Why is it that HIV-1 RT can have a mutation in p66 and still have the...

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Why is it that HIV-1 RT can have a mutation in p66 and stillhave the exact same mutation in p51? help me

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Previous work on mutations in the thumb of HIV1 reversetranscriptase RT showed that the majority of the mutant RTs weredegraded by the viral protease to various extents in virionsThis degradation was in most cases temperature sensitive andpresumably was due to a partial unfolding of the protein at37CWe used recombinant proteins to investigate the effects of themutations on the thermal stability and proteolytic degradation ofRTBoth subunits contribute to the stability of RT In general thedifferences in stability between the mutants and WT were greater ifthe mutation was in p51 rather than p66 Expressing the Polpolyprotein containing the RT mutants in E coli producedresults similar to what was seen in virions the mutant RTs weremisfolded andor degraded at 37C but were better folded andprocessed at 30CThe structural proteins found in mature HIV1 virions aresynthesized as parts of the Gag polyprotein the viral enzymes aremade as parts of the GagPol polyprotein Pol consists of proteasePR reverse transcriptase RT and integrase IN Uncleaved Gagand GagPol coassemble with viral RNA and the envelopeglycoprotein in producer cells to form the immature virionMaturation which is concomitant with virus budding occurs when PRcleaves the Gag and GagPol polyproteins into the structuralcomponents and enzymes found in the mature virionPR is a homodimer dimerization is essential for PR activityHowever in an immature virion PR is embedded within GagPolAlthough PR carries out the majority of the cleavages that areinvolved in maturation the initial processing of GagPol musteither involve cleavages made by PR when it is still embedded inGagPol or be made by some unidentified host protease present inamounts too small to detect PR is known to be one of the lastproteins to be cleaved from GagPol in an in vitro systemThere are reports that either a single or multiple pointmutations within RT destabilizes RT in virions and mutations in RTcould also have more global effects on the processing of GagPol orGag We tested a number of mutations in RT most of which are inthe thumb subdomain for their effects on the replication of aoneround vector A large fraction 80 of the mutations wetested affected the stability of RT in virions The mutant virionscontained reduced amounts of intact p66 and p51 and for some ofthe RT mutants the virions had no intact RT The amounts of IN andPR which are also derived from GagPol in the virions werenormal or nearly normal We showed that the viral PR wasresponsible for the degradation of three of the RTs with mutationsin the thumb Wapling et al showed that PR was responsible for thedegradation of a connection subdomain mutant Although in ourexperiments there was evidence that some of the mutations in RTcaused a small portion of GagPol to be misfolded andormisprocessed the fact that the mutant virions contained normal ornearly normal amounts of PR and IN suggested that the majority ofthe mutant GagPol was appropriately processed and that RT wasdegraded by PR after it was properly cleaved from the polyproteinprecursor Several of the mutations conferred atemperaturesensitive phenotype on both viral replication and RTdegradation the triplemutant described by Huang et al wastemperature sensitiveBecause the fraction of the RT mutations that we tested that ledto PR susceptibility was large we also proposed that thesusceptibility of RT to PR cleavage could be an importantconsideration for determining which RT mutations are and are notacceptable to the virus an idea that has been supported by workfrom the Dykes laboratoryWe have developed E coli expression systems in whichvarious forms of HIV1 RT can be expressed and purified Theseconstructs can be used to express either the p66 subunit or p51subunit by themselves or a p66p51 heterodimer in which only oneof the two subunits is mutated subunit selective expressionAnother expression vector that contains both the p66 subunit codingregion and a separately expressed protease coding region can beused to express and isolate p66p51 heterodimers with the mutationsin both subunits The most recent construct described hereexpresses the entire Pol coding region in E coli seeMethods and is similar to the construct used by Wrobel et alWrobel et al 1998 We show that the purified mature p66p51form of each of the four mutant RTs with mutations in bothsubunits is temperature sensitive Although the E coliexpression systems do not match the conditions either in aninfected eukaryotic cell or in an assembled virion the Ecoli expression systems make it possible to dissect theeffects of the mutations on RT stability in ways that cannot beeasily accomplished using a eukaryotic viral expression system Ingeneral the mutant proteins behaved similarly in virions and inthe E coli expression systems When the mutant RTs wereexpressed in E coli as a component of Pol their behaviorwas similar to what had been seen when the same mutant RTs wereexpressed as part of GagPol and incorporated into virionssuggesting that the behavior of the mutant RTs in virions is due tothe thermal stability of the mature mutant RTs and to theirinteractions with PRMaterial and MethodsPreparation of the expression plasmids and growth of theEcoli strainsThe E coli expression systems used to generate thevarious forms of the HIV1 RT have been described Boyer et al1994 Boyer et al 2001 Hizi et al 1988 The constructs thatexpress the p66 subunit alone leading to p66 homodimers p51subunit alone leading to p51 homodimers and the subunitselective p66p51 which creates heterodimers with the mutation inonly one subunit are based on the vector pUC12N and the proteinsare constitutively expressed The constructs are transformed intoDH5 cells The cells are allowed to grow overnight and areharvested the next day Bacteria were collected by centrifugationfor 5 minutes at 325 g Media was removed and the pellet wasresuspended in PBS For protein purification 1 L of cells wereused and purified as described below For Western blot analysis 4ml cultures grown for two hours were used the OD600 was measuredand equal amounts of E coli were pelleted gently by centrifugationfor 5 minutes at 325 g The supernatant was removed and the pelletwas vortexed and stored 70CThe vector that encodes and expresses the p66 subunit andseparately encodes and expresses protease was used to generatep66p51 heterodimers with the mutations in both subunits For theexperiments described here we generated a new plasmid thatexpresses the entire Pol coding region in E coli The Polregion was derived from the HIV1 clone BH10 Genebank HIVBH102A glycine codon located in p6 nucleotide 1545 22 codons fromthe beginning of the protease coding region was selected as thestart position PCR amplification was used to add a methionineinitiation codon and the sequence around the ATG was modified tocreate an NcoI site CC ATG GGT AGA GAC The same PCRreaction was used to add a HindIII site 3 of the See Answer

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