadenylated) that All retroviruses have three major genes (Fig. 36.3).
serve as the retroviral genome (retroviruses therefore Gag (for group-associated antigen) encodes the GAG
have diploid genomes). The RNA is bound by the posi- polyprotein. The pol gene encodes the polymerase
tively charged (basic) nucleocapsid protein (NC). Also (POL) polyprotein and env encodes the envelope
within the capsid are a few molecules of the enzymes (ENV) polyprotein. Retroviruses encoding only gag,
reverse transcriptase (RT), integrase (IN), and protease pol, and env genes are sometimes called “simple” retro-
(PR). Two molecules of tRNA are also found within the viruses. The order of these genes is always gag-pol-env.
virion. They are associated with RT and are base paired The oncogenic retroviruses of rodents and chickens are
to the genomic RNA near its 50 end, at sequence called simple retroviruses. The so-called “complex” retro-
the primer-binding site (PBS). A molecule of tRNA is a viruses such as HIV and human T-cell leukemia virus
primer for DNA synthesis (Box 36.2). (HTLV) have additional genes encoding a variety of
Some complex retroviruses, such as HIV, package regulatory and accessory proteins (Fig. 36.4).
additional proteins within their virions. Proteins such In addition to protein-coding regions, the retroviral
as HIV-1 viral protein R (VPR), viral protein U (VPU), genome contains a number of important regulatory
and virus infectivity factor (VIF) play important roles in sequences. We will start with a discussion of the

VIRUSES
290 36. FAMILY RETROVIRIDAE

BOX 36.2

T H E F A M I LY R E T R O V I R I D A E C O N TA I N S S E V E N G E N E R A
C O N TA I N E D I N T W O S U B F A M I L I E S
Subfamily: Orthoretrovirinae Genus: Gammaretrovirus. Examples include:
Genus: Alpharetrovirus. Examples include:
Feline leukemia virus
Avian leukosis virus Murine leukemia virus
Rous sarcoma virus Gibbon ape leukemia virus
Reticuloendotheliosis virus
Genus: Betaretrovirus. Examples include:
Genus: Lentivirus. Examples include:
Mouse mammary tumor virus
Mason-Pfizer monkey virus Human immunodeficiency virus 1
Jaagsiekte sheep retrovirus Human immunodeficiency virus 2
Simian immunodeficiency virus
Genus: Deltaretrovirus. Examples include:
Equine infectious anemia virus
Bovine leukemia virus Caprine arthritis encephalitis virus
Human T-lymphotropic virus types 1, 2 Feline immunodeficiency virus
Simian T-lymphotropic virus types 1, 2, 3 Visna/maedi virus

Genus: Epsilonretrovirus. Examples include: Subfamily: Spumavirinae

Genus: Spumavirus. Examples include:
Walleye dermal sarcoma virus
Walleye epidermal hyperplasia virus 1 Simian foamy virus.

FIGURE 36.3 Genome of a simple retrovirus. Simple retroviruses have three open reading frames that encode three polyproteins.

VIRUSES
 GENOME ORGANIZATION 291

FIGURE 36.4 Comparison of retroviral genomes.

FIGURE 36.5 Comparison of RNA and DNA versions of a retroviral genome. During the process of reverse transcription the LTR is
formed when U3 and U5 sequences are duplicated.

genomic RNA (Fig. 36.5). At each end of genome are sequence called U5 (for unique at the 50 end). Just
3040 nucleotide long direct repeats (R) essential for upstream of R at the 30 end of the genome is a region
the process of reverse transcription. One copy of the R called U3 (unique at the 30 end). Two additional cis-acting
sequence is found at the very 50 end of the genome and sites are present near the 50 end of the genome. The short
the R sequence at the 30 end precedes the poly(A) tail. primer binding site (PBS) is base paired with a molecule
Directly following R at the 50 end of the genome is a short

VIRUSES
292 36. FAMILY RETROVIRIDAE

of tRNA. Just following the PBS is the RNA packaging

site or RNA encapsidation signal (also called ψ).
The product of reverse transcription, the DNA ver-
sion of the genome, is longer than the RNA genome
(Fig. 36.5). This occurs during the process of reverse
transcription (described later) when the U5 sequence is
copied to the 30 end of the genome and the U3
sequence is copied to the 50 end of the genome. This
process forms the long terminal repeats (LTRs) that
flank the protein-coding sequences. LTRs are orga-
nized as follows: U3-R-U5. LTRs are critical elements
for transcription and polyadenylation of retroviral
mRNA. The promoter and enhancer elements are often
found in U3 and the upstream LTR the promoter for
transcription of viral mRNA. A poly(A) addition site is FIGURE 36.6 Molecular structure of RT. Cocrystal structure of
present at the R-U5 junction of each LTR, but polyade- HIV-1 RT and an RNA/DNA substrate. The polymerase and RNase
nylation is suppressed at the upstream LTR. H domain of p66 are shown in blue and red. The connection domain
is yellow. p51 is shown in gray. The RNA template and DNA primer
strands are shown in green and purple respectively. From Schultz, S.
J., Champoux, J.J., 2008. RNase H activity: structure, specificity, and func-
tion in reverse transcription. Virus Res. 134, 86103.
OVERVIEW OF THE RETROVIRAL
REPLICATION CYCLE
The steps up to and including integration are some-
The retroviral replication cycle begins with binding times called the early steps in the retroviral replication
of SU to the receptor. Some retroviruses enter cells by cycle, while those that occur after integration are called
fusion at the plasma membrane (for example, HIV-1) the late steps. In some cases integration may occur
but others are endocytosed and the trigger for fusion is months to years before virions are ever produced, as
low pH. In either case, the hydrophobic amino termi- expression of the provirus may require a specific set of
nus of the TM protein mediates membrane fusion. circumstances. In the case of HIV, the infection of a
When the retroviral core is released into the cytosol, T-cell may be silent until the cell is stimulated to
viral RT synthesizes a DNA copy of the RNA genome. divide upon recognizing a foreign antigen.
The ds DNA molecule is then integrated into the host Some key steps in the retroviral replication cycle are
chromosome. The DNA genomes of some retroviruses now described in greater detail.
(members of the genus Lentivirus) actively cross
nuclear pores to gain access to the chromosome, but
many retroviruses require a dividing cell, as their THE PROCESS OF REVERSE
DNA genomes can only reach the host chromosome TRANSCRIPTION
during mitosis (when the nuclear membrane breaks
down). The next key player is the retrovirus replication The process of reverse transcription is carried out
cycle is IN. Recall that IN is present in the infecting by a polymerase called RT. RT is a heterodimer. The
virion. Dimers of IN interact with each end of the newly longer polypeptide in the dimer contains the polymeri-
synthesized ds DNA genome. IN also interacts with zation domain, a short linker domain, and an RNaseH
host DNA and catalyzes cleavage and ligation steps that domain. The shorter polypeptide in the dimer contains
result in the covalent linkage of the viral DNA to the only the polymerization domain. The longer polypep-
host DNA. The viral DNA is now, in essence, a cellular tide provides the enzymatic functions of RT while the
gene. Once integrated, the retroviral genome is referred shorter polypeptide appears to play a strictly structural
to as the provirus. In order for the retroviral replication role. The structure of HIV-1 RT is shown in Fig. 36.6.
cycle to continue, the provirus must be transcribed by HIV p66 folds to form the typical palm, fingers, and
cellular RNA polymerase II to produce capped, polyade- thumb domains of polymerases. The catalytic site is
nylated mRNA. Spliced and unspliced viral mRNAs are located in the palm and contains three critical aspartic
transported out of the nucleus and are translated to acid residues and two coordinated Mg 21 ions.
produce a full complement of retroviral proteins. As the Reverse transcription is key to the replication cycle
concentration of structural proteins increases, unspliced of a retrovirus. As described earlier, retroviral genomes
mRNAs are packaged to serve as the genomes of prog- are 50 capped and 30 polyadenylated mRNAs. The RNA
eny virions. genome serves as the template for the synthesis of a

VIRUSES
 THE PROCESS OF REVERSE TRANSCRIPTION 293
molecule of ds DNA. Although two copies of RNA enter RNase H domain of RT. Note that the RNA:RNA
the cell, only one copy of ds DNA is synthesized. duplex formed by the tRNA primer and the PBS is not
The steps in reverse transcription are outlined in digested as RNase H only degrades RNA that is part
Fig. 36.7. Note that in this model only one copy of RNA of an RNA:DNA hybrid. The short DNA product is
is shown. However there is good evidence that during called minus-strand strong-stop DNA.
the process of reverse transcription, RT may jump from The next step in the process is sometimes called a
one of the packaged RNA strands to another. “jump” or a “strand transfer.” In the infected cell this
Reverse transcription begins with synthesis of a step probably requires the NC protein (although this is
piece of DNA from the tRNA primer. The first strand not shown in Fig. 36.7). Key to the jump is that the
of DNA synthesized is called “negative strand” newly synthesized R DNA base pairs with the R RNA
because it is a copy of the mRNA (the positive strand). at the 30 end of the genome. The negative strand of
The first piece of DNA synthesized is rather short DNA is then synthesized through to the 50 end of the
because RT reaches the 50 end of the mRNA and can RNA. Not shown in Fig. 36.7 is that most of the RNA
go no further. The RNA strand of this RNA:DNA template is degraded during the process of DNA syn-
hybrid molecule is digested away by the activity of the thesis. One piece of RNA remains undigested

FIGURE 36.7 Steps in the reverse transcription process.

VIRUSES
294 36. FAMILY RETROVIRIDAE

however: the so-called polypurine tract (PPT). The PPT from the 30 ends of the linear, unintegrated ds DNA
is the RNA primer for synthesis of plus-strand DNA molecule. Second, it is an endonuclease that makes a
(Fig. 36.7, step 7). As we saw with minus-strand DNA staggered cut in host cell DNA. Third it has ligase
synthesis, the first product of plus-strand DNA synthe- activity to join viral DNA to host cell DNA (ligation).
sis is a short fragment. As shown in step 8, part of the Successful integration also requires host cell enzymes
tRNA is copied, generating the PBS. Now the tRNA is to repair gaps at the ends of the newly integrated mol-
digested away by RNase H and a second jump occurs: ecule. The sites in the chromosome that serve as tar-
The PBS region on the plus strand is base paired to its gets for integration are generally considered to be
complement on the minus strand. The ds DNA mole- random. However some types of retroviruses preferen-
cule is completed by extension of the two partial DNA tially integrate in region of active gene transcription,
strands to form the completed linear ds DNA. As we while others do not.
see later, the LTRs generated during reverse transcrip- Integration can be considered the last step in the
tion are key regulatory regions of the provirus. It is early phase of the retrovirus replication cycle. The viral
important to note that RT is not the only viral player genome is now a host cell gene that can be passed to
in the RT process. NC and other virion proteins (CA daughter cells. The provirus may remain silent for
and IN) remain associated with the DNA as it is syn- some time, but successful completion of the replication
thesized, forming the so-called preintegration complex cycle requires that it eventually be transcribed.
(PIC). The PIC traffics to the chromosome. Many retro-
viruses replicate only in mitotically active cells as they
require breakdown of the nuclear membrane to reach TRANSCRIPTION OF RETROVIRAL MRNA
the host chromosome. However lentiviruses, such as
HIV, can replicate in nondividing cells because the PIC The retroviral LTR controls transcription of the pro-
actively crosses the nuclear pore. virus. U3 contains a core promoter as well as enhancer
elements. Transcripts are synthesized by the host RNA
Pol II complex. The efficiency of transcription is regu-
lated by sequences in the LTR. Some retroviral promo-
INTEGRATION ters are active in many cell types while other
promoters are only active in specific cells at specific
IN is bound to the very ends of the linear ds DNA times. An instructive example is the retrovirus mouse
molecule (Fig. 36.8). IN polypeptides interact to form mammary tumor virus (MMTV). The MMTV promoter
dimers and tetramers, bringing the two ends of the lin- is active only in the mammary glands of a lactating
ear provirus together. IN carries out three enzymatic mouse. Virus is produced during lactation, when it
activities: First it is an exonuclease that cleaves 24 nt can be successfully transmitted to the suckling mouse
pups. Some retroviral promoters, for example HIV, are
relatively weak and require virally encoded transcrip-
tion factors to increase transcription to sufficient levels
for successful completion of the retrovirus replication
cycle [Chapter 37: Replication and Pathogenesis of
Human Immunodeficiency Virus (HIV)]. Oncogenic
retroviruses often have strong promoters that drive
transcription of not only the provirus, but of nearby
cellular genes, a process called insertional activation.
All retroviral transcripts begin at the R region of the
upstream LTR and are cleaved and polyadenylated at
a site in the downstream LTR (Fig. 36.5). (Recall that
the R sequences are found at the 50 and 30 ends of the
RNA genome.) However before any RNA is packaged,
structural proteins must be synthesized. The GAG
polyprotein is translated from unspliced mRNA
(Fig. 36.3). The pol gene products are also synthesized
FIGURE 36.8 Integration. The ends of linear retroviral DNA are
from the unspliced mRNA, but this requires either a
associated with IN protein. IN makes a staggered cut in target chro- ribosomal frame shift or suppression of a stop codon
mosomal DNA and ligates each strand. DNA repair (to fill gaps) is to produce the GAG-POL polyprotein.
performed by host enzymes. This process generates short direct The ENV precursor is synthesized from a spliced
repeats at each end of the insertion. mRNA. (A mixture of spliced and unspliced mRNAs is

VIRUSES
 ASSEMBLY, RELEASE, AND MATURATION 295
needed to generate the full complement of retroviral pro- allows specific incorporation of genomes into budding
teins.) ENV is translated on rough ER and the precursor particles. GAG and GAG-POL polyproteins associate
is processed in the ER and Golgi. Processing includes with a cell membrane (the PM in the case of HIV).
cleavage to produce SU and TM, as well as glycosylation. That interaction is facilitated by a fatty acid molecule
Complex retroviruses also encode accessory and that is linked to the 50 end of the MA domain of the
regulatory proteins from a variety of singly and multi- GAG and GAG-POL polyproteins. These polyproteins
ply spliced mRNAs (see Fig. 37.3). In the case of HIV also contain interaction domains that allow them to
and other lentiviruses two key regulatory proteins are specifically interact with the cytoplasmic tail of TM
TAT and REV. TAT is a small protein that increases (Fig. 36.9).
transcription from the HIV LTR. REV is a small protein Mutational analysis of retroviral gag genes has
that regulates transport of spliced and unspliced revealed the presence of short amino acid motifs that
mRNAs from the nucleus. These two proteins and promote the release of the budding particles from
their activities will be described in more detail in the PM. These motifs are called “late domains”
Chapter 37, Replication and Pathogenesis of Human because they are required in the very last steps of
Immunodeficiency Virus (HIV). release from the cell. Three types of retroviral late
domains have been identified: Pro-Thr/Ser-Ala-Pro
(PTAP or PSAP), Pro-Pro-Pro-Tyr (PPPY), and Tyr-
STRUCTURAL PROTEINS Pro-Xn-Leu (YPXnL, where X is any amino acid and
n 5 13 residues). The motifs are found in various
The full-length (unspliced) viral transcript is trans- places in GAG: The HIV late domain is in p6 (near
ported to the cytoplasm where it is translated to pro- the carboxyl terminus of GAG) while the HTLV-1
duce two precursor polyproteins, GAG and GAG-POL. late domain is in MA. Mutation of these motifs dis-
GAG contains the domains for MA-CA-NC while the rupts virion budding and release. Retroviral late
GAG-POL polyprotein also contains the RT and IN domains function by interacting with components of
domains. As show in Fig. 36.4, the PR domain is some- the cellular endosomal sorting machinery. The so-
times part of the GAG precursor and sometimes part called ESCRT complexes (endosomal sorting com-
of GAG-POL. Thus retroviruses have evolved a variety plexes required for transport) are multiprotein com-
of strategies for producing PR. The GAG and GAP- plexes that sort cellular proteins into vesicles; in
POL precursors remain uncleaved within the infected addition to their “sorting” function they promote
cell. As we see later, PR is not active until the budding budding and membrane scission. There are four dif-
process is complete. ferent ESCRT complexes that function together in
The ENV polyprotein is translated from a singly cells; only a subset of ESCRT machinery is required
spliced mRNA that lacks gag and pol sequences. The for retrovirus budding.
ENV polyprotein is synthesized on rough ER. Within HIV is an example of a retrovirus that buds from
the ER lumen, ENV is cleaved (by furin-like proteases) the plasma membrane. During budding, GAG and
and the cleavage products are glycosylated as they GAG-POL polyproteins become densely packed,
travel through the ER and Golgi. For many retro- triggering activation of the PR domain. PR cleaves
viruses, the final destination of the ENV glycoproteins the precursor proteins into their final products. MA
is the plasma membrane. Complex retroviruses like remains associated with the inside of the viral
HIV use multiply spliced mRNAs to produce addi- envelope, CA rearranges and assembles to form a
tional accessory proteins. Some of these are nonstruc- capsid that surrounds the genome, NC, RT, and IN.
tural proteins while others are packaged into new The virion is now ready to infect a new cell
virions to exert their effects during the next cycle of (Fig. 36.9). During an HIV infection, the virus infects
replication (Chapter 37: Replication and Pathogenesis several types of cell that are key players in the
of Human Immunodeficiency Virus (HIV)). immune response. Disruption of the activities of
these cells leads to immune suppression and
disease. These processes will be discussed in more
ASSEMBLY, RELEASE, AND MATURATION detail in Chapter 37, Replication and Pathogenesis of
Human Immunodeficiency Virus (HIV). The remain-
In the cytosol the RT domain of the GAG-POL pre- der of this chapter will focus on retroviral
cursor binds the tRNA that will be used to prime oncogenesis.
DNA synthesis in the next cell. Full-length retroviral
mRNAs have a “packaging signal” near the 50 end that

VIRUSES
296 36. FAMILY RETROVIRIDAE

FIGURE 36.9 Assembly of a retrovirus. This figure illustrates assembly of HIV-1 at the plasma membrane of the infected cell. The imma-
ture virion assembles from polyprotein precursors that are cleaved to form infectious virions after release from the cell.

MECHANISMS OF RETROVIRAL and kinases that activate signaling pathways to trans-

ONCOGENESIS mit messages to the cell nucleus.
Insertion of a provirus within a proto-oncogene may
Retroviruses must integrate (insert) their genomes result in production of an aberrant protein: perhaps
into host DNA for the purposes of replication, and dis- one with unregulated growth-promoting potential. But
ruption of host DNA is an inherently mutagenic event many insertions are near, not within, a proto-
(Box 36.3). Early studies of avian retroviruses revealed oncogene. These types of insertions may increase the
different mechanisms of transformation: Some retro- amount of normal protein product. The expression of
viruses (for example, Rous sarcoma virus (RSV)) are growth-promoting proteins is tightly regulated and
acutely transforming and rapidly give rise to polyclonal insertion of a provirus, with its own promoter,
tumors (each tumor is derived from a different enhancer, splicing, and polyadenylation signals can
infected cell). These so-called “acute” transforming ret- disrupt that regulation. The overexpression of a
roviruses carry an oncogene into every infected cell growth-promoting protein is often the first step in cell
(Fig. 36.10). In fact, these retroviral oncogenes were transformation. Retroviral insertions may also promote
originally “captured” from cells. cell transformation by inactivating a growth-suppressing
Other oncogenic retroviruses are less “potent”: gene. However, this is much less common, as the inac-
Tumors are slower to develop and those that do are tivation of one allele leaves a second, intact allele to
usually monoclonal. These tumors are caused by a pro- produce an active protein product. Finally, it is impor-
cess called insertional activation or mutagenesis tant to understand that cell transformation is a readily
(Fig. 36.10). Many cells are infected, but only rarely observable event: most insertional events are silent.
does chromosomal insertion provide a trigger for cell Jaagsiekte sheep retrovirus (JSRV) is an acutely
transformation. Studying tumors caused by insertional transforming betaretrovirus that causes a contagious
activation led to the discovery of dozens of “growth- lung cancer in sheep called ovine pulmonary adeno-
promoting” cellular genes (so-called proto-oncogenes). carcinoma. JSRV brings an oncogene into every
These genes fall into two broad classes: Cell receptors infected cell. However the JSRV “oncogene” is not a
that respond to extracellular signals to promote growth captured cellular gene, it is the viral TM protein. JSRV

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 MECHANISMS OF RETROVIRAL ONCOGENESIS 297

B O X 3 6 . 3 A B R I E F H I S T O RY O F R E T R O V I R A L O N C O G E N E S I S
Studies of oncogenic retroviruses date back to the Nobel Prize in Physiology or Medicine jointly to David
early 1900s with descriptions of “filterable agents” capa- Baltimore, Renato Dulbecco,3 and Howard Temin for
ble of causing avian leucosis and avian sarcoma. Over their discoveries concerning interactions between cells
the next 20 years, many distinct avian cancers were and tumor viruses. In 1980, the first human oncogenic
attributed to transmissible agents (viruses). By the 1950s, retrovirus (HTLV-1) was described.4 HTLV-1 was dis-
a group of related, tumor-causing RNA viruses had covered in the laboratory of Robert Gallo at the US
been identified from birds, rodents, and other mammals. National Cancer Institute. Also in the early 1980s a retro-
They shared biochemical features and were morphologi- viral etiology was ascribed to lung tumors in sheep.5
cally similar, but displayed different phenotypes as There are currently seven genera in the family
regarded host specificity, tumor types, and transforming Retroviridae, five of which contain oncogenic
ability. For many years, these viruses were collectively retroviruses.
called “RNA tumor viruses.” In general they caused
1. Temin, H.M., Mizutani, S., 1970. RNA-dependent
noncytopathic infections, required dividing cells for
DNA polymerase in virions of Rous sarcoma virus.
establishment of productive infection, and were tumori-
Nature 226, 12111213.
genic; once tumor cells were formed, the transformed
2. Baltimore, D. 1970. RNA-dependent DNA
phenotype was stably inherited by daughter cells.
polymerase in virions of RNA tumour viruses.
In the 1960s, Howard Temin (working at the
Nature 226, 12091211.
McArdle Laboratory for Cancer Research at the
3. In 1975 the Karolinska Institute awarded the Nobel
University of Wisconsin-Madison) began to suspect that
Prize in Physiology or Medicine jointly to David
after infection, the genetic information of RNA tumor
Baltimore, Renato Dulbecco, and Howard Temin for
viruses became stably associated with the cell genome.
their discoveries concerning interactions between
Temin thought that process might be similar to DNA
tumor viruses and cellular DNA. Dulbecco studied
bacteriophage such as lambda phage, known to integrate
oncogenesis of the DNA virus SV40, a polyomavirus.
into bacterial genomes. But the RNA tumor viruses had
Also in 1975, the family name Retroviridae was
RNA genomes and synthesis of DNA, from an RNA tem-
adopted at the first meeting of the International
plate was unheard of! Temin performed a variety of
Committee on the Taxonomy of Viruses.
studies to support his idea, but the breakthrough came
4. Poiesz, B.J., Ruscetti, F.W., Gazdar, A.F., Bunn, P.A.,
when he demonstrated that RNA tumor viruses package
Minna, J.D., Gallo, R.C., 1980. Detection and isolation
a DNA polymerase. Temin published this seminal work
of type C retrovirus particles from fresh and cultured
in 1970,1 side-by-side with a paper from David
lymphocytes of a patient with cutaneous T-cell
Baltimore.2 Thus in 1970, two laboratories independently
lymphoma. Proc. Natl. Acad. Sci. USA 1980, 77,
and convincingly demonstrated that RNA tumor viruses
74157419.
package an enzymatically active DNA polymerase.
5. Safran, N., Zimber, A., Irving, S.G., Perk, K., 1985.
Discovery of the polymerase we now call reverse tran-
Transforming potential of a retrovirus isolated from
scriptase (RT) revolutionized the study of RNA tumor
lung carcinoma of sheep. Int. J. Cancer 35, 499504.
viruses. In 1975 the Karolinska Institute awarded the

TM directly activates cell-signaling cascades to pro- cats do not transmit virus, nor do they develop FeLV-
mote cellular proliferation; additional mutations in associated tumors. Infection of kittens often has a very
proliferating cells lead to malignant transformation. different outcome. Kittens fail to control virus replication
Experimental infection of day-old lambs with JSRV and become persistently infected (PI) cats. They shed
induces tumor formation within 36 weeks. virus and most will eventually become sick. Some will
Feline leukemia virus (FeLV) (genus Gammaretrovirus) develop tumors, initiated by insertional mutagenesis.
causes a variety of diseases in infected cats, among However the most interesting feature of PI cats (from
them, tumors induced by insertional mutagenesis. The the perspective of viral pathogenesis) is the emergence
pathogenesis of FeLV is complex because the virus of mutated viruses with altered cell tropism. These var-
mutates during infection and can recombine with endogenous iants arise by point mutations in SU and/or by recombi-
FeLVs to generate variants with different cell tropisms. nation of the env gene of exogenous FeLV with env
FeLV infection of adult cats is well controlled by the genes of endogenous copies of FeLV-like viruses. One
immune system (Fig. 36.11) and very little, if any, repli- variety of mutated FeLV is called the T subtype. FeLV-T
cating virus is detected after the acute infection; these is tropic for, and causes cytopathic infection of T cells,

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298 36. FAMILY RETROVIRIDAE

FIGURE 36.10 Two methods of retroviral oncogenesis. On the left, a chicken is infected with an acutely transforming retrovirus (for exam-
ple, RSV). The viral genome encodes an oncogene that is delivered to every infected cell. Many cells become transformed to generate tumors.
The tumors in the chicken on the left are polyclonal (each tumor originated from a different cell). The chicken on the right is infected with a
retrovirus that has the potential to insertionally activate a chicken proto-oncogene. During infection the retrovirus inserts into chicken chromo-
somal DNA. In rare cases, insertion will activate a growth-promoting gene and the cell may be transformed. There are a variety of mechan-
isms by which a proto-oncogene can be activated. Often the gene is overexpressed by the strong retroviral promoter. The tumors in the
chicken on the right at monoclonal. One cell was initially transformed.

leading to immunosuppression. Another variety, FeLV- that modify cell transcription and signaling pathways to
C, causes fatal anemia. FeLV-T and C are rarely trans- promote cell growth. HTLV-1 is an oncogenic human ret-
mitted, but instead, arise from virus mutations within rovirus. HTLVs cause life-long infections that are largely
PI cats. asymptomatic but can increase risk of developing leuke-
Some retroviruses that cause cancers do so by a mia. Oncogenesis of HTLV-1 is thought to require two
completely different mechanism. They encode proteins small viral proteins, TAX and HTLV-1 bZIP (HBZ)

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 MECHANISMS OF RETROVIRAL ONCOGENESIS 299

FIGURE 36.11 Pathogenesis of FeLV is complex. Infection of an adult cat often results in development of protective immunity. The cat is
antibody positive but not viremic (viral antigen cannot be detected in the blood). If a kitten is infected with FeLV, it may not develop protec-
tive immunity. Instead it becomes persistently infected. The PI kitten or cat sheds virus and is identified by the presence of viral antigen in
the blood. The antigen positive cat is a source of infectious virus and may transmit FeLV to other cats. The antigen positive cat may develop
any number of diseases including cancers or acute (fatal) anemia. The viruses found in the sick cat often contain mutations (specific mutations
are linked to different disease manifestations).

(Fig. 36.4). Both are multifunctional and interact with a Walleye dermal sarcoma virus (WDSV) is a member
variety of host cell proteins. TAX is thought to be a of the genus Epsionretrovirus. Infection is associated
key player in tumor initiation but by the time cells are with proliferative lesions in walleye, a freshwater fish
transformed, TAX is silenced, and HBZ is expressed (see native to Canada and the Northern United States. In
Box 36.4). infected fish, proliferative lesions are seasonal. The

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300 36. FAMILY RETROVIRIDAE

BOX 36.4

H U M A N T- C E L L L E U K E M I A V I R U S - 1
Oncogenic animal and bird retroviruses were discov- transcription. The mechanism of transcription activation
ered and characterized several decades before the dis- seems to involve changes to chromatin structure.
covery of HTLV-1 in 1980. HTLV-1 and its close relative HTLV-1 bZIP factor (HBZ) is also encoded in the X
HTLV-2 are complex retroviruses in the genus region but is transcribed in the opposite direction to all
Deltaretrovirus. other transcripts. The promoter for HBZ transcription is
HTLV-1 is endemic in parts of Japan, the Caribbean the 30 LTR. HBZ is a Basic Leucine Zipper Domain
Islands, Africa, and South America. Mechanisms of virus (bZIP) protein that interacts with many cellular proteins
transmission include sexual contact, breast-feeding, blood including several important transcription factors. HBZ
transfusion, and contaminated needles. It is estimated activities are complex: (1) HBZ is required for efficient
that 1020 million people worldwide are infected. Most viral infectivity and persistence in a rabbit model; (2)
infections are asymptomatic; however, 3%5% of infected HBZ is dispensable for HTLV-1-mediated cellular trans-
persons develop HTLV-1-associated T-cell tumors. A formation in cultured cells but (3) HBZ by itself pro-
smaller percentage of infected individuals develop a motes proliferation of T-cell lines; (4) HBZ suppresses
demyelinating disease called HTLV-1-associated myelop- TAX-mediated viral gene transcription.
athy (HAM) or tropical spastic paraparesis (TSP). Adult T-Cell Leukemia
In vivo HTLV-1 infects primarily T-lymphocytes but Adult T-cell leukemia (ATL) is a very aggressive
in vitro tropism is broader and the virus infects a variety T-cell cancer that may arise decades after infection with
of other cell types including B-lymphocytes, monocytes, HTLV-1. The leukemic cells of ATL are monoclonal and
endothelial cells, and fibroblasts. HTLV-1 is a unique harbor HTLV-1 proviral DNA at random chromosomal
retrovirus in several respects. First, HTLV-1 is a highly integration sites. This suggests an important role for
cell-associated virus. Whether in vitro or in vivo, few HTLV-1 in tumor development but also suggests that
cell-free virions are released; for the most part, virus is insertional activation of a cellular proto-oncogene does
transmitted directly from cell to cell via a virological not drive cell proliferation. Models of ATL focus on the
synapse. Second, within an HTLV-1-infected individual, activities of TAX and HBZ. The process is clearly com-
there is very little virus expression. Polymerase chain plex and multistep, but some common findings are: (1)
reaction (PCR) is required to detect very low levels of ATL cells are genetically unstable and TAX promotes
viral RNA; viral proteins are undetectable. (Note the chromosomal damage. This suggests an early role for
contrast with HIV, another T-cell tropic human retrovi- TAX in tumor development. (2) TAX expression is
rus.) When peripheral blood mononuclear cells from an largely silenced in ATL cells. As TAX is a target of cyto-
infected patient are cultured, levels of viral RNA and toxic T cells, lack of expression would protect proliferat-
protein increase significantly. This suggests that chroni- ing tumor cells. (3) HBZ mRNA and protein are found
cally infected patients maintain a pool of “latently” in most ATL tumors. (iv) Continued expression of HBZ
infected cells and HTLV-1 is passed to daughter cells via is necessary to maintain tumor cell proliferation.
mitosis. HTLV-1-Associated Myelopathy/Tropical Spastic
The HTLV-1 genome is shown in Fig. 36.4. Gag, pro, Paraparesis
pol, and env genes overlap and their arrangement is remi- It is estimated that between 0.25% and 3% of HTLV-
niscent of simple retroviruses. However, an open reading 1-infected persons may eventually develop HAM/TSP,
frame called X, found at the 30 end of the genome, a slowly progressive, chronic disease of the spinal cord.
encodes several additional proteins. The best studied of Spinal cord damage results from inflammatory
these are TAX and REX. TAX is a transactivating protein responses directed against infected cells. Unfortunately,
while REX increases nuclear export of unspliced and sin- nerve cells are also damaged in the process. The main
gly spliced viral mRNAs. TAX interacts with cellular pro- symptoms are painful stiffness and weakness of
teins and forms a complex on the LTR thereby increasing the legs.

highest incidence of disease occurs in the Fall/Winter believed to be responsible for tumor development.
seasons and lesions regress as water temperatures One of these is a cyclin-like protein [retrovirus (rv)-
warm up in the Spring/Summer seasons. WDSV is a cyclin] that may stimulate cell growth. The other (ORF
complex retrovirus that encodes auxiliary proteins B) protects cells from apoptotic stimuli. The specific

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 MECHANISMS OF RETROVIRAL ONCOGENESIS 301
factors involved in the seasonality of tumor formation many are defective. About 8% of the human
are unclear and this interesting retrovirus has yet to genome consists of endogenous retroviruses (or
reveal all of its secrets. their remains).
In this chapter we learned that: • Insertional activation describes the process by
which integration of a retrovirus activates a
• The major steps in the retrovirus replication cycle
cellular proto-oncogene. Proto-oncogene is a
are: attachment, penetration, reverse transcription,
general term to describe any growth-promoting
integration, transcription, translation, assembly,
gene. Retroviral integration can directly mutate a
release, and maturation.
proto-oncogene or it can increase the level of
• Integration of the retroviral genome (the ds DNA
expression of a normal protein product. Expression
version) is a unique to the family Retroviridae.
of growth-promoting genes is tightly regulated.
Reverse transcription is almost unique to this family
Insertion of a provirus may provide a strong
but the hepadnaviruses also use reverse
promoter, may provide enhancer elements, may
transcription for genome synthesis (see Chapter 38:
change splicing patters, change use of a
Family Hepadnaviridae).
polyadenylation signal or RNA stability element.
• Retroviral PR cleaves the GAG and GAG-POL
Insertional activation of a proto-oncogene is often
precursors to generate MA, CA, NC, PR, RT, and
the first step in the transformation process. Often
IN. PR is active after budding. Budded virions are
additional mutations are required to confer a fully
initially immature and become mature after PR
transformed phenotype.
cleavages are complete.
• The acutely transforming retroviruses of chickens
• Exogenous retroviruses are transmitted horizontally,
encode oncogenes. These growth-promoting genes
by infectious particles, from one animal or person to
were picked up from the host during the process of
another. Endogenous retroviruses are transmitted
reverse transcription. Thus a retroviral oncogene
through the germ-line (sperm and eggs) from parent
was originally a host proto-oncogene.
to offspring. They are seldom found as particles and

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