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MOLECULAR GENETICS OF
MAMMALIAN RETROVIRUS REPLICATION
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Judith
G. Levin, PhD, Head, Section on Viral
Gene Regulation Tiyun
Wu, PhD, Research Fellowc Susan
L. Heilman-Miller, PhD, Postdoctoral Fellowb Yasumasa
Iwatani, PhD, Postdoctoral Fellow Margaret
R. Caplan, BA, Postbaccalaureate
Fellowb Swathi Gopalakrishnan, BA, Postbaccalaureate Fellowc |
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The
goal of our research is to define the molecular mechanisms involved in the
replication of HIV and related retroviruses, studies that are critical for developing
new strategies to combat the AIDS epidemic. We have developed reconstituted
model systems to investigate the individual steps in HIV-1 reverse
transcription, a major target of HIV therapy. Much of our work focuses on the
viral nucleocapsid protein (NC), which promotes highly efficient and specific
viral DNA synthesis. NC is a nucleic acid chaperone, which means that it can
facilitate nucleic acid conformational rearrangements that lead to formation
of the most thermodynamically stable structure. This activity is essential
for viral DNA synthesis. In other studies, our efforts are directed toward
understanding the function of the viral capsid protein (CA) in HIV-1 assembly
and early post-entry events during the course of virus replication in vivo.
Role
of nucleocapsid protein in HIV-1 strand transfer Guo, Heilman-Miller,
Wu, Iwatani, Levin; in collaboration with Gorelick, Musier-Forsyth HIV-1
NC is a small basic protein with two zinc fingers, each containing the
invariant CCHC zinc-coordinating residues. The function of NC in virus
replication is dependent on the protein’s dynamic interaction with
nucleic acids; NC plays a critical role in the two-strand transfer steps that
occur during viral DNA synthesis. We have shown that during minus-strand transfer,
NC nucleic acid chaperone activity destabilizes the highly structured
complementary TAR stem-loop (TAR DNA) at the 3´ end of (-) strong-stop DNA
and inhibits TAR-induced self-priming, a dead-end reaction that competes with
annealing of (-) strong-stop DNA to acceptor RNA, i.e., the strand transfer
reaction. Mutational analysis has demonstrated that both NC zinc fingers are
required for this function. Our recent work has focused on measurement of the
NC-induced conformational changes in (-) strong-stop DNA that form the basis
for the inhibition of self-priming, the influence of nucleic acid structure
on NC nucleic acid chaperone activity, and the removal of the 5´ terminal RNA
fragments generated during RNase H degradation of genomic RNA. In
further studies on NC-mediated inhibition of self-priming, we have
collaborated with Karin Musier-Forsyth and colleagues, who developed a
fluorescence resonance energy transfer (FRET) assay that makes it possible to
monitor directly conformational changes in TAR DNA. The results demonstrate
that, when NC binds to TAR DNA alone, only a modest shift occurs toward less
folded conformations, with little effect on self-priming. However, if
acceptor RNA is present, NC binding to TAR DNA results in a shift of the
majority of molecules to the unfolded state, and self-priming is blocked.
Taken together, the findings suggest that NC-mediated annealing of nucleic
acids is a concerted process involving a destabilization step that occurs in
synchrony with hybridization. In other
work on nucleic acid chaperone activity, we are investigating the structural
and thermodynamic requirements for NC interaction with strand transfer
nucleic acid intermediates. We studied a series of synthetic (-) strong-stop
DNA and acceptor RNA truncation mutants by in vitro assay of
minus-strand transfer and self-priming, enzymatic structure probing, and
analysis of secondary structure using RNA and DNA structure prediction
algorithms. Truncations that disrupt the TAR DNA structure in (-) strong-stop
DNA completely eliminate DNA self-priming. However, the elimination of
self-priming does not necessarily result in an increase in strand transfer
efficiency; the structure of the acceptor RNA is also important. Thus, we
have demonstrated that NC-mediated strand transfer is efficient only when (-)
strong-stop DNA and acceptor RNA are both moderately structured.
Collectively, the data demonstrate that a delicate thermodynamic balance
between (-) strong-stop DNA and acceptor RNA must be maintained for efficient
minus-strand transfer. Recently, we also obtained evidence suggesting that,
for annealing, NC nucleic acid chaperone activity ultimately depends on the
stability of local structure at the nucleation site, not on the stability of
the overall structure. As
(-) strong-stop DNA is synthesized, RNase H catalyzes degradation of the
genomic RNA template and generates short 5´ terminal RNA fragments, which are
initially annealed to the 3´ end of (-) strong-stop DNA. The resulting
hybrids have high Tm values, yet these RNAs must be removed to
allow annealing of (-) strong-stop DNA to acceptor RNA. To assess the roles
of RNase H and NC in this reaction, we have modeled fragment removal in the
context of minus-strand transfer by heat-annealing a 5´ terminal RNA oligonucleotide
to a longer synthetic (-) strong-stop DNA and then adding acceptor RNA,
reverse transcriptase (RT), and NC. Our results demonstrate that, under these
conditions, the efficiency of minus-strand transfer catalyzed by either RNase
H-minus or wild-type RTs is highly similar. Thus, NC nucleic acid chaperone
activity alone can facilitate terminal fragment removal without requiring
secondary RNase H cleavage (as previously thought). Interestingly,
coordination of zinc by the CCHC motifs in NC is required for terminal
fragment removal. Guo J, Wu T, Kane BF, Johnson DG, Heilman-Miller SH, Wu T, Levin JG. Alteration of nucleic acid
structure and stability modulates the efficiency of minus-strand transfer
mediated by the HIV-1 nucleocapsid protein. J Biol Chem 2004;279:44154-44165. Hong M, Harbron EJ, O’Connor DB, Guo J, Barbara PF, Levin
JG, Musier-Forsyth K. Nucleic acid conformational changes essential for HIV-1
nucleocapsid protein-mediated inhibition of self-priming in minus-strand
transfer. J Mol Biol 2003;325:1-10. Nucleic
acid and protein requirements for initiation of HIV-1 reverse transcription Iwatani, Guo, Levin;
in collaboration with Gorelick, Musier-Forsyth We
have been investigating the initiation step in HIV-1 reverse transcription,
an event that is primed by the host tRNA, tRNA3Lys,
which is annealed to the 18-nt primer binding site (PBS) near the 5´ terminus
of the viral RNA genome; extension of the primer leads to synthesis of the
short DNA product known as (-) strong-stop DNA. In earlier work, we demonstrated
that, when NC is present, an additional 24 bases in the template downstream
of the PBS are dispensable for synthesis primed by tRNA, but not for
synthesis primed by an 18-nt RNA complementary to the PBS. We proposed that
NC abrogates this requirement by facilitating stable formation of extended
interactions between the full-length tRNA and the RNA template, which are not
possible with an 18-nt RNA. Mutational analysis supports the possibility that
NC promotes an interaction between the 3´ arm of the anticodon stem, part of
the variable loop of tRNA3Lys, and nt 143 through 149
in viral RNA. To
analyze further the effect of NC on initiation, we used a band-shift assay to
measure the affinity of RT for the viral RNA-tRNA complex in the presence or absence
of NC. In the absence of dNTPs, NC does not affect RT binding to complexes
constituted with either wild-type or mutant templates (substitution of the
complementary bases in nt 143 through 149). In contrast, in a functional
assay with a +1 extension of the tRNA primer, NC stimulates incorporation
with the wild-type templates, but not with the mutant templates. The results
provide further evidence for a specific NC-dependent interaction outside the
PBS region. In additional experiments conducted with low concentrations of
dNTPs (5 microM), we found that NC significantly stimulates tRNA-primed (-)
strong-stop DNA synthesis, even with a template with more than 24 downstream
bases. Taken together, our data suggest that NC-facilitated interactions are
also important in vivo, where dNTP concentrations are thought to be
relatively low. Interestingly, studies with NC zinc finger mutants
demonstrate that zinc coordination is not required for NC stimulation of (-)
strong-stop DNA synthesis, regardless of dNTP concentration. Iwatani Y, Rosen AE, Guo J, Musier-Forsyth K, Levin JG.
Efficient initiation of HIV-1 reverse transcription in vitro:
requirement for RNA sequences downstream of the primer binding site abrogated
by nucleocapsid protein-dependent primer-template interactions. J Biol
Chem 2003;278:14185-14195. Functional
analysis of HIV-2 reverse transcriptase activities Post, Guo, Levin; in
collaboration with Powell, Le Grice, Hizi HIV-2
infection is a significant public health problem in Under
standard assay conditions, the RNase H and DNA- and RNA-dependent DNA
polymerase activities of the two enzymes are comparable. However, when the RT
concentration is significantly reduced, HIV-2 RT is less active than the
HIV-1 enzyme. HIV-2 RT is compromised in its ability to catalyze secondary
RNase H cleavages and to initiate plus-strand DNA synthesis by the polypurine
tract (PPT) primer. Previously, we showed that HIV-1 plus-strand initiation
is dependent on nucleic acid contacts with “primer grip” residues
in the palm subdomain of the p66 RT subunit. We have proposed that the
reduced plus-strand priming activity of HIV-2 RT may reflect architectural
differences in the primer grip regions in the two enzymes. We have also
suggested that HIV-2 RT may have a lower binding affinity for the RNA PPT and
other substrates used in our assays. Taken together, our findings should be
useful for the development of specific high-throughput screening assays of
potential HIV-2 inhibitory agents. Post K, Guo J, Howard KJ, Powell MD, Miller JT, Hizi A, Le Grice
SFJ, Levin JG. Human immunodeficiency virus type 2 reverse transcriptase
activity in model systems that mimic steps in reverse transcription. J
Virol 2003;77:7623-7634. Function
of HIV-1 capsid protein in virus assembly and early post-entry events Tang, Levin; in
collaboration with Freed Our
laboratory has been investigating the role of the HIV-1 capsid protein (CA)
in early post-entry events, a stage in the infectious process that is still
not completely understood. In our initial study, we described the unusual
phenotype associated with single alanine substitution mutations in a group of
N-terminal, conserved hydrophobic residues (including Trp23 and Phe40), using
genetic, molecular, and ultrastructural approaches. We found that mutant
virions are not infectious and lack a cone-shaped core. Moreover, despite
their possessing a functional RT enzyme, the mutants are blocked in the
initiation of viral DNA synthesis in infected cells. The findings demonstrate
the intimate connection among infectivity, proper core morphology, and the
ability to undergo reverse transcription. The data also suggest that the
mutations result in a defect in an early step preceding reverse
transcription, which is correlated with a defect in the assembly of viral
cores. We
modeled disassembly (i.e., viral uncoating) in vitro by generating
viral cores from particles treated with mild detergent and isolating the
cores by sedimentation in sucrose density gradients. We observed two striking
differences in the protein profiles of the mutants and wild type: mutant core
fractions displayed a marked deficiency in RT protein and enzymatic activity
(less than 5 percent of total RT in gradient fractions) and a substantial
increase in the retention of CA. The high level of core-associated CA suggested
that mutant cores may be unable to undergo proper disassembly. Taken together
with the almost complete absence of RT in mutant cores, the findings account
for the failure of the CA mutants to synthesize viral DNA following virus
entry into cells. We
have also performed studies to determine whether substitutions other than
alanine result in a different phenotype. We made 13 additional substitutions
at position 23, two at position 40, and one double mutation. Only one mutant,
W23F, exhibited infectivity, albeit at a very low level. The phenotype of
W23F appears to be intermediate between wild-type virus and the original W23A
mutant. The W23F mutant (unlike W23A) is able to replicate during long-term
culture in MT-4 cells, but with delayed replication kinetics. With continued
passage, we eventually isolated two second-site suppressor mutants, which
replicate like wild-type virus in MT-4 cells. We have also found that the
original W23A mutant has trans-dominant inhibitory activity, i.e.,
W23A can reduce the infectivity of wild-type virus produced by cells
co-transfected with a mixture of wild-type and mutant particles.
Interestingly, the trans-dominant inhibitory activity of W23F is
approximately one fifth of the corresponding activity of W23A. The results
indicate that the virions resulting from co-transfection contain a
co-assembled population of wild-type and mutant CA molecules. Moreover, the
findings imply that collaboration of many Tang S, Murakami T, Cheng N, Steven AC, Freed EO, Levin JG.
Human immunodeficiency virus type 1 N-terminal capsid mutants containing
cores with abnormally high levels of capsid protein and virtually no reverse
transcriptase. J Virol 2003;77:12592-12602. COLLABORATORS Eric O. Freed, PhD, HIV Drug Resistance
Program, NCI, Robert J. Gorelick, PhD, AIDS Vaccine
Program, SAIC Frederick, Inc., NCI, Frederick, MD Amnon Hizi, PhD, Stuart Le Grice, PhD, HIV Drug
Resistance Program, NCI, Frederick, MD Karin Musier-Forsyth, PhD, Michael D. Powell, PhD, Morehouse aDeparted in 2004,
currently Special Volunteer. bDeparted in 2004. cArrived in 2004.
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