Bennett, R. P., S. Rhee, R. C. Craven, B. Hunter, and W. J.W.. 1991. Amino Acids ..... Arthur. 1993. The Two Zinc Pingers in the Human Immunodeficieney Virus.
•
Mechanism of Selective Incorporation and Genomic Placement of Primer tRNALys3 into Buman Immunodeficiency virus Type 1
by
Johnson Mak
A the sis submitted to ths Faculty of Graduate Studies and Research, McGill Univsrsity, in partial fulfillment of the requirements of the degree of Doctor of Philosophy
Department of Medicine, Division of Experimental Medicine McGill University, Montréal, Canada. March 1996
•
~
Johnson Mak, 1996
i
1+1
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ISBN 0-612-19747-6
Canada
• Ta rny parents, for their infinite support, understanding, and encouragement.
Tc my sister, for her humors, and for jusl being there.
To my brother. has
for his achievement, which
encouraged me
ta strive
for my own
success.
But above aIl,
this thesis is dedicated ta
aIl those who lost their lives or a
one in this tragedy called AlOS .
• ii
loved
•
Abstract
During
HIV-l
assembly.
the
major
marnmalian
isoacceptors,
tRNALys
tRNA Lyol.2 and tRNAL y n3, are selectively incorporated into the virus. project has been to study the mechanism involved in this process.
My This
was done by transfecting COS-7 cells with wild type and mutant HIV-1 proviral
process. is
DNA,
the
effects
of
these mutations
on
this
In the wild type virus, 60% of viral low molecular weight RNA
tRNALy..
packdging
and analyzing
compared
to
6%
tRNALys
is
selective.
of
moleculcs of tRNAL y s3,
Mutations
which
including
the
of
the
tRNA
in
HIV-l
the cytop1asm. cantains
i. e,
the
approximately 8
the primer for reverse transcriptase, per virion.
either
primer
remove
binding
the
S'
site,
portion
or
which
of
the
reduce
RNA
genome, l~~A
genornic
incorporation approximate1y la fo1d, have no effect upon select tRNALys incorporation,
indicating that genonic RNA does not play a role in this
prr)cess.
the
On
Pr160Qag-pol
reduces
molecule/virion, evidence
carrying
other
i.e,
implicating
hand,
tRNALys) inhibits
a
large
deleticn
of
RT
sequences
incorporation
to
approximately
select packaging
of
tRNALys.
in 1
Initial
Pr160gag-pol as being the viral precursor protein
tRNALYs
into the virus is as fol1ows: 1) Viral partic1es composed on1y of PrSS gog do not se1ective1y package tRNALys, whi1e viral partic1es composed of both prSS gog and Pr160gog-pol do. 2) Immature viral particles,
unable to process these precursor proteins due to an
inactive viral protease. that
processing
of
still show select tRNALys packaging,
precursor
proteins
is
not
indicating
required.
We
next:
investigated regions within Pr160gog-pol which may play a ro1e in the tRNALy. packaging process
using mutationa1
ana1ysis.
The
carboxy1
deletion of IN sequences in Pr160gog-po1 does not significantly affect select tRNALys packaging, but carboxy1 de1etions which include both the IN sequence and the RNase H and connection domains of RT do.
Smal1
ami no acid insertions(2-3) placed into various domains of RT show no effect upon tRNALys packaging when p1aced inco the fingers. palm, part of the thumb.
and RNase H domains
• but mutations within or just N-
terminal to the connection domain inhibit select tRNALys packaging.
•
A
direct correlation has been found between these mutations which affect tRNALys) packaging and the absence of mature Gag and Gag-Pol pro teins in
iii
•
the
virus.
tRNALYs
Since
precursor
packaglng,
it
seems
incorporation into the virus packl;Jing
into
the
virus
as
processing
likely
is
that
the
is due to the
a
result
not
of
required
inhibition
inhibi tion of
these
for
select
of
tRNAL~'l1
Pr160gaa-pol
connection
domdin
mutations. We have aiso done preliminary experiments on the effect of sorne of these mutations upon tRNALys3 :>lacement on the viral RNA genome.
Genomic placement of tRNALysJ in vivo was detected by measuring reverse transcription in vitro, source
of
using exogenous RT and total viral RNA as the
primer/ternplate.
Our
results
indicate
placement of tRNA Lys3 occurs independently of viral
that
the
genomic
precursor protein
processing and the presence of IN sequences. Genomic placement was found to be significantly reduced when one of the connection domain mutations, SVC21 GR, was tested .
• iv
•
Résumé Lors de l'assemblage du virus VIH de type I, de
mammi fère
l' ARNtLysl, 2 et l' ARN t Ly s3
l' ARN t Lys.
façon sélective par le virus. mécanismes
les isoaccepteurs majeurs sont
incorporés
de
Le but de mon projet était d'étudier les
impliqués dans ce processus d'incorporation en transfectant
des cellules COS-7 avec de l'ADN proviral de type sauvage ou muté et en
analysant
les
effets
cl incorporation.
Chez
1
l'ARNtLYS
de
le
sélective
est
de
de
mutations type
celui-ci
sur
sauvage,
compose
la
de
60%
ce
processus
présence
de
l' ARN viral de
tandis que dans le cytoplasme on en retrouve
6%. Chaque particule virale contient environ 8 l' ARN t Ly s3. l'amorce nécessaire pour la transcriptase
propart ion
molécules
virus
car
faible poids moléculaire. une
différentes
de
inverse (TI).
Des mutations qui enlèvent le bout 5' du génome viral et
en
le
incluant
l'incorporation
site de
de
liaison
l'ARN d'un
de
l'amorce
facteur
de
10
ou
n'ont
qui pas
réduisent d'effet
sur
l'incorporation sélective de l'ARNtLYS, ce qui semble indiquer que llAkN génomique n'affecte pas ce processus.
Cependant une délétion dans la
séquence de la TI du préc\\rseur Pr160 Qa g-pol r(dui t
a
l'ARNtLYs
environ
impliquaient transportant
déjà
le
molécule
l'ARNtLYS
l'ARNtLYS alors que
Pr160 0AO -pol le font. le
par virion.
pr160gag-pol dans
uniqueme~1t du
contenant
maturer
une
le
PrSS gag
comme
virion:
l'incorporation du
Plusieurs
étant
le
évidences
précurseur
1)
Les
particulp.s
n'incorpore
pas
de
viral
virales
façon
sélective les particules virales contenant le PrSS gag et le
2) Les particules virales qui sont incapables de
précurseur parce qu'ils possédent une protéase
inactive,
démontrent une incorporation sélective de l'ARNtLys indiquant que cette maturation nlest pas nécessaire. au
niveau
des
régions
Nous avons donc investigué davantage
Pr160gag-pol
Douvant
jouer
un
l'incorporation de l'ARNtLyS en utilisant des mutations.
du
bout
C-terminal
de
la
séquence
intégrase(INI
du
rôle
dans
Des délétions
Pr1600AO-pol
n' af fectent pas l'incorporation de l' ARNtLys tandis que des délétions éliminant la séquence IN, la RNase H et le domaine de la liaison avec la
•
TI affectent ce processus.
De petites insertions (2-3) d'acides aminés
lorsque placées dans les différents domaines de la TI comme la paume, les doigts, une partie du pouce et le domaine RNase H produisent aucun
v
•
effet
alo~s
que des insertions situees â
terminale du domaine de
liaison inhibe
l'int~ri~ur
ou du
cot~amino
l'incorporation selective des
Une corrélation direct.e fut démontrée entre les mutations qui
ARNtLys.
affectent l'incorporation de l'ARNtLYs dans le virus et la maturation du précurseur Pr160 0a q-pol dans le virus. Pr160gb.g-pol n'est pas nécéssaire à
virus,
il
Comme la maturation du précurseur l'incorporation des ARNtLYs dans
semble donc que cette inhibition est causée par
It:'
l'absence
d'incorporation du précurseur Pr160gag-pol dans le virus de à la présence des mutations dans le domaine de liaison.
Nous avons aussi conduit des
études sur l'effet de ces mutations pour la mise en place de l'ARN t LysJ
sur le génome dlARN viral. transcriptase
In vivo nous avons mesuré l'activité de la
inverse alors qu'in vitro nous avons utilisé de
la 'rI
exogène et de l'ARN viral comme source cl 1 amorce/patron. Nos résultats indiquent que la mise en place de l'ARN t Lys3 se produit indépendamment de
la maturation du précurseur ou de
Cependant celle-ci
est
fortement
la présence des séquences
réduite
domaine de liaison. soit la SVC21 GR .
• vi
IN.
avec une des mutations du
•
Acknowledgements
l would like ta
expres~
my appreciation tO aIl the past and the present
staff members in the Lady Davis Institute. to
the
prin~ipal
5nvestigators.
A
from the house keeping staff
simple
thanks
or
a
list
individuals' names will not be sufficient to express rny gratitude.
of
The
last five years at the LDI has not only been a time for science, but it was aiso a personal enrichrnent process.
There were times that l wish l
was r.ot there to wi.tness or to participate in, but there were aise times
that 1 was glad to partake in.
For aIl those people with whom 1 have
come across over the past five yeal's,
l
thank you.
In my heart,
are bits and pleces from each and everyone of you which l with me no matter where l go.
there
will bring
In a way you have aIl shaped me to be the
persan 1 am today(No ius;.\l.t intenùed).
1 will net provide a list of individuals te whom l ewe my gratitude, but
you know who you are.
For those who have helped me along the way, a
brief mention can never repay for aIl their assistance.
However,
individuals have to be singled out
They are my
supervisor,
Dr.
Lawrence Kleiman,
from this process. and my colleague,
as
weIl
two
as my
friend, Dr. Mark A. Wainberg.
Larry,
how
~hould
l
begin?
In the past five years,
tremendously great superviser as weIl
you have been a
a Hreat friend.
dS
wou Id have
1
hoped that certain things could have been different, but above aIl 1 am glad that 1 had the privilege to obtain my Ph.D. supervision.
l
training under your
would be honored te study under your supervision if l
had to do it aIl over again.
1 thank you for your faith,
trust, and those not so great pep-talks.
confidence,
1 especially tbank you for
giving me the freedom to take on my research direction and to establish coll~boration
with members of the institute in my own free will.
importantly, you have talked me the essence of intelligence. shawn me that
You have
the essence of intelligence is not ba.sed on how much
knowledge one has,
•
Most
but rather,
how willingly one has the courage to
openly accept how little we aIl know, criticism with open arrns.
and be humble enough to greet
For that, 1 thank you!
vii
•
Mark,
l
thank you!
Without
them.
possible.
a
Thank you for yaur Eaith, lot
of
But especially,
work l
in
this
thesis
confidence, would
and support.
never
have
been
thank you f_r yaur helping hands, without
which 1 would have never been able to complete my Ph.D. training in this institute.
l aIse wish to acknowledge Health and Welfare Canada for having provided me with a Pre-Doctoral Fellowship for rny graduate training.
A special thanks goes to Normand Pepin for translating the abstracto
Last,
but certainly not least.
Thanks to Sunita de Tourreil for her
support and friendship during the writing of this Thesis .
• viii
•
preface
In
with
dccc)l"danCB
the
Guidelines
for
Thesis
Preparation
fram
Department of Graduate Studies and Research at McGill University. candidate
has
exercised
n~n~script-based
for
option
of
writing
the
thesis
the option of including,
as
part of
the
the text of a paper(s) submitted or to be submitted
publication,
published
or
paper (s).
the
clearly-duplicated
These
texts
must
be
text
bound
of
as
a
an
integral part of the thesis.
1f
this
option is
lo~ical brid~es
chosen,
connecting texte
that
provide
between the different papers are mandatory.
1'he thesis must be written in such a way that it is more than
a
mere
collection
of manuscr ipts;
in
ether words,
results of a series of paper must be integrated.
'rhe thesis must still conform to aIl ether requirements of
the "Guidelines for Thesis Preparation". include:
A Table of Contents,
The thes!s must
an abstract in English and
French, an introduction which clearly states the rationale and objectives of the study, a comprehensive review of the literature,
a
final conclusion and summary,
and a
through
bibliography or reference list.
Additional material must be provided where appropriate(e.g. in appendices) and in sufficient detail to allow a clear and precise
judgment
to
be
made
of
the
importance
and
originality of the research reported in the thesis.
In the case of rnanuscripts co-authored by the candidate and
•
others,
the
candidate
is
required
to make
an
explicit
atatement in the theaia as to who contributed to auch work and to what extent.
Supervisors must attest to the accuracy
ix
The as
thesis.
Candidates have
thesis,
the
the
a
•
of such SLdtement dt the doctoral
or~l
d~t~nse.
task of the exarniners is made more ditticult it 1S in
the candidate's
interest
Und.er
no
circumstanc8S
CdS~S,
these
ln
tht""'
to make per::ectly clear
the responsibilities of aIL the authors of papers.
Si.nC'~
cau
a
th~
c0-author~d
co-author ot
Any
component of Buch a th8sia serve .s an examiner t'or that
thesis.
l
have included,
as chapters of
paper which has been published.
this thesis.
the texts of an originel1
one original rnanuscript which
hdS
b~en
subrnitted for publication, and sorne preliminary data which are included
in the format of a short communication. own Abstract,
and
Chapters 2 and 3 include their
Introduction, Materials and Methods,
Reference
sections.
Chapter
4 has
its
Results,
Discussion.
own abstract,
and
the
Introduction, Materials and Methods, Results, and Discussion are written in a single section. the
thesis
have
respectively.
A General Introduction and General Discussion to
been
inl..:luded
and
represent
Chapters
In order to bridge connecting papers,
1
'lnd
5,
Chapters 2 to 4
each contain a preface.
The manuscripts presented in the thesis are the following:
Chapter 2.
Mak, J., Jiang, M., Hammarskjold, M.L., Rekosh, D" Kleiman,
L.
1994.
Incorporation Particles.
of
Role of tRNALys
Pr160gag-pol
into
Journal of Virology.
Human
in
Wainberg, M.A"
Mediating
the
and
Selective
Immunodef iciency Virus
Type
1
68:4. 2065-2072.
Chapter 3.
Mak, J., Cao, Q., Huang, Y., Lowy, I., Prasad, V.R .. Wainberg, M.A., and Kleiman, L.
1996.
Reverse Transcriptase Connection Domain Mutations in
Prl60gag-pol Inhibit the Select Incorporation of Primer tRNALy.J
•
HIV-I.
Journal of Virology.
Submitted for Publication .
x
into
•
Chapter 4 . Huang, Y., Li, Z., Wainberg, M.A., and Kleiman, L.
Mak, J.,
Genomic
Placement of HIV-l
Func~i~nal
1996.
The
Primer tRNALyo3 Occurs in the Absence of a
Protease or Integrase Sequences.
The candidate was responsible for all the research described in Chapters 2 to 4.
Dr.
M.Jiang provided initial assistance in the 2-D PAGE
analysis in chapter 2,
Dr.
Q.Cao provided technical assistance in sorne
of the plasmids isolation and virus isolation in chapter 3, Dr. Y.Huang provided genomic
technical advice on the western analysis and placement
in
Assay
chapter
3
&
4
the
respectively.
in
vi tro
Dr.
Z.Li
provided the enzyme reverse transcriptase used in the in vitro genomic
placement assay in chapter 4.
Ors.
M.-L.Hammarskjëld,
O.Rekosh,
V.R.Prasad, I.Lowy provided sorne of the mutants used in chapters 3 & 4. Dr. M.A.Wainberg provided the containment facilities, many reagents, and
many helpful advice throughout this study.
AIl this work was done under
the supervision of Dr. L.Kleiman. Other relevant studies that the candidate was involved with, but are not included as part of the thesis. 1. Jiang, M., Mak,
J.,
Wainberg,
M.A.,
Parniak,
M.,
Cohen,
E"
and
Kleiman, L. 1992. A Variable tRNA Content in HIV-1 IIIB. Biochemical and Biophysical Research Communication. 185:3, 1005-1015. 2. Jiang, M., Mak, J., Ladha, A., Cohen, E., Klein, M., Rovinski, B"
and Kleiman, Type
and
L. 1993. Identification of tRNAs Incorporated into Wild
Mutant Human
Immunodeficiency Virus Type
1.
Journal
of
Virology. 67:6, 3246-3253. 3. Arts, E., Mak.
J.,
Kleiman,
L.,
and Wainberg, M.A.
1993. Mature
Reverse Transcriptase (p66/p51) is Responsible for Low Levels of Viral
•
ONA Found in Human Immunodeficiency Virus Type l(HIV-l). Leukemia. 8:1, 175-178 .
xi
•
4.
Jiang,
M ..
Mak,
J ••
Wainberg,
M.A.
and Kleiman,
L.
1993.
Reverse
Transcriptase is an Important Factor for the Primer tRNA Selection in
HIV-1. Leukemia. 8:1, 149-151.
5. Arts, E., Mak, J., Kleiman, L., and Wainberg, M.A. 1994.
DNA Found
in Human Immunodeficiency Virus Type 1 Particles 1s Not Required for Viral Infectivity. Journal of General Virology. 75, 1605-1613.
6.
Li,
X.,
Parniak,
Maki
M.A.
J.,
1994.
Arts,
Effects
E.J.,
Kleiman,
L.,
of
Alterations
of
Wainberg,
Primer
Sequences on HIV-1 Replication. Journal of Virology.
M.A.,
and
Binding Site
68:10, 6198-6206.
7. Huang, Y., Mak, J., Cao, Q., Li, Z., Wainberg, M.A., and Kleiman, L. 1994. Incorporation of Excess Wild Type and Mutant tRNALys3 Into HIV-1. Journal of Virology.
68:12, 7676-7683.
8. Huang, Y., Shalom, A., Li, Z., Wang, J., Mak, J., Wainberg, M.A., and Kleiman,
L.
1996.
Effects
of Modifying
the
Initation of HIV-1 Reverse Transcription.
tRNALys3
Anticodon upon
1996. Journal of Virology.
Submitted for Publication.
9. Mak, J., Wainberg, M.A., and Kleiman, L. 1996. The Incorporation of Cyclophilin A into Human Immunodeficiency Virus Type 1 Is Independent from Primer tRNA and Genomic RNA Packaging. Biochemical and Biophysical Research Communication. Submitted for Publication.
10. Li, Z., Arts, E., Huang, Y., Mak, J. Kleiman,
L.
1996.
Multiple Forms of
Publication .
• xii
f
Cao, Q., Wainberg, M.A., and
tRNALys3
in HIV-l.
Sumitted for
•
Table of Contents Page ..
iii
R6sum6 ••••••••.•••••••••••••••.•••••••••••••••••••••••••
v
Acknowledgemsnts ••••••••••••••••••••.•••••••••••••••••••
vii
Preface to the Thesis ••••••••••••••••••••.••••••••••••••
ix
Abstract
..
xiii
List of Figures and Tables ••••••••••••••••••••••••••••••
xvi
List of Abbreviations •••••••••••••••••••••••••••••••••••
xviii
Table of Contents
Chapter 1: Literature Review ••••••••••••••••••••••.••••
1
1.1. Introduction ••••••••••••••••••••••••••••
2
1.2. Retrovirus Classification •••••••••••••••
3
1.3.1. Human Immunodeficiency virus Structure and Genome organization•••••••••••••••••
4
1.3.2. OVerview of HIV-l Replication Cycle ••••
7
1.4. HIV-l Viral Assembly: Viral Proteins, Viral RNA, and Their Roles in Viral Assembly ••
13
1.4.1. Viral Proteins •••••••••••••••••••••
14
1.4.1.1. PrSS craCl
..
14
1.4.1.1.1 Matrix ••••••••••••••••••••••
16
1.4.1.1.2. Capsid ••••••••••••••.••••••
17
1.4.1.1.3. p2 •••••••••••••••••••••••••
18
1.4.1.1.4. Nucleoeapsid •••••••••••••••
18
1.4.1.1.5. p6
21
..
1.4.1.2. Pr160 gag-pol
•
..
22
1.4.1.2.1. Expr.ssion •••••••••••••••••
22
1.4.1.2.2. Enzymes coded by Pr160gall'-pol
24
1.4.1.2.2.1. Prot••so ••••••••••••••••
24
1.4.1.2.2.2. Reverse Transcriptase •••
25
1.4.1.2.2.3. Int.gr.sa
.
26
1.4.1.3. Env.lape •••••••••••••••••••••••
28
1.4.1.4. Naf
..
29
1.4.1.5. vif
..
30
xiii
•
1.4.1.6. vPr •••••••••••••••••••••••••••• 1.4.2. Virion Associated RNA •••••••••••••• 1.4.2.1. Ganomic RNA •••••••••••••••••••• 1.4.2.2. tRNALy.3 •••••••••••••••••••••••• 1.4.2.2.1. Retrovira1 Primer tRNAs ••••
32 33 33 35 35
1.4.2.2.2. rnteraction of Primer tRNALy.3 and Ganomic RNA ••••••••••••
38
1.4.2.2.3. rnteraction of Primer tRNALY·3 with Viral Proteins •••••••••••••••
40
1.4.2.2.4. Ro1e of RT in packaging and placement of tRNALy.3 ••••••••••••• 1. 5. References •••••••••••••••••••••••••••••
Preface
42 44
86
..
ehapter 2: Ro1e of Pr160gag-po1 in Mediating the Selective rncorporation of tRNALyo into Human rmmunodeficiency virus Type 1 Partic1es ••••••••••••••••••••••••••
87 .
88
2.2. rntroduction •••••••••••••••••••••••••••
89
2.3. Materia1s and Methods ••••••••••••••••••
91
2.4. Resu1ts ••••••••••••••••••••••••••••••••
105
2.5. Discussion •••••••••••••••••••••••••••••
109
2.6. Acknowledgaments .•••••••...•••••••••••.
113
2.7. Refemces ••••••••••••••••••••••••••••••
114
2.1. Abstract
Preface
119
..
Chapter 3: Reverse Transcriptase Connection Domain Mutation. in Pr160 gag-pol rnhibit the Select rncorporation of
•
Primer tRNALy.3 into HXV-1 ••••••••••••••••••
120
3.1. Abstract
..
121
3.2. Introduction •..••.••••.•.••••••••.••••
122
3.3. Materia1. and Method••••••••••••••••••
124
3.4. Resulta
..
143
3.5. Di.cussion
..
148
3.6. Acknowledgements ••••..••••••••••••••••
152
3.7. Ref.r.nc••.•..•.......•........•.......
153
xiv
•
Preface
160
..
Chapter 4: The Ganomic Placement of HIV-l Primer tRNALy.3 Oceurs in the Absence of a Punctional Protease or IntegraB8 Sequences ...••••.•....•••....•..
161
4.1. Al:>stract •••••••••••••••••••••••••••••
162
4.2. Results and Discussion •••••••••••••••
168
4.3. References •••••••••••••••••••••••••••
172
Chapter 5: General Discussion
5.1. General Discussion •••••••••••••••••••
175
5.2. References
.
180
Original Knowledge •••
185
Chapter 6: Contribution to Original Knowledge 6.1. Contribution
t~
• xv
•
List of Figures and Tables P.g.. Chapter 1. Figure 1. Organization of viral Sequences in vir.l DNA and Genomic RNA ••••.•.•.•.••••••••••.•••.••••. Figure 2. Schematic Diagram of The Matur.. HIV-l
Vi~ion
5 6
Figure 3. overview of The HIV-l Replication Cycle •..•.
B
Figure 4. Mechanism of R..troviral Revers.. Transcription.
9
Figure 5. Maturation of Human Immunodeficiency Virus Typ.. 1 Viral Particl..s ••••••••••••••••••••••..
12
Retroviruses and Their Primer tRNAs •••••••••••
36
Table 1. Chapter 2.
Figure 1. Structures of Wild-Type and Mut...'t HIV-l Plasmids •••••••••••••••••••••••••••••••••••
95
Figure 2. 2-D PAGE Patterns of Low-Molecular-Weight Vir.l RNA •••••••••••••••••••••••••••••••••••••••• Table 1.
Percentag.. of
tRNA~
Isoacc..ptors in Total
Cellular and Viral tRNA ••••••••••••••••••••• Figure 3. Quantitation of
97
tRNAL~3
99
in viral RNA ••••••.•
100
Figure 4. Quantitative RT-PCR for Viral Ganomic RNA •••
102
Table 2.
Molecules of tRNALy.3 per Two Molecules of Genomic RNA ••••••••••••••••••••••••••••..•••
104
Chapter 3. Figure 1.
Schema~ic
Representations of wild Type and
Mutant Pr160 g • g -pol Pr..cursor Proteins ••••••• Table 1.
Mutations in HIV-l pr160 g • g -pol ••••••••••••••
Figure 2. 2-D PAGE Patterns of Low Mol ..culer
..
•
132
Characterization of Wild Typ" and Mutant HIV-l Viral Partic188
Table 3.
131
w.. ight
viral RNA
Table 2.
129
..
135
Eff..cts of Mutations upon 2-D PAGE vir.l tRNA Patterns and tRNALy.3 Incorpor.tion ••••••••
Figure 3. Quantit.tion of Genomic RNA in Viral RNA
xvi
136
•
Samples •••••••••••••••••••••••••••••••••••• Figure 4. Quantitation of tRNALy.3 in viral RNA Samples
137
Figure 5. Western Analysis of Viral Proteins •••••••••
141
139
Chapter 4. Figure 1. Schamatic Representations of wild Type and Mutant HIV-l Viral DNA Sequences •••••••••••••••••• Figure 2. In vitro Primer Extension Assay ••••••••••••
163 165
Figure 3. Quantitation and Primer Extension of Total Viral RNA in Wild Type and Mutant Particles ••••••
• xvii
167
•
List of Abbreviations Ab
:Antibody
ASLV
:Avian Sarcoma L8Ukosis virus
AMV
:Avian Myeloblastosis Virus
CA
:Capsid
CMV
: Cytomegalovirus
env
:Envelope
Gag
:Group Antigen(Viral structural Protein precursor)
Gag-Pol
:Group Antigen-Polymerase Fusion protein(Viral Enzymatic Protein Precursor)
HIV
:Human Immunodeficiency Virus
IN
: Integrase
LTR
:Long Terminal Repeat
MA
:Matrix
MoMuLV
:Moloney Murine Leukamia virus
Ne
:Nucleocapsid
PBS
:Primer Binding Site
P.R
:Protease
RSV
:Rous Sarcoma virus
RT
:Reverse Transcriptase
SDS-PAGE
:Sodium Dodecyl Sulfate-Polyacrylamide Gal Electrophoresis
•
SIV
:simian Immunodeficiency virus
TCIDs.
:Dosage of 50% Infection of Tissue Culture Cella
2-D PAGE
:TwO Dimensional-Polyacrylamide Gal Electrophoresis
• Chapter 1 Literature Review
• l
•
1.1. Introduction
The discovery of a filterable,
transmissible agent associated with
avian leukosis sarcoma by Peyton Rous in 1911 is recognized as the first landmark in modern retrovirology(2S4).
This
transmissible agent was
shown to have the ability to transform celis and to cause turnors, while extracts of transplantable turnors in rats, mice, and dogs failed(254). During the 19305, high frequencies of spontaneous leukernia were observed in severai inbred mi ce strains.
lt was speculated that the etiological
agent of leukemia in mice and birds had a viral origin(92, 247).
It
WC1S
la ter shown that a significant portion of turnors is associated with RNA tumor viruses(retroviruses), and that mast RNA tumor viruses capable of
transforming cells in culture are also defective for replication(3131. Baltimore and Temin
independently pubUshed
protein reverse transcriptase (RT)
the
discovery of
in the spring of 1970(9, 292).
viral RT is
a viral protein which has RNA-dependent and DNA-dependent DNA polymerase
activity, and it is capable of synthesizing viral DNA using viral RNA as the template(10).
The discovery of RT challenged the central dogma of
unidirectional information transfer, fram DNA tn RNA ta protein(lO) . In the early 1980s, Gallo and his colleagues isolated the first human
retrovirus
established
from a
T-lymphoblastoid
cell
Une
fram cells of a patient diagnosed with
lymphoma(232,
233).
Later,
a
related
retrovirus
that
had
been
cutaneous T-cell
with
significant
differences in immunological cross-reactivity was isolated fram a T-cell variant of a hairy cell leukemia cell line(148).
These viruses are now
known as human T-cell leukemia virus l(HTLV-l) and human virus 2 (HTLV-2), respectively.
T-~ell
leukemia
Subsequent studies have shown that HTLV-
land HTLV-2 are the etiological agents of adult T-cell leukemia, and atypical hairy-cell leukemia, respectively. On Oecember 10, 1981, three independent studies were published in
The New England
Journ~l
of Medicine to describe a new class of acquired
immunodeficiency syndrome (AlOS) drug users(10S,
199, 268).
among homosexual men and intravenous
In 1983, Moltcagnier and colleagues at the
Pasteur Institute in Paris reported the isolation of a T cell retrovirus
•
from a patient with l:nnphadenopathy, one of the early signs in patients progressing toward AIOS(13, 282).
This virus was later shown to be the
2
•
etiological agent of AlOS. and it was renamed as human immunodeficiency
virus, HIV.
1.2. Retrovirus Classification
Based
on
the
pathogenici ty
of
the
viruses.
divided into three subfarnilies: 1) Oncovirinae. oncogenic rnernbers of the retroviruses.
viruses.
retroviruses
are
These include aIl the
2) Lentivirinae,
or the -slow·
Members of this subfamily are characterized by their ability
ta cause slow progressive degenerative disease Spumavirinae,
or the "foarny· viruses.
In cell culture,
effects
of
the
formation
vacuolation in infected calis.
3)
They are the least characterized
subfamily of the retroviruses. with
in hast animaIs.
giant
they cause cytopathic
multi-nucleated
cells
and
Foamy viruses are alsa known ta induce
persistent infections without Any clinical disease.
Retroviruses are alsa categorized basad on their morphological characteristics, and these different morphologies may reflect different modes of viral assembly in vivo.
By virtue of their morphologies seen
with the electron microscope, retroviruses are classified into type-A, type-B, type-Co and type-O particles(290) .
Type A particles form intracellular viral particles, and they have not been shawn ta have any infectivity. intracytoplasmic
forms
Thë;..re are intracisternal and
of type A particles.
Intracisternal
type A
particles are often found in cells producing type C or type 0 particles, but their functions are not known.
Intracytoplasmic type A particles
have been shown ta be the precursor of sorne type B particles. Type
B particles
morphological
features:
are
particles
which
1)
Particles
at
conta in
the
two
striking
plasma membrane show
doughnut shape(hollow-sphere in 3-D) and long spikes are seen at the cell surface.
2)
The budded mature particles have electron-dense
nucleoids that are eccentrically located within the envelope particles. Intracytoplasmic particles are found in the cytoplasm of the infected cells. e.s. Mouse Mammary Turnor Virus(MMTV). No
•
intracytopla"mic viral
structure
particle-producing cells until viral budding. electron-dense
crescent-shaped(dome-shaped
3
can
be
found
in Type
C
During viral budding. an in
3-D)
structure,
the
•
precursor of the: viral core, plasma membrane.
is loacted on the cytoplasmic side of thlO>
Less prominent spikes than type B particles are tound
on the cell membrane surfaces where crescent-shaped(dome-shaped in 3-D) structures are found.
electron-lucent
cores
Budded viral particles contain centrally locdted
in
immature
particles,
electron-dense cores in mature particles 1
e. s.
or
centrally
located
avian leukosis-sarcoma
Virus (ALSV) . 'IYPE: P particle3 lJav.a bath intracellular and extracellular forms
of viral particles, e.s. Mason-Pfizer Monkey Virus (M-PMV) . particles,
Like type 8
they produce ring-shaped intracellular particles, and contain
eccentrically-located electron-dense nucleoid in mature extracellular particles.
But unlike type B particles,
they contain shorter surface
spikes which are sirnilar ta type C particles.
1.3.1. Human
~unodeficiency
virus Structure and Genome Organization
HIV is a type-C-like retrovirus, and it belongs to the family of Retroviridae and the subfamily of Lentivirinae.
Like all retroviruses,
HIV-l con tains of two copies of positive-sense single strand genomic
RNA, and each copy of the viral RNA is 9.2 kb in length(238). genomic
RNA
encodes
viral
precursor
proteins (Pr5S gag
1
HIV-l
Pr160gag-pol.
envelope protein(gp160», viral regulatory proteins(Tat, Rev, and Nef), and viral auxiliary pro teins (Vif, Vpr, and Vpu) (Figure 1 & 2). PrSS o• o contains the sequences for matrix(MA), capsid(CA) , p2, nucleocapsid(NC), pl and p6. In addition to MA, CA, p2, and NC, PrI600· o- pol contains the viral enzymatic proteins(protease(PR), reverse transcriptase (RT), and integrase(IN».
Envelope(Env) protein is made up of the gpl20 surface
protein(SU) and the gp41 transmembrane protein(TM).
HIV-I genomic RNA
is associated with the viral NC in the viral core along with the primer tRNALys3, and the viral enzymatic proteins(PR, RT, and IN). core is def ined as
the viral
The viral
structure enclosed by CA structural
proteins and components within (Figure 2).
Immediately outside of the
viral core is the MA, which is found underneath the viral envelope. Like other members of the retrovirus family, HIV consists of a lipid-
•
containing envelope derived from the plasma membrane during budding . Viral Env proteins are inserted into this membrane, and this membrane
4
.'
• Proviral DNA PBS
1
U3
~
gag
1 1
pol
~::;env :Pd~ï~_3~[E
Viral Genomic RNA PBS Ul
r ..v
env
o
TF1 PR
RT
IN
U3
Mature HIV-1 Virion
•
Legend ~ matrix
•
III capsid
cyclophilln. Vpr, or vn
o protease • •
reverse transcriptase •
surface ~ transmembrane
~ primer tRNA
' \ genomic RNA
•
~ nucleocapsid
integrase
•
surrounds a viral structure composed of the MA proteins and the viral
core within(313).
Viral auxi1iary proteins(Vpr and Vif) and ce11u1ar-
derived protein cyclophilin A are founà ta associate with HIV-l virus, but it is not c1ear if the subvira1 loca1ization of these proteins is
between the MA prote in and the viral core or inside of 'the viral core structure (Figure 2).
Electron microscope studies estimate that most
retroviruses are rough1y spherica1 structures with a diameter between 80 to 130 nm(209). re~rovirus
The 1ength of the envelope pro teins varies from one
ta the next, and theyare found on the surfaces of the viral
particles.
Envelope proteins are glycoproteins, and they contain type-
specifie epitopes.
These envelope glycoproteins are used to bind to the
target cell which the virus will infect.
1.3.2. OVerview of HIV-l Replication Cycle
Attachment of a retrovirus to its target cell requires specifie i.nteraction of the viral Env gp120 SU protein and a cellular surface receptor on the target cell(Figure 3).
Cellular surface receptor CD4
was the first identified receptor for a retrovirus, and the interaction between CD4 After
the
receptor and the HIV-l Env has been intensely studied. binding
of
the
retrovirus
protein
to
its
corresponding
receptor, receptor-mediated endocytosis(120, 198, 326), or fusion of the viral envelope and cellular membrane(201, 279) (for review see (162)) is required for the virus to gain access into the target cell.
For HIV,
after binding of the Env protein to CD4 receptor, the viral envelope and the
cellular
cytoplasm(45, proteins (1%
memorane 279),
fuse
to
release
the
the
core
into
the
and the envelope proteins and the majority of MA
of MA pro teins are associated wi th the viral core)
thought to remain at the plasma membrane (96) . enters
viral
cytoplasm of
the
target cell,
Once
are
the viral core
viral encoded protein RT
initiates reverse transcription and converts viral genomic RNA into ~
double-stranded DNA.
The current model for the reverse transcription of genomic RNA
•
into double strand cDNA is shown in figure 4.
Synthesis of HIV-l viral
cDNA requires the usage of a packaged cellular tRNALys3 as the primer, and the elucidation of the mechanism of selective tRNA packaging and
7
•
• Overview of HIV-1 Replication Cycle Adsoijltlon
r-
1..
Provirus
r::wrI
'0
--= """"""-
~ ~..-.--
-=:.. ' =- """"""Transcription
--
cDNA
.................................. /
,
~uclear Localization and Integration
~
~
.oo,:ranslatlon
tRNA 't
F
lcRNA
1 ' \Gag·Pol Gag
Assembly
Maturation
Mechanlsm of Retrovlral Reverse TranscrlptJon 1
•
U3
A
U3
A
III A' U5'ëIP
A US PBS
····-1-1- - - - - - - - - - - - - - - f l - H I J A'US'
A US PBS
U3
9P
A
...·+-1==============::::t1=t:11-1.. . , ...
1
PBS'
11
U3'
....
A' uS' I r
A US PBS U3 A ...................................................
11
1 PBS'
PPT
...
1( PBS'
•••
~
genomic RNA ANase H degraded viral ANA anllsense cDNA sensecDNA primer !RNA
U3
A US PBS
U3 U3'
U3'
A' US' 'iP
U3
A
1
PSS
)
11 U3'
A' US'
U3
AUS
A A'
~;==================1==t+=I U3' A' US' PBS'
U3' A'US'
•
J\
•
genomic placement have been the objectives of HIV-I virus,
rny
studies.
Inside the
tRNALys3 is placed onto the primer binding site(PBS), a
region located very near the 5' end of the RNA genome(239). reverse transcription,
During
viral protein RT binds to primer tRNALyo3 and
uses genomic RNA as the template for DNA polyrnerization(45).
Synthesis
of viral DNA begins with the synthesis of the minus-strand strong-stop DNA(Figure 4).
Minus-strand strong-stop DNA synthesis initiates from
the 3' end of the primer tRNA, and is the first discrete intermediate in retroviral DNA synthesis(step 1).
Minus-strand strong-stop DNA is then
translocated from its site of synthesis at the 5' end of the genomic RNA to a
pos i tion on
the
3'
end of
the genomic
RNA R sequence via
complementary binding between the sense R sequence and the antisense R sequence in the minus-strand strong-stop DNA(step 2). transferring minus-strand strong-stop DNA from 5' template is known as the first template switch. synthesized by the RT,
This process of to 3' end of the
As the viral DNA is
the RNA template is degraded by the RT-encoded
RNase H activi ty which 00150 facili tates the template swi tching (24) . Minus-strand DNA synthesis continues until it advances past the plus strand RNA PBS sequence(step 3 & 4), and it is followed by the synthesis of plus-strand viral DNA.
The synthesis of plus-strand viral DNA is
primed by a stretch of RNA sequences, a poly purine tract(PPT) fragment, near
the
3'
end
of
the
genome,
degradation' of the genomic RNA. viral DNA yields
that
remains
.of ter
the
RNase
H
Continued synthesis of the plus-strand
the plus-strand strong-stop DNA,
and this strand
con tains a copy of sense PBS sequence that is dbrived from the 3' 18 nucleotides of the tRNA primer used(step 5).
The 3' end of the plus-
strand strong-stop DNA hybridizes to the complementary sequences at the 3' end of the elongated minus-strand DNA, second temp1ate switch(step 6).
and this is known as the
DNA synthesis of both plus and minus
strand DNAs continues, and yields the full length proviral DNA(step 7) (for review see (291». During the synthesis of the full length double-stranded HIV-1 DNA sequences,
the cDNA becomes part of the pre-integration comp1ex(91).
Studies have shown that in Oncovirinae,
•
nuclear entry of the pre-
integration complex correlates with cell division, membrane break-down(18l, 249).
Le. with nuclear
Lentivirinae, on the other hand, can
10
•
infect non-dividing cells, which appears to be due to the ability of the pre- int.egration complex to be carried into the nucleus by 2 viral proteins MA and Vpr(37,
119,
301).
The vltdl protein integrase is
responsible for viral DNA integration into the host cell genome. (32, 33, 74) . The integrated proviral DNA is recognized as part of the host cell sequence. complex
The transcription of the HIV-l ge"ome is regulated by a interplay between viral
regulatory proteins
and cellular
transcription factors
that interact with the viral LTR region(for
reviews see:
2531 i.
(6,
143,
trans-activator prote!n, Tat,
direct
interaction wi th
a
For example,
the expression of viral
enhances the viral RNA transcription via
viral
RNA sequence,
the
Tat
responsive
element(TAR) (83), and cellular transcription factors (88, 143, 153, 253). The primary RNA transcript from the HIV-1 genome is 9.2 kb in length, and this RNA is the source not only of genomic RNA in newly produced virus and full length mRNA, but it also gives rise to smaller mRNAs as a result of single and double splicing events.
During HIV-l
viral replication,
9.2 kb,
three major classes of viral RNA,
4.3
kb(singly spliced), and 1.8 kb(doubly spliced), are found in the viralinfected cells,
but only the 1. 8 kb class of viral RNA enters the
cytoplasm and is translated at the early stage of HIV replication(159). The 1.8 kb class of viral RNA is known as the early class of viral RNA, and it encodes viral regulatory proteins Tat, Rev, and Nef.
Tat enters
the nucleus, and it binds to the TAR sequence to enhance the elongation of viral RNA.
Rev enters the nucleus to facilitate the transport of the
4.3 kb and 9.2 kb mRNAs from the nucleus to the cytoplasm.
The 4.3 kb
singly spliced mRNA encodes the Env protein and auxiliary proteins(Vif, Vpr, Vpu) , while the 9.2 kb unspliced mRNA codes for the Gag and Gag-Pol precursor proteins.
Expression of Rev allows the translocation of the
unspliced and the singly spliced viral mRNA from the nucleus to the cytoplasmic domain of the infected cells by redirecting the viral RNAs to a non-mRNA export pathway, such as is used for the export of 5S rRNA and spliceosomal U snRNAs(84).
•
This Rev-mediated redirection of viral
mRNA export requires the interaction of Rev with the
Rev responsive
element(RRE) that is found in the 3' region of the Env-coding sequences in the unspliced and singly spliced viral rnRNA.
11
The singly spliced and
'.
• Maturation of Human Immunodeficlency Virus-1 Virai Particles
~I Pr55geg
Pr1 &OlI8lIlIOl
gp160
gag
1
1
CVifl~~~=:I2~
pol
I!!I!!:Î
1
vpu
1
env
--
118 matrlx Il capsId lm nucleocapsid 0
;.:>:. ,:late LoopLoop
Interaction
in
the
Initiation
Complex
of
HIV-l
Reverse
Transcription. J. Biol. Chem. 268:25269-25272. 136.
Jacks,
T.,
H.
D. Madhani,
P.
R. Masiarz,
and H. Varmu..
1988.
Signals for Ribosomal Framshifting in the Rous Sarcoma Virus gag-pol Region. Cell. 55:447-458. 137.
Jacks, T., and H. B. varmus. 1985. Expression of the Rous Sacroma
Virus Pol Gene by Ribosoma1 Frameshifting. Science. 232:1237. 138.
Jacobo-Molina, A., and B. Arnold. 1991. HIV Reverse Transcriptase
Structure-Function Re1ationships. Biochemistry. 3016351-6361. 139.
Jaco~o-Molina,
A., J. Ding, R. G. Nanni, J. A. D. Clark, X. Lu, C.
Tantillo, R. L. William., G. Xamer, A. L. Perri., P. Clark, A. Hizi, S. H.
•
~ughas,
and
E.
Arnold.
1993.
Crystal
Structure
of
Human
Immunodeficiency Virus Type 1 Reverse Transcriptase Comp1exed with Doub1e-Stranded DNA at 3.0 A Resolution Shows Bent DNA. Proc. Nat1 . Acad. Sei. USA. 90:6320-6324.
60
•
Jiang, li., J. Mak, A. Ladha, E. Cohen, M. Klein, B. Rovinski, and
140.
L. Kl ..iman. 1993.
Identification of tRNAs Incorporated into Wild-Type
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141.
Johnson,
M.
s.,
M. A. McClure,
O.-P.
Fang,
J.
Gray,
and R.
P.
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Enzyrnatic
Functions
te
Specif~c
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and
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wit~:
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142.
Jan.... J. S •• R. W. 'llan. and H. M. Temin. 1994. One Retroviral
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143.
Jon.... K. A•• and B. M. Peterlin. 1994. Control of RNA Initation
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144.
Jowett. J. B•• V. Planelle•• B. Paon. N. P. Shah. M.-L. Chen. and
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145.
Jowett. J. B. M•• D. J. Hockley. M. V.
NeL~ut.
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Kalpana. G. V•• and S. P. Goff. 1993. Gentic Analysis of Homomeric
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G. V••
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•
148.
Kalyanaraman.
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the
Type
Formation
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Partial
Protease
Inhibition of the Human
Resul ts
Noninfectious
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Murine Leukf'!mia Virus Protease ls Encoded by the Gag-Pol Gene and Is Synthesized through Suppression of An Amber Termination Codon.
Proe.
Natl. Acad. Sei. USA. 82:1618-1622.
328.
Yoshinaka.
Morphogenesis:
Y••
and R.
B.
Luftig.
1977.
Murine
Leukemia
Virus
Cleavage of P70 In Vitro Can be Accompanied by a Shift
Erom a Concentrical1y Coiled Interna1 Strand ("Immature") to a Col1apsed ("Mature") Form of the Virus Core. Proc. Nat1. Acad. Sei. USA. 74:3446-
3450.
329.
Yoshinaka.
Y ••
and
R.
B.
Luftig.
1977.
Properties
of
a
P70
Proteolytic Factor of Murine Leukemia Viruses. Cel1. 12:709-719.
330.
Yu. X.,
of Sirnian
Z. Matsuda, Q.-C. Yu. T.-H. Lee, and M. Essex. 1993. Vpx
Immunodeficiency Virus
Is
Localized Primarily outside
the
Virus Core in Mature Virions. J. Virol. 67:4386-4390.
331.
YU. X., X. Yuan.
Z. Matsuda, T. H. Lee. and M. Essex. 1992. The
Matrix Protein of Human Irnrnunodeficiency Virus Type 1 Is Required for
Incorporation of Viral Envelope Protein inta Mature Virions. J. Virol. 66:4966-4971.
332.
Yu.
X.,
X. Yuan,
M.
P. McLane.
T.-H. Lee.
and M.
Bssex.
1993.
Mutations in the Lytoplasmin Domain of Hurnan Immunodeficiency Virus Type l
•
Transmernbrane Protein Impair the Incorporation of Env Proteins into
Mature Virions. J. Viro1.
~7:213-221.
84
•
333.
Yuan, X.,
Human
Z. Matusda, M. Matsuda, M. Essex, and T. H. Laa.
Immunodeficiency
Virus
Vpr
Gene
Encodes
d
lQ9D .
VirlOn-t\~--:socidtl..·d
protein. AlOS Res. Hum. Retroviruses. 6:1265-1271.
334.
Yuan, X., X. Yu, T.-H. Lee,
and M. Essex.
1993. Mutations in the
N-Terminal Region of Human Immunodeficiency Virus Type 1 Matrix Protein Black Intracellular Transport of the Gag Precursor. J.
Viral.
~7:6387
6394.
335.
Zapp, M. L., and M. R. Green. 1989. Sequence-specifie RNA binding
by the HIV-l Rev protein. Nature. 342:714-6.
336.
Zhou,
W.,
L.
J.
Parent,
J.
W.
willB,
and IC.
D.
ReBh.
1994.
Identification of a Membrane-Binding Domain within the Amino-Terminnl Region
of
Human
lnununodeficiency
Virus
Type
1
Gdg
Prote in
Interacts with Acidic Phospholipids. J. Viral. 68:2556-2569 .
• 85
Which
•
preface(Chapt~r
In tRNALyo
this
1,0.': quantitatively dernonstrate
chapter,
incorporation
2)
into HIV-l by comparing
the
the
selective
concentrations
of
tRNALyn in low molecular weight cellular RNA and low rnolecular weight viral RNA.
We then examined the viral factor.s required for selective
tRNALyu incorporation inta HIV-l hy addressing the following questions: 1) T5 the processing of Gag and Gag-Pol precursor proteins necessary for the selecti,.. e
incorporation of viral
precursor pcotein will
alon~
the expreesion of
sufficient
information
tRNA?
2}
If expression
is sufficient for
vir~l
Gag
alane
and Gag-Pol
for
selective
or Gag
particles
tRNA packaging?
Gag
format~~n.
alane
3)
0:
provide
Nill
the
removal of PBS sequences or inhibition of genomic RNA packaging affect the selective incorporation of viral tRNA?
4) Since mature RT prote in
has been shown to specifically bind to primer tRNALys3 in vitro, will the deleticn of RT sequence alter the selective packaging of tRNALys3
into the virus'",
5) How many primer tRNALys3 malecules are incarparated
inta the virus?
• 86
• Ch&pter 2 The Role of Prl60gag-pol in Mediating the Selective Incorporation of tRNALya into BIV-l Particles
• 87
•
2.1. Abatract
cos-7 cells transfected with HIV-l proviral DNA produce virus in which three tRNA species are mast abundant in the viral tRNA population. Theze
tRNAs have been identified through RNA sequencing
tRNALy"3,
the
isoacceptor
primer
family.
tRNA
in HIV-l,
These RNAs
and
members
of
60% of
the
represent
techniques as
the
tRNALY"1.2
low rnolecular
weight RNA isolated from virus particles, while they represent only 6% of the low molecular weight RNA isolated from the COS cell cytoplasm.
Thus tRNALyn is se!ectively incorporated inta HIV-l particles. measured the
We have
ra tio of tRNALys3 molecules to copies of genomic RNA in
viral RNA samples. and have calculated that HIV-l con tains approximately 8
molecules
tRNALys3/2
copies
genomic
RNA.
We
have
aiso
obtained
evidence that the Pr160 0 3.g-pol precursor is involved in primer tRNALys3
incorporation into virus.
First,
selective tRNALys
incorporation and
wild type amounts of tRNALys3 were maintained in a protease-negative virus unable to process the PrSS gag and Pr160gag-pol precursors, indicating that precursor processing was not required for primer tRNA incorporation. Second, viral particles containing only unprocessed gag PrSS protein do not selectively incorpora te tRNALYs, while virus containing both unprocessed PrSS gag and Pr160gag-pol proteins demonstrate selective
tRNALys packaging.
Third,
studies with a
proviral mutant
containing a deletion of most of the reverse transcriptase sequences and approximately
113
of
the
precursor resulted in the
integrase
1055
sequences
in
the
Pr160g a g-pol
of selective tRNA incorporation, and an 8
fold decrease in the amount of tRNALys3/2 copies genomic RNA.
We have
also confirmed herein a previous study which indicated that the primer binding site is not required for the selective incorporation of tRNALys .
• 88
•
2.2. Introduction
A limited nurnber of tRNAs derived fram the infected hast cell packaged into retrovirus during virus assembly (9, Il, 24. 31, 33).
ctr~
One of
these i5 termed the primer tRNA because it 15 used to prime the reverse transcriptase(RT)-~atalyzed synthesis
of
retroviral
strand DNA.
minu~
AlI rnembers of the avian sarcorna and leukosis virus group examined ta date
use
tRNATrp
(10,14,26,33,39,40), (13,24.37).
as
primer
for
reverse
transcript ion
whereas the murine leukemia virus employs tRNAho
and mouse rnanunary
tumar
tRNA Lys3
virus utilizes
(25,38).
The J'-terminal 18 nucleotides of primer tRNA are complementary ta regian
near
site(PBS). tRNA
in
the
5'
end
of
the
RNA,
termed
the
primer
binding
The sequence of this site in HIV-l suggests that the primer
this virus
is also
tRNALj'o3,
isoacceptors in manunalian cells either
355
d
a
-free-
(8,10,11,38,39).
state
or
(28,29).
the
three major
tRN~Lj'u
Retroviral tRNA is found in
associated
with
the
viral
genome
When the viral 70S RNA complex is used ta direct DNA
synthesis cata1yzed by RT associated
one of
than
the
(11,18,24,37,39),
one of
others,
ln HIV-l,
serves
these bound tRNAs, as
a
primer
for
more
tightly
DNA synthesis
the tightly associated tRNA
is
tRNALya3
(16) .
During viral assembly,
tRNA packaging is selective,
and in HIV-l
produced by transfecting COS cells with HIV-1 proviral DNA, isoacceptors,
packaged (16), isoacceptor
tRNALyol.2 and tRNALyo3.
the tRNALyo
are the major abundance tRNAs
These tRNAs are selected from over 100 different tRNA
species
in
mechanism responsible
the
for
cytoplasm.
and we are
this process.
investigating
Previous work has
the
indicated
that the reduction or absence of viral genomic RNA incorporation does not
affect
primer
retroviruses
are
tRNA
incorporation
into
virions(20,26).
PrSS gag and Pr160gag-pol precursors
assernbled using
which are not c1eaved until after viral budding that
the selection of
•
sequences processed
for
and
Pr160 gag -pol
NC and RT proteins, have
it is likely
PrSS gag cantains the amina acid sequence for
prote in (NC),
forms)
(17,41),
tRNALyo may occur by the binding of tRNALya to
either of these proteins.
nucleocapsid
Because
been
shown
89
conta in
Both Ne and RT to
bind
to
the amino acid
1in their fully
tRNALya3
in
vitro
•
(1,27.32). the
The absence of RT protein has a1so been reported to affect
incorporation of primer tRNA in both murine
(21)
and avian
(26)
retrovirus.
In
this
report,
we deterrnine
the
incorporated into wild type HIV-1, and binding
site(PBS),
selective
viral
incorporation
protease,
of
tRNALys
number
of
tRNALYs
molecules
examine the role of the primer
PrSS gsg •
and Pr160gsg-po1 on the
into viral particles.
This
is
accomplished through the analysis of the tRNA population in wild type and mutant HIV-l particles produced by transfecting COS-7 cells with wild type and mutant HIV-l pr"viral DNA. supporting evidence for the involvement of tRNALys packaging .
• 90
The data obtained provide uncleaved Pr160 gs g-po1 in
•
2.3. Materials and Methods
A schema tic representation of the plasmids
Plasmid construction.
used for the transfections carried out in this study are shown in Figure 1.
SVC21 BH10 contains wi1d type HIV-l proviral DNA sequence.
SVC2l
P(-) differs from SVC21. BHlO by a single point mutation at position 25 of
the protease region,
converting Asp25
ta Arg25.
Transfection of
SVC21 P(-) produces noninfectious viral particles containing wild type genomic RNA and unprocessed precursor proteins PrSSoao and Pr1600ag-pol
(12).
Both
SVC21
BH10
and
SVC21
P(-)
are
gifts
from
E.
Cohen,
University of Montreal. SVC21 MSCI contains an in-frame deletion of RT and approximately 1/3 of
integrase,
and was derived from SVC21 BHlO.
completely digested with Biolab). and
deleted
a
fragment
restriction
enzyme MSc1
(New England
The resulting 12 kb fragment, containing the plasmid vector H:i.V-l
electrophoresis, using
the
SVC21 BH10 was
was
fractionated
using
and after extraction from the gel,
mini-spin was
genome,
""luron,
self-1igated
and pheno1-ch10roforrn with
T4
DNA
ligase
agarose
further purified extraction.
The
(Pharmacia).
DNA
pSVGAG-RRE-R
and
sequencing was perforrned to confirrn the deletion. The
construction
and
characterization
of
pSVGAGPOL-RRE-R and pSVGAGPOL-RRE-R P(-) were as previously described (34,35).
Viral
production
from
these
two
plasmids
transfection with the REV protein expression vector,
requires pCMV-rev.
coCo-
transfection of pSVGAG-RRE-R with pCMV-rev produces virus-like particles containing unprocessed PrSS gag precursor prote in, while co-transfection of pSVGAGPOL-RRE-R P (-)
with pCMV-rev produces virus-like particles
containing both unprocessed PrSS lIAlI and
Pr1601lAlI-pol.
AlI restriction and modification enzymes are used according to the specifications of the manufacturer. Production of wild type and mutant BrV-1 virus.
Transfection of
COS-7 cells with wi1d type and mutant proviral DNAs using the calcium
•
phosphate method was as previously described (17). from
the
cell
culture
medium
63
hours
Virus was isolated
post-transfection.
The
supernatant was first centrifuged in a Beckman GS-6R rotor at 3000 rpm
91
•
for
30
minutes,
t~ie
and
virus was
then pelleted from the resulting
supernatant by centrifuging in a Beckman Ti45 rotor at 35,000 rpm for one hour.
The viral pellet was then purified by centrifugation at
26,500 rpm for l hour through
l5~
sucrose cnte a
sucrose cushion,
65~
using a Beckman SW4l rotor. Isolation of viral RNA, and human placental tRNALyo isoacceptors.
Total viral RNA was extracted from viral pellets using the guanidinium isothiocyanate procedure (4). The purification of tRNALys3 and tRNALysl,2 from human placenta was performed as previously described (16).
The term tRNALysl,2 refers to a
population of two tRNALys species which differ by one base pair in the anticodon stem (28).
In this paper, spots 1 and 2 are collectively
referred to as tRNALysl,2 since we have not sufficiently characteraed these two species to distinguish tRNALysl from tRNALys2 (16). RNA labeling.
The fractionated RNA samples were labeled using the
32 pCp 3' end-labeling technique (3). 32pCp was made as follows: 5 mCi 32 of garnma- P-ATP (specific activity 3000 Ci/mmole, Dupont, Canada) was dried down in a microcentrifuge tube using N2 .
100 ul of the following
reaction solution was added (reaction solution: 50 mM Tris-HCl, pH 9.2, 5 mM MgC12' 3 mM di thiothrei tol, 5~ bovine serum albumin, 1 uM 3'cytidine monophosphate,
and 10 units T4 kinase).
The reaction was
incubated at 37°c for 3 hours, and the conversion of 3'-CMP to 32pCp was monitored using PEI thin layer chromatography in 0.8 M NH 2S0 4 , which separa tes 32pCp from AT32p. Labeling of the RNA with 32pCp was as previously described (3,18). After labeling, free 32pCp was removed from the labeled macromolecules either
using
G-50
Sephadex
(Pharmacia)
home-made
spin
columns,
equilibrated with TE buffers (10 mM Tris, 7.5; 1 mM EDTA) , or during the electrophoresis run.
Before analysis by polyacrylamide electrophoresis,
the samples were heated at 90°C for 2 minutes. One- and two-dimensional polyacrylamide gel electrophoresis (lDPAGE and 2D-PAGE).
Electrophoresis of viral RNA was carried out at 4°C
using the Hoeffer SE620 gel electrophoresis apparatus.
•
cm x 32 cm.
The first dimension was run in a
10~
Gel size was 14
polyacrylamide/7 M
urea gel for approximately 16 hours at 800 volts, until the bromephenol blue dye was beginning to elute from the bot tom of the gel.
92
After
•
autoradiography,
the
piece
of
gel
c:"ntaining
RNA was
eut
out
and
embedded in a second gel (20% polyacrylamide/7 M urea) and run for 30 hours
(25
watt
limiting),
followed
by
autoradiography.
All
electrophoret:c runs were carried out in 0.5 x TBE (1 X TBE = 100 mM Tris; l mM borie acid; 2 mM EDTA-Na2)' The electrophoretic gel patterns shown in this paper show only low molecular weight RNA, since the high molecular weight viral genomic RNA cannot enter the polyacrylamide gels. Furthermore,
these patterns represent only the most abundant
tRNA
species present, since the high specifie activities of the labeled tRNAs
used
will
reveal
more
minor
aboJndance
species
with
longer
film
exposures.
The signal intensity of each radioactive low molecular weight RNA species in the 2D-PAGE RNA pattern was determined using phosphor-imaging (BioRad, Toronto, Ont.). Measurement of tRNALyo3 using RNA-DNA hybridization. amount of tRNALys3 in viral RNA,
To measure the
we have synthesized an 18-mer DNA
oligonucleotide complementary to the 3' 18 nucleotides of tRNALys3 - (5' UGGCGCCCGAACAGGGAC 3'). This probe hybridizes specifically with tRNALys3 (16,17),
and was hybridized to dot blots on Hybond N (Amersham) of either purified human placental tRNALys3 or total RNA from wild type and mutant virus.
The DNA oligomer was first 5' -end labeled using T4 polynucleotide kinase and gamma- 32 P-ATP (3000 Ci/mMol, Dupont Canada), and specifie activities lOS to 10 9 cpm/ug were generally reached.
Approximately
10 7
cpm oligomer was
generally
used
per
blot
in
hybridization reactions. Me&surement of viral genomic RNA using quantitative peRo Labeling of primer: l nmol of sense primer is mixed with 50 uci of
32P-gamma-ATP (Dupont), and end labeled with 3 ul of T4 PNK (10 U/ul, Pharmacia) in kinase buffer (70 mM Tris HCl (pH 7.5), 100 mM MgC1 2 , and 50 mM DTT) at 37°C for l hour.
Free radioactive label is removed by
passing through a G2s spin column. Reverse transcription: 10 pmol of cold antisense primer is mixed with a known volume of viral RNA or in vitro-transcribed RNA in a total
•
volume of 16.5 ul. Three drops of oil layer is placed on top of the primer/template
mix
to
avoid
evaporation.
Tubes
containing
primer/template mix are heated at 85°C for 3 min and they are slowly
93
•
cooled down to room temperature to allow primer/template annealing.
8.5
ul of RT master mix (30 mM Tris-HC1, pH 8.3, 5 mM MgC1 2 , 6 mM DTT, 1.5 uM dNTPs, 0.5 ug BSA, 10 units RNase inhibitor (Promega), and 5 units MMLV RT (Pharmacia»
is added to the primer/template mix.
tubes are incubated at 37°C for 1 hour.
Reaction
is terminated by
RT activity
incubation at 95°C for 5 minutes.
Polymsrass chain rsaction:
75 ul of PCR masteL mix (10 ul 10X Taq
DNA polymerase buffer (BRL/GIBCO), 10 ul 2 mM dNTPs,
l ul 32p labeled
primer mix, and 1.25 units of Taq DNA polymerase (BRL/GIBCO»
is added
to the reaction tubes containing reverse transcription products which
are incubated at 95°c. amplification
elongation,
Reaction tubes are subjected to 19 cycles of
(denatu:c:ation,
72°C,
3 min).
94°C,
1 min; annealing GOoe,
1 min; and
Aliquots of PCR products are run on a 8%
polyacrylamide gel with 7 M urea to separate amplification products from excess radioactive primers.
The gel is dried at
soce
for 2 hours, and
then exposed to a phosphor-imaging screen for quantitative measurement on a phosphor-imager.
Quantitativs dstsrmination viral
~
antisense
samplss:
primer
amplification.
o~
gsnomic
Sense primer (JS1) (JAl)
~
in wild typs and mutant
50 ATTCGGTTMGGCCAGGGGG3 0 and
50 GGGATGGTTGTAGCTGTCCC3'
A 148 bp
fragment,
corresponding
are
used
for
to HIV-l
PCR
3B DNA
sequence 843-990, ic amplified. A standard curve is established by using known amounts of in vitro transcribed RNA for RT-PCR reaction.
A 957 base RNA fragment (sense) is
made from a linearized DNA plasmid, pEA2, with T7 RNA polymerase (pEA2 plasmid is kind gift from E.J. Arts of the McGill AIDS Centre).
This
RNA fragment corresponds to HIV-l 3B DNA sequence 473-1420. Quantitation
of genomic
RNA
in wild
type,
mutant viral RNA
samples, and in vitro transcript RNA are done by phosphor-imaging (BioRad).
The relative intensity of amplified signals are used to determine
the concentration of genomic RNA in the viral samples using the standard curve .
• 94
Figure 1. Structure of wi1d type and mutant HIV-1 p1asmids. (B) SVC21 BH10 p1asmid contains the wi1d type HIV-1 provira1 DNA sequence. (C) SVC21 P(-) contains a protease-deficient HIV-1 provira1 DNA due to a point mutation at amino acid 25 in the protease region (12). (D)SVC21 MSC1 is an in-frame RT de1etion mutant derived from SVC21 BH10 as described in Materia1s and Methods. The dashed 1ine of SVC21 MSC1 indicates the de1etion region. (E)pSVGAG-RRE-R and (F)pSVGAGPOL-RRE-R P(-), which serve as temp1ates for the synthesis of either Pr55ga g or unprocessed Pr160ga g-pol, respectively, were constructed as has been previously de~cribed (32,33).
•
•
•
• ~::;
B. lI1IC21 BRl.
1
U3
C.
5... ~
gag
pol
""".hg"
lI1IC21 P(-)
~ U3 5...
1 1
gag
1
LI
pol
'Yffurvpu
-
:Ff-J 031fJ 1
~~:~ OllE I:œ!:.I 'i'fh '0' ... , vpu
D, SVC21 MSC:r PBS
1 U3
~I.lJ
MSCI
gag
1
Jpro
11
-
~,1 int r wl:œ!:.Ir~i:'; :p~ vpu _net env
E, pSVGAG-RRE-R Sac l
!J.-------,g==a:-=g;----.,\ PPE 1
F, pSVGAGPOL-RRE-R P(-)
lUIp~5-Gly~5
Sac l
li
gag
1
Ll
Sal l
pol
ryn!1 ME
1
03
1
~
Figure 2. Two-dimensional PAGE patterns of low molecular weight viral RNA. Electrophoretic conditions are as described in the text .. A, uninfected COS cell; B, wild type virus-SVC21 BHI0; C-F, mutant viral particles: C, SVC21 P(-); D, SVC21 MSC1; E, pSVGAG-RRE-R; and F, pSVGAGPOL-RRE-R P(-).
fi
•
•
..
• ü
w
.. ft
•
,,
.. ~it-:
•
c
c(
98
•
• Table 1. Percentage of tRNALys isoacceptors in total cellular and viral tRNA *
tRNA source A. COSCELL
%tRNALysl,2 (spot 1)\1
%tRNALysl,2 (spot 2)t
%tRNALys3 (spot 3)6
%tRNALys
%!RNALysl,2 %tRNALys3
2.1
1.0
2.6
5.7
1.2
B. SVC21 BHIO
24.7
5.3
31.2
61.2
1.0
C. SVC21 P(-)
19.6
5.6
25.7
50.9
1.0
1.9
0,4
1.9
4.2
1.2
E. pSVGAG-RRE
2.0
0.6
2.1
4.7
1.2
F. pSVGAGPOLRRE-R P(-)
20.5
5.2
24.4
50.1
1.1
D. SVC21 MSCI
• Determined through phosphor-imaging analysis of the 2-D PAGE patterns in figure 2. y Spot 1 in figure 2, t Spot 2 in figure 2. 6 Spot 3 in figure 2.
•
Figure 3. A. Quantitation of tRNALys3 in viral RNA. The specificity of a tRNALys3 DNA oligomer probe has been described previously (15). Purified human placenta tRNALys3 is used as an external standard. Aliquots from the sarne viral RNA sarnples used to quantitate genomic RNA in Figure 2 were used to de termine the arnount of tRNALys3 present. Volumes of viral RNA sarnples used for hybridization are: B, 2 ul; C, 2 ul; D, l ul; E, 2 ul; F, 2 ul.
•
• A
(ng)
0.1 0.2 0.6 1.0 5.0
tRNALys3 Viral RNA
••
samples
B
C
•• 0
E
•• F
125
B
~ 100
-... >.
:=
~ QI
-
.5 QI
~
75 50
.!li QI
a:
25
o
1
234
tRNALys3 (ng)
• 101
5
6
Figure 4. A. Quantitative reverse transcription polymerase chain reaction. Reverse transcription and PCR is performed on in vitro transcribed genomic RNA (used as an external standard) or on known volumes of RNA samples isolated from virus produced by transfecting COS cells with constructs B-F, as designated in Figure 2. Volumes of viral RNA samples used are: B, 0.1 ul; C, 0.1 ul; D, 0.1 ul; E. 1 ul; F. 1 ul. Amplified products are run on a 8% PAGE with 7 M urea to separa te amplified products from excess radioactive primers. Equal amounts of viral RNA samples are also used for PCR amplication without reverse transcription as a negative control. B. Genomic RNA RT-PCR standard curve. The relative intensities of amplified signaIs from in vitro transcribed RNA are used to plot a standard curve. Concentrations of genomic RNA from the viral RNA samples are calculateà from this curve.
•
•
• ï=" a:
-
IL
:::1
0
W
:!
0
a:
0
::
III
r-
IL
oC
w
0
a:
:-! a:
lates,
and
sites
were
R6,
H2
sense
primer and
R7 (35)
primer RT3 'Pa as
DNA
HIV-l RT sequences containing the
amplified.
Using
RT3' Ps (5' GTCAGTGCTGGAATCAGG3'), RT(Del)a(5'CCCGCCCACCAACAGGCG3'),
H2, and R7) were
Using
anti-sense
PHRTRX2: R3 ,
fragments of 1673bp(Fl)
mutational
R6,
sense
antisense
primer
primer
and SVC2l BHIO as DNA templates,
fragment of 430bp(F2) was amplified.
a
The desired FI and F2 fragments
were then mixed, joined, extended, and reamplified.
The final secondary
PCR
and
products
were
phenol-chloroform
restriction enzyme MSCI.
extracted
digested
with
The digested secondary products were separated
by running on a 1% agarose gel, and the 2kbp MSCI fragment was purified and subcloned back to SVC2l MSCI. SVC21 GR was also cons tructed through recombinan t amplified
by
using
sense
primer
RT5'OS,
PCR.
FI was
antisense
primer
RT3'IA(5'TGTTTCCTTTTGTATGGGCAGTTT3'), and SVC2l BHlO as DNA template. F2 was amplified by using antisense primer RT(Del)A, SVC2l BHlO as DNA template, and a 99 nucleotides long oligo sense primer corresponded to HIV-l DNA sequence 3716 to 3814 with the alternation of nucleotides 3740-2,
3749-51,
3752-4,
3764-6,
TGG(Trp) to GAG (Glu) ( see Fig. 1). reamplified as
3776-8,
and 3788-90,
which
change
FI and F2 were joined, extended, and
previously described(15).
The
final
secondary PCR
products were phenol-chcloform extracted and digested with restriction enzyme MSCI.
The digested secondary products were separated by 1%
agarose electrophoresis,
and the 2kbp MSCI fragment was purified and
subcloned back to SVC2l MSCI.
Production of wild-type and mutant Hrv-l COS-7 cells were transfected with wild-type and mutant proviral
•
DNAs by the calcium phosphate method, and virus was isolated from the culture medium 63 h post-transfection as previously described(27) .
126
•
RNA analysis Isolation of viral RNA, purification of human placental tRNALys isoacceptors,
RNA labeling, one and two dimensional polyacrylamide gel
electrophoresis (ID and 2D PAGE), and measurement of tRNALys3 by RNA-DNA hybridization
~ere
performed as previously described(271 .
Tc quantitate the amount of genomic RNA in viral RNA samples. an
antisense oligo 790H(5'CTGACGCTCTCGCACCC3') was used.
790H hybridized
specifically to HIV-l genomic RNA sequence 338-354 (DNA sequence 791807) .
Dot
blot
hybridizations
were
performed
using
Hybond
N
paper(Amersham) with either in vitro-transcribed HIV genomic RNA as a standard or total RNA from wild type and mutant viral particles.
The
DNA oligomer was first S'-end labeled using T4 polynucleotide kinase and gamma- 32 p-ATP (3000 Ci/mMol, Dupont Canada), and specifie activities 10 8 to 10 9 cpm/ug were generally reached.
Approximately 10 7 cpm oligomer
was generally used per blot in hybridization reactions.
A standard
curve is established by using known amounts of in vitro-transcribed RNA for hybridization reaction.
A 957 base RNA fragment
(sense)
from a linearized DNA plasmid, pEA2, with T7 RNA polymerase. fragment corresponds to HIV-l 3B DNA sequence 473-1420. genomic RNA in wild type,
is made This RNA
Quantitation of
mutant viral RNA samples,
and
transcript RNA are done by phosphor-imaging (Bio-Rad).
in vitro
The relative
intensity of amplified signaIs is used to determine the concentration of genomic RNA in the viral samples using the standard curve. Characterization of wild-type and mutant viral particles 63 h post transfection, the cell culture supernatant was clarified by centrifuging in a Beckman Gs-6R rotor at 3,000 rpm for 30 min at 4°C. Aliquots
of
clarified
viral
transcriptase activity Assay,
supernatant
were
saved
for
p24 antigen captured Assay,
reverse
and viral
infectivity assay. (il Reverse transcriptase activity assay 50ul of clarifed supernatant was mixed with 50ul RT mix containing 50mM Tris-Cl, pH8.0, 5mM MgC12, 150mM KCl, 0.5mM EGTA, 0.05\ Triton X100, 2\ ethylene glycol, 5mM dithiothreitol. 0.3mM reduced glutathione.
•
50ug/ml poly(rA)ooligo(dT). 80Ci/mmol).
and 20uCi
[3HldTTP(spec.
activity 50 to
The reactions were incubated at 30°C for 22 h.
l ml of
cold 10\ TCA(in 20mM sodium pyrophosphate) was added to the reaction
127
•
mixed
ta
terminate
the reaction,
and precipitated for
2 h at 4°C .
Incorporated [3HldTTP was precipitated cnte Whatman GF/C filters, which
were then counted for radioactivity in a Packard tri-Carb scintillation analyzer.
(ii) p24 antigen captured assay Clarified supernatant was diluted and assayed using p24 ELISA
detection kit(Abbott, Diagnostics division).
Assay conditions were clone
according ta the manuafacturerls recommandation. (iii) Tissue culture infective dosage-SO%(TCIDSO) assay SOul of clarified supernatant was used for each infectivity assay to
infect MT4
target
cells.
Each viral
sample was
tested at
10
different dilutions,each dilution being replicated eight times.
The
virus-induced cytopathic effect(CPE) was used to score
The
reaction
conditions
described(19) .
of
The assay
the
TCIDso
assay
were
postives.
as
previously
allows for the detection of the wild type
HIV-l virion, SVC21 BHlO, at concentrations
as low as 0.2ng
01
p24/ml
of clarified supernatant.
Western Analysis Transfected cells were washed twice with phosphate buffer saline, and
cellular prote in was
extracted with
IX
RIPA buffer.
Sucrose
cushion-purified viral particles were washed with IX TNE and viral protein was extracted with IX RIPA buffer.
viral
prote!n,
described(S).
and
Western
analysis
Extraction of cellular and
were
clone
as
previously
AlOS patients sera and 12SI-protein A/G(ICN) were used to
detect viral proteins .
• 128
• Figure 1. Schematic representations of wi1d type and mutant Pr1Gogag-pol precursor proteins. DrdI, DrdII, and RdI are de1etion mutants that express truncated Gag-Pol precursor pro teins up to the connection domain of the RT. BspMI is an integrase deletion mutant that expresses truncated Gag-Pol precursor up to the first 30 ami no acids in the integrase sequence. R3a, R3b, RG, A2a, H2, R7, Ha4, Dr2, Ra, and A4 are in frame linker insertion mutants, and the sizes of nucleotide inserts are indicated in the parenthesis. GR is a replacement mutant. A stretch of trytophan residues is replaced with glutamic acids as indicated in the figure. * represents the mutations that affect selective tRNALys3 incorporation, and viral content of processed Gag and Pol proteins.
•
•
Pr160 gag-pol
MA
CA
Ne
RT
PR
IN
:;:;:;:;:;::: lingers palm fingers
I)r
palm
thumb
.. '2'.yT...y.:!'2'yT."'yT.d.... connectlon
IN --'
RNaseH
dl"
Dr dU" R dl"
8spMI ~;:;:;:;:;:;:;~T~Tgrg:?'C
1
fingers palm fingers
palm
thumb
ccnnectlon
.....J
RNaseH
T(6)
R3a T(6)
R3b
-..TyTiP;,TZ!lL
~~.:.:.:::.:.::~'Y'T.'T;:r.''''' _ _. . . J ~ " " " •••••
_-----J __
fingers paim Rngers
palm
thumb
connectlon
l
----J
RNaseH
_
GlnLysGluThrTrp GluThrTrpTrp ThrGluTyrTrp GlnAlaThrTrp lIeProGluTrp GluPheValAsn
•
GR"
GlnLysGluThrGlu G1uThrGluGlu ThrGluTyrGlu GlnAlaThrGlu lIeProGluGlu GluPheValAsn
•
Table 1. Mutations in HIV·1 Pr160g a9-pol Amino acjd changes Mutants a
•
Location b
Mutations(bp}b
From
Ta
B, SVC21 Drdl
358S
D(i29},1(5}
C, SVC21 Drdll
358S
D(947}, I(u,
D, SVC21 Rdl
3827
D(397)
E, SVC21 BspMI
4317
D(724), 1(8)
F, SVC21 R3a
2752
I(S)
S
SEF
G, SVC21 R3b
2752
I(S}
S
SAG
H, SVC21 RS
3270
I(S)
V
VNS
l, SVC21 A2a
3300
I(S)
S
RNS
J, SVC21 H2
3472
1(9}
a
alEF
K, SVC21 R7
3489
I(S)
V
VNS
L, SVC21 Ha4
3552
1(12)
G
GANSR
M, SVC21 Dr2
3715
I(S)
F
FAG
N, SVC21 R8
3827
D(1},1(7}
Y
SRF
0, SVC21 A4
3884
I(S)
A
AGIP
P, SVC21 GR
3740
R(18)
W
E
a Mutant names are preceded by letter designations identical to those used to label panels in Figure 2, b The nucleotide location is based on the HXB2 HIV·l proviraJ DNA sequence. D, deletion; l, insertion; A, replacement. The first four mutations in the table(B,C,D, E) contain 3' deletions of Pr160gag-pol coding .sequence. The position of the 5' terminus of each deletion is IIsted under 'Location'. In the 'mutations' column, the length of the deletion is listed in the flrst parenthesis, and any added inserted bases are Iisted in the second parenthesis. The last mutation in the table, P. designates a cluster of 6 tryptophan amino acids(beginning at 3740}, separated on average by 3 amino acids, which have been replaced with 6 glutamic acids(a tolal of 18 bases replacement). The remaining mutations, except for N, represent 2·4 amino acid insertions at the location designaled. N was constructed by deleting one base and inserting 7 bases at the location Iisted.
Figure 2. 2-D PAGE patterns of low molecular weight viral RNA. Electrophoretic conditions were as described in the text. (A) wild type virus(SVC21 BHI0); B to P, mutant viruses: (B),SVC21 DrdI ; (C),SVC21 DrdII ; (D),SVC21 RdI ; (E),SVC21 BspMI ; (F),SVC21 R3a ; (G),SVC21 R3b; (H),SVC21 R6 ; (I),SVC21 A2a ; (J),SVC21 H2 ; (K),SVC21 R7 ; (L),SVC21 Ha4 ; (M),SVC21 Dr2 ; (N),SVC21 RB ; (O),SVC21 A4 ; (P),SVC21 GR .
•
•
•
•
....
.. ~
Q
":'.,
t'", -
.'",."
'. :i,:.'.".. H:
~
v'l "' Q) "oS' "ct:J ..Y "e': oS'.
"oS' ~
{()~ 1
oS'.
0
"oS'
"~ b
"ct:J
"%,...~ ""1iS'.
~
-j,
...",,'10' •
"'10''";.
• ••
•
"0
•• • •
"'0
~
!J' "E!
~.g
a"-a
OUl
• .66-
,
c9/~0
0.(:
~~'1Q1 •
'YQ) 0"1 0 ~~
\S'I. 0"1 '"
.--.
\S'I~
~lt.:t.>nct.>s.
pr..Jt02in th~
t0r
selectivt:-'
5uggt'stin\.j th'lt
pdC"kd,::11ng
L't
pI'lnlt'f
tRNALyn3 and proteolytic cleavage of the vlrd: prote in pI-ècursurc.
1 RT is di"'ided into the fingers, H domains.
toJhile
packaging,
a
palm,
IN sequences drt'
selective viral
ct
C"0nnectiùn.
dispensàblt>
carboxyl deletion which
the RNase H demain and
thumb.
tor
includes aIl ot
tiIV
dnd the RNrloSl'
selectlvt.·
the
thl.'
IN
tRN/\l.y:;
sequènct.~S,
portion of the connection dotndin ot RT inhibits
packaging
of
into the connection damain of
insertiLm~~
Small amino acids
tRNAl.yn3.
the RT or amino deid sequences
just
N~
terminus of the RT connection damain inhibits the selective pdckaging ot primer
tRNALys3
finger,
into HIV-l virus.
but amino dcids insertions inta the
palm. part oE the thumb. and th€ RNase H demains oE RT does not
afEect the selective viral packaging eE primer tRNALyu3 inte the HIV-l. A direct correlation is observed between mutations that aEfect tRNALyuJ packaging ar.d the absence of mature Gag anà Gag-Pol virus.
proteins
in
the
It is possible that mutations in the connection demain of RT
inhibits
selective
efficiency
of
the
tRNALys3 Gag-Pol
packaging precursor
by
hindering
proteins.
and
the
packaging
that
selective
packaging oE tRNALysJ into HIV-l does not occur in the absence of CagPol protein packaging.
Chapter 4
From an in vitro RT assay that uses total viral RNA as the source
oE primer/ternplate. we have shown. that a protease deEective HIV-l which contains unprocessed Gag and Gag-Pol genomic placement of primer tRNA.
precursors still
has wild
type
This suggests that both viral protein
and genomic RNA maturation are not required for the genomic placement of primer
tRNALysJ.
probably occurs
and that
the genornic placement of HIV-l primer tRNA
prior
the maturation of
to
the vir.3.1
core.
An
IN
deletion mutant virus containing a truncated Gag-Pol precursor lacking
90% of
the
IN sequence from the carboxyl
genomic placement of primer tRNA.
•
end also shows wild type
This suggests that the IN sequences
is dispensable for the genomic placement of primer tRNA. SVC21 GR containing a
rnut~tion
Mutant virus
in the connection domain of RT, has been
shown to have non-selective viral tRNA packaging. and lacks mature Pol
186
•
gene sequences,
i.e.
p66 RT and p32
IN sequences.
Primer extension
analysis of SVC21 GR viral RNA shows a significant reduction of primer tRNA Ly.3 placement cnte the HIV-I genome. several possibilities:
genomic
plc!lcement.
1}
2}
This cou Id be explained by
Select primer tRNA may be a perquisite for
Genomic
placement
does
not
Pr160g·g-pol is not packaged into the viral particles.
oceur
because
3) The mutated
sequences in the SVC21 GR mutant(tryptophan repeat sequence) directly
blacks the mechanism of primer tRNA genornic placement in vivo .
• 187
