Prototype Foamy Virus (PFV)
Intasome
Clara Fischman '13 and Katie Adlam '13
Contents:
I.
Biological Motivation
Human
Immunodeficiency Virus (HIV), considered a pandemic by the World Health
Organization, is a retrovirus known to cause acquired immunodeficiency
syndrome
(AIDS). Retroviruses have the
disturbing
ability to stay dormant in the host cell, allowing infected individuals
to live
symptom free for prolonged periods of time. Due
the silent transmission of HIV, AIDS exploded onto the
scene from
near anonymity, and has killed over 25 million people worldwide since
its discovery
in 19811.
While
treatments such as HAART, highly active antiretroviral therapy, have
prolonged
the lifespan of infected individuals, a clear remedy for HIV has yet to
be
discovered. The mechanism by which
retroviruses
infect and propagate within cells still marks a wide area of research,
with the
hope that further understanding will yield a cure to HIV and other
viruses of
its kind.
Due
to its
availability, the prototype foamy virus (PFV) has been used as a model
of
retroviral activity. All retroviruses
are single stranded RNA viruses that contain reverse transcriptase and
integrase enzymes. Reverse transcriptase
produces viral DNA (vDNA) from an RNA genome, which is then inserted
into the
host cell chromosome by integrase (IN). The
intasome, vDNA-IN complex, engages with target chromosomal DNA (tDNA)
and participates
in strand transfer, irreversibly joining the viral and
target
cellular
DNA. Figure 2a The mechanism of retroviral
integration
1"Global
Statistics." AIDS.gov.
Globalhealthfacts.org, 20 June 2011. Web. 27 Nov. 2011.
<http://aids.gov/hiv-aids-basics/hiv-aids-101/overview/global-statistics/>.
II.
General Structure
The
PFV forms an intasome composed of a
tetrameric nucleoprotein complex of
integrase proteins around the ends
of
replica viral DNA.
The
PFV contains both alpha
helices
and
beta
sheets
.
The
interface between
symmetrical
dimers
each
comprised of four subunits,
creates the active site of DNA insertion. Upon
interaction with the target DNA, the intasome retains its shape,
forcing the
tDNA into a severely bent conformation within the cleft between the IN
dimers. This allows the IN active
sites to
access the
phosphodiester bonds necessary for vDNA insertion.
The
subunits, amino-terminal domain (NTD)
, the
NTD extension domain (NED)
,
carboxy-terminal
domain (CTD),
and
inner (CCDi)
and
outer (CCDii)
catalytic
core domains
associate
to bind the transfer
and
non-transfer
strands
of the viral DNA
and
the tDNA
to
form the tetramer
. The
inner CCD, NTD, and CTD subunits are
involved in the binding of the
viral DNA
and in tetramerization, while the outer CCD subunits only provide
support to
the intasome. In addition,
intermolecular interactions between the NTD and CCD domains stabilize
the dimer,
causing rigidity of the intasome. The
NED, smallest of the domains,
contacts
viral DNA, and is a
concerted
element in other spumaviral and potentially gammaretroviral integrases.
III.
DNA Binding
Each
of the subunits participates in
protein-protein and protein-DNA interactions. The closest protein-DNA
contacts
occur within the terminal six nucleotides of the viral DNA, where the
most
deviation from the ideal B form is seen. NTD
and NED subunits
interact with the vDNA that is located on the
opposing CCD domain. The NTD, CTD, CCD, as well as the NTD-CCD and
CCD-CTD
linkers interact with the DNA bases, while the NED interacts with the
phosphodiester backbone.
Target DNA
The
pre-catalytic target capture complex (TCC) and the post-catalytic
strand transfer complex (STC) are stabilized by
hydrogen bonds between the amide
groups of Thr163, Gln186, Ser193,
and Tyr212
and
oxygen
atoms
from the tDNA
phosphodiester
backbone
.
These
rigid bonds stabilize
the tDNA to prevent its movement in the active site of the
intasome. The target DNA
phosphodiester
backbone also has a pair of salt bridges formed by Arg362 within the
inner
chain
CTD.
Due
to IN not being site sequence
specific, interactions
between IN and target DNA bases are
rare. One
occurs with Arg329,
which forms a hydrogen bond to
guanine 3, guanine -1, and thymine -2
in the
major groove
of
the
target
DNA
.
This
intercalation of Arg329
widens the major groove, which severely bends the tDNA,
making it more accessible for viral genome invasion.
In
a second sequence specific interaction, the methyl of Ala188
within the
inner chain CCDi contacts the oxygen of
cytosine 6
in the minor groove through Van der
Waals attractions
.
Viral DNA
More
sequence specific interactions occur between IN and the viral DNA than
do between IN and the target DNA. The
carbonyl group of Gly218
forms a
hydrogen bond with the fourth guanine of the non-transferred
viral strand
. The
protein-DNA interactions continue into the CCD α4
helix and into the minor grove at the
terminal end of the viral DNA. The
side chain of Arg222
forms a
hydrogen bond with the bases of the
fifth thymine and sixth cytosine
of
the non-transferred
strand
. The
side chain of Asn106
interacts with
thymine 8 of the non-transferred
strand, which widens the
minor groove
of the
viral DNA allowing easier access to
the phosphodiester bond
.
Not
only are the subunits themselves involved in sequence specific
interactions, but the
NTD-CCD and CCD-CTD linkers also contact bases in the viral genome.
Within
the minor groove,
the side chain of Arg313
intercalates its guanidinium group, base
stacking it against adenonsine 12 from
the reactive
strand to form a hydrogen
bond with
cytosine
11
. Together,
these interactions
primarily aid in the
stabilization of the viral DNA within the protein
subunits.
The
active-site loop, which includes residues Pro214 and Gln215, separates
the viral DNA2
by a mechanism highly similar to HIV-1.
Gln215 dislodges thymine 3 of the
non-transferred strand, which causes it to turn away from the
inside of
the DNA Figure 3 Retroviral
intasome assembly .
2 For
successful crystallization of the pre-catalytic target complex (TCC), a
viral
DNA mimic lacking the reactive 3'-OH was used. Therefore, separation of
the
viral DNA strands at the catalytic site cannot be shown.
IV.
Active Site
The
active site, located deep within the dimer-dimer interface,
contains Asp128,
Asp185,
and Glu221
,
which engage with
the reactive 3’ termini of
the viral DNA2.
Two
magnesium or manganese atoms
promote catalysis in the active site3.
Metal A, coordinated by Asp128
and Asp185,
destabilizes the
phosphodiester bond in the tDNA,
while metal
B
,
coordinated by Asp128 and
Glu221,
activates the 3' hydroxyl of
the viral DNA
for strand transfer. Figure 2b The mechanism of retroviral
integration
Both ions facilitate the SN2
nucleophilic substitution reaction by
which the viral strand invades the host genome. Upon
integration of the
nucleic acids, the intasome shifts from the pre-catalytic target
capture
complex (TCC) to the post-catalytic strand transfer complex (STC),
ejecting the
newly-joined DNA from the active site, thus preventing a
reversal of the
strand transfer.
3For
successful crystallization of the pre-catalytic target complex
(TCC), a viral DNA mimic lacking the reactive 3'-OH was used with only
one Mg2+
ion. This Mg2+
corresponds to metal B in Fig. 2b.
V.
References
"Global
Statistics." AIDS.gov. Globalhealtfacts.org, 20 June 2011. Web. 27 Nov.
2011.
<https://aids.gov/hiv-aids-basics/hiv-aids-101/overview/global-statisctics/>.
Hare,
Stephen, Saumya Shree Gupta, Eugene Valkov, Alan
Engelman, and Peter Cherepanov. "Retroviral Intasome Assembly and
Inhibition of DNA Strand Transfer." Nature
464.7286 (2010): 232-36.
Maertens, Goedele N.,
Stephen Hare,
and Peter Cherepanov. "The Mechanism of Retroviral Integration from
X-ray
Structures of Its Key Intermediates." Nature
468.7321 (2010):
326-29.
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