From owner-btk-mca@hgmp.mrc.ac.uk Wed Jun 14 12:58:59 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 9399617B5B; Wed, 14 Jun 2000 12:58:58 +0100 (BST) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 6024) id 0A73B17A9C; Wed, 14 Jun 2000 12:58:56 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id 6BE8A415DC for ; Wed, 14 Jun 2000 12:58:56 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id MAA26638 for btk-mca-list@hgmp.mrc.ac.uk; Wed, 14 Jun 2000 12:58:55 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Leonard Pattenden X-Newsgroups: bionet.metabolic-reg Subject: Re: Drug design for HIV-1 PR. Date: Wed, 14 Jun 2000 21:58:50 +1000 Organization: University of Queensland Lines: 69 Message-ID: References: <393D6667.B5C164C6@biokin.com> <3940E7A3.59CB46E0@biokin.com> <394126A0.B500AEC1@biokin.com> <3942971B.CDAC475C@biokin.com> <39442CA2.7D79B8FE@biokin.com> Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Trace: bunyip.cc.uq.edu.au 960983932 6699 130.102.2.14 (14 Jun 2000 11:58:52 GMT) X-Complaints-To: news@uq.edu.au X-Sender: ddlpatte@dingo.cc.uq.edu.au In-Reply-To: <39442CA2.7D79B8FE@biokin.com> To: btk-mca@hgmp.mrc.ac.uk Sender: owner-btk-mca@hgmp.mrc.ac.uk Precedence: bulk G'day Petr; Sorry to not have replied sooner, but I have been doing some lit chasing based on your last e-mail and considering what the micro-environment of a virion may be. Firstly, I have found nothing to suggest that a virion needs to be a discreet "delivery bag". Contrary to this there is great evidence a virus can exchange ions and solvent with its' surrounds, some possibly having ion channels to aid this not unlike a dialysis bag I imagine. Under such conditions the viral particle can still be a densely packed, semi-solid body as you describe, or in the words of Tuma and Thomas ["Mechanisms of virus assembly probed by Raman spectroscopy: the icosahedral bacteriophage P22." Biophys Chem 68: 17-31 (1997)] "A molten globule". In this state the virion has all the properties that help it survive in extreme conditions such as heat, acid/base, oxidation and even time. However, even with the ability to exchange I think it occurs relatively slowly as shown by deuterium exchange in model SDS micelles, so your point is still valid, but there may be a window of time for the protease to be active before it is trapped in a "static" state (after reading the literature I hypothesise the gelatenous state is a desired state that is reached to enhance longivity of the viral core - perhaps even from the protease as well as environmental factors). The host-cell protein, cyclophilin A, is also incorporated into HIV-1 particles. Once the viral core gains entry to the cell, the protective envelope about the core is degraded by the absconded cellular enzyme cyclophilin A. This allows the release into the cytoplasm of the pre-integration complex (PIC), which contains the machinery necessary for reverse transcription of the RNA viral sequence and integration into the host genome. But what about the protease? If there is very little of the protease present as you have suggested, what happens? I believe after, or at budding the protease becomes active and starts its little work of chewing up the gag and gag-pol precursors in and acidic and salty microenvironment which is exchanging with the surround to move away from these conditions and more towards a neutral pH (at the viral surface where the protease is active). Proteins that carry out cysteine oxidation reduction can modify the protease to enhance and later stop activity. The protease will also chew itself to pieces, and at more neutral pH, the sequence element in p2 (a critical element for HIV-1 packaging between the NC and gp120 and is close to the virion surface where the pH will be closest to the outside environment), becomes a potent inhibitor of the protease. I believe all these elements conspire against the protease to destroy it once it has completed it's task. Indeed as the particle moves towards an ordered icosahedral shape through polyprotein processing, the protease would be predicted to be less likely to function. So I would think the protease sees optimum conditions for activity for a period, which deteriorates as it's function is realised before inactivation and/or destruction. However, we obviously do not see a huge sink of protease numbers, but rather a very discrete number and quantifiable number of protease molecules - that perhaps, as you say - many of our laws such as the law of mass transfer do not occur, but these laws are also measured in large volumes compared to a virion {(if we take your values of [E] = 1.0 nM in 1.0 mL, having 10^(-9) x 10^(-3) x 6 x 10^(+23) = 6 x 10^(+11) or 600,000,000,000 molecules in 1 x 10^(-6)m^3 compared to 100-120nM or volume of V[t] = 4/3 Pi r[t]^3, r[t] = 9.04 x 10^(-22)m^3 with 20 odd molecules)} I think it all equals about the same thing, so perhaps it is irrelevant and the enzyme behaves the same? Therefore features we see in our assays such as "rate constants" and "equilibrium constants", still apply? Len... .