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.jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117 J+Virol
2014 ; 88
(23
): 13722-31
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Induced maturation of human immunodeficiency virus
#MMPMID25231305
Mattei S
; Anders M
; Konvalinka J
; Kräusslich HG
; Briggs JA
; Müller B
J Virol
2014[Dec]; 88
(23
): 13722-31
PMID25231305
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HIV-1 assembles at the plasma membrane of virus-producing cells as an immature,
noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the
viral protease (PR) activates the viral enzymes and results in dramatic
structural rearrangements within the virion--termed maturation--that are a
prerequisite for infectivity. Despite its fundamental importance for viral
replication, little is currently known about the regulation of proteolysis and
about the dynamics and structural intermediates of maturation. This is due mainly
to the fact that HIV-1 release and maturation occur asynchronously both at the
level of individual cells and at the level of particle release from a single
cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on
protease inhibitor (PI) washout from purified immature virions, thereby
temporally uncoupling virus assembly and maturation. Drug washout resulted in the
induction of proteolysis with cleavage efficiencies correlating with the off-rate
of the respective PR-PI complex. Proteolysis of Gag was nearly complete and
yielded the correct products with an optimal half-life (t(1/2)) of ~5 h, but
viral infectivity was not recovered. Failure to gain infectivity following PI
washout may be explained by the observed formation of aberrant viral capsids
and/or by pronounced defects in processing of the reverse transcriptase (RT)
heterodimer associated with a lack of RT activity. Based on our results, we
hypothesize that both the polyprotein processing dynamics and the tight temporal
coupling of immature particle assembly and PR activation are essential for
correct polyprotein processing and morphological maturation and thus for HIV-1
infectivity. IMPORTANCE: Cleavage of the Gag and Gag-Pol HIV-1 polyproteins into
their functional subunits by the viral protease activates the viral enzymes and
causes major structural rearrangements essential for HIV-1 infectivity. This
proteolytic maturation occurs concomitant with virus release, and investigation
of its dynamics is hampered by the fact that virus populations in tissue culture
contain particles at all stages of assembly and maturation. Here, we developed an
inhibitor washout strategy to synchronize activation of protease in wild-type
virus. We demonstrated that nearly complete Gag processing and resolution of the
immature virus architecture are accomplished under optimized conditions.
Nevertheless, most of the resulting particles displayed irregular morphologies,
Gag-Pol processing was not faithfully reconstituted, and infectivity was not
recovered. These data show that HIV-1 maturation is sensitive to the dynamics of
processing and also that a tight temporal link between virus assembly and PR
activation is required for correct polyprotein processing.
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