| Summary: | 博士 === 國立陽明大學 === 臨床醫學研究所 === 106 === Abstract
HIV-1 protease (PR) is encoded by pol, which is initially translated as a
Pr160gag-pol polyprotein by a ribosomal frameshift event. PR functions as a
homodimer mediating virus maturation following virus budding. Gag-Pol
dimerization is believed to trigger embedded PR activation by promoting PR
dimer formation. Early PR activation can lead to markedly reduced virus yields
due to premature Gag cleavage. Within Gag-Pol, truncated p6gag is replaced
by a transframe region (referred to as p6pol or p6*) located directly upstream of
PR. The p6* peptide is believed to ensure virus production by preventing early
PR maturation. Overlapping reading frames between p6* and p6gag present a
challenge to researchers using genetic approaches to studying p6* biological
functions. To determine the role of p6* in PR activation without affecting the
gag reading frame, we constructed a series of Gag/Gag-Pol expression
vectors by duplicating PR with or without p6* between PR pairs, and observed
that PR duplication eliminated virus production due to significant Gag cleavage
enhancement. This effect was mitigated when p6* was placed between the two
PRs. Further, Gag cleavage enhancement was markedly reduced when either
one of the two PRs was mutationally inactivated. Additional reduction in Gag
cleavage efficiency was noted following the removal of p6* from between the
two PRs. Next, we engineered multiple constructs derived from Dp6*PR (an
assembly- and processing-competent construct with Pol fused at the
inactivated PR C terminus). The data indicated that a p6* deletion adjacent to
active PR significantly impaired virus processing. We also observed that the
insertion of a leucine zipper (LZ) dimerization motif in the deleted region
eliminated virus production in a PR activity dependent manner, suggesting that
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the LZ insertion triggered premature PR activation by facilitating PR dimer
formation. As few as four C-terminal p6* residues remaining at the p6*/PR
junction were sufficient to restore virus yields, with a Gag processing profile
similar to that of the wild type. To clarify the involvement of C terminal p6*
residues in mitigating enhanced LZ-incurred Gag processing, we engineered
constructs containing C-terminal p6* residue substitutions with and without a
mutation blocking the p6*/PR cleavage site. The p6*-PR cleavage blocking did
not significantly reduce the LZ enhancement effect on Gag cleavage when
only four amino acid residues were present between the p6* and PR. This
suggests that the potent LZ dimerization motif may enhance PR activation by
facilitating PR dimer formation, and that PR precursors may trigger sufficient
enzymatic activity without breaking off from the PR N-terminus. We also
observed that a proline substitution at the P3 position eliminated the ability of
p6*-deleted Gag-Pol to mediate virus maturation, thus emphasizing the
importance of C-terminal p6* residues to modulating PR activation.
Supporting evidence for the assumption that p6* retards PR maturation in
the context of virus assembly is lacking. We found that replacing p6* with a
leucine zipper peptide abolished virus assembly due to the significant
enhancement of Gag cleavage. However, C-terminal p6* tetra-peptides
remaining in the deleted region were sufficient for significant PR release, as
well as for counteracting leucine zipper incurred premature Gag cleavage. Our
data provide evidence that (i) p6* ensures virus assembly by preventing early
PR activation and (ii) four C-terminal p6* residues are critical for modulating
PR activation.
Key words: HIV-1 , virus maturation, protease, ribosomal frameshift,
transframe region (p6pol or p6*), leucine zipper (LZ), tetra-peptide
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