Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins 1 1Edited by J. Karn

Cherry, Elana; Liang, Chen; Liang, Rong; Quan, Yudong; Inouye, Phil; Li, Xuguang; Morin, Nicolas; Kotler, Moshe; Wainberg, Mark A.

doi:10.1006/jmbi.1998.1968

Cited by 42 publications

(38 citation statements)

References 59 publications

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“…However, few studies have attempted to define the functional consequences at the virus level of mutating these PR-recognized cleavage sites in the Pol polyprotein. Mutation of the PR-RT junction prevented cleavage at this site and resulted in virions containing RT subunits of 77 kDa (corresponding to the PR-RT p66 fusion) and 62 kDa (corresponding to the PR-RT p51 fusion) (11,12). The mutant virions showed wild-type levels of RT activity and were only slightly attenuated in infectivity.…”

Section: Fig 2 Western Blot Analysis Of Pr160mentioning

confidence: 99%

Virion Instability of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Mutated in the Protease Cleavage Site between RT p51 and the RT RNase H Domain

Abram

Parniak

2005

J Virol

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Each of the human immunodeficiency virus type 1 (HIV-1) pol-encoded enzymes, protease (PR), reverse transcriptase (RT), and integrase (IN), is active only as a dimer (or higher-order oligomer in the case of IN), but only RT comprises subunits of different masses. RT is a heterodimer of 66-kDa and 51-kDa subunits. The latter is formed by HIV PR-catalyzed cleavage of p66 during virion maturation, resulting in the removal of the RNase H (RNH) domain of a p66 subunit. In order to study the apparent need for RT heterodimers in the context of the virion, we introduced a variety of mutations in the RT p51-RNH protease cleavage site of an infectious HIV-1 molecular clone. Surprisingly, rather than leading to virions with increased RT p66 content, most of the mutations resulted in significantly attenuated virus that contained greatly decreased levels of RT that in many cases was primarily p51 RT. IN levels were also reduced in several mutants. However, most mutants showed normal levels of the Pr160 gag-pol precursor polyprotein, suggesting that reduced virion RT arose from proteolytic instability rather than decreased incorporation. Mutant virion p24 Gag levels were equivalent to wild type, indicating that Gag incorporation and processing were not affected. Repeated passage of MT-2 cells exposed to mutant viruses led to the appearance of virus with improved replication capacity; these virions contained normally processed RT at near-wild-type levels. These results imply that additional proteolytic processing of RT to the p66/p51 heterodimer is essential to provide proteolytic stability of RT during HIV-1 maturation.The human immunodeficiency virus type 1 (HIV-1) genome encodes a variety of different proteins, including the essential viral enzymes protease (PR), reverse transcriptase (RT), and integrase (IN). These viral enzymes are translated not as discrete units but rather as segments of a much larger polyprotein termed Pr160 gag-pol , and nascent virions assemble using these polyprotein precursors. The individual active enzymes are subsequently formed by proteolytic cleavage at specific sites on Pr160 gag-pol during virion assembly and budding, a "maturation" process catalyzed by PR (25,34).The monomeric subunits of the HIV-1 enzymes are inactive; each enzyme must oligomerize to at least a dimer for enzymatic activity (3,16,64). PR and IN are homodimers (or perhaps higher-order homo-oligomers in the case of IN) with subunits of the size predicted from their genes. The mature active form of HIV-1 PR is a symmetrical homodimer released from Pr160 gag-pol upon autoprocessing carried out by the polyprotein form of the enzyme (45). RT and IN are formed after activation of PR, but it is still unclear whether the PRmediated proteolytic processing of these Pol proteins occurs in cis, in trans, or in some combination of these (56, 57). Like HIV-1 PR, active HIV-1 IN is at least a homodimer, although higher-order homo-oligomers may play a role in the multiple activities of this enzyme (30, 31). In contrast, RT in mature infectio...

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Section: Fig 2 Western Blot Analysis Of Pr160mentioning

confidence: 99%

Virion Instability of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Mutated in the Protease Cleavage Site between RT p51 and the RT RNase H Domain

Abram

Parniak

2005

J Virol

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“…1). The N-terminal cleavage is critical for protease (PR) maturation (14,15,19), while the C-terminal cleavage has negligible influence on protease activity (5,14). The mature protease exists as stable dimers (dimer dissociation constant [K d ], Ͻ10 nM) with the catalytic site formed at the dimer interface by two aspartic acids, each contributed by one monomer (13).…”

mentioning

confidence: 99%

Modulation of Human Immunodeficiency Virus Type 1 Protease Autoprocessing by Charge Properties of Surface Residue 69

Huang

Sayer

Swinford

et al. 2009

J Virol

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Mature, fully active human immunodeficiency virus protease (PR) is liberated from the Gag-Pol precursor via regulated autoprocessing. A chimeric protease precursor, glutathione S-transferase-transframe region (TFR)-PR-FLAG, also undergoes N-terminal autocatalytic maturation when it is expressed in Escherichia coli. Mutation of the surface residue H69 to glutamic acid, but not to several neutral or basic amino acids, impedes protease autoprocessing in bacteria and mammalian cells. Only a fraction of mature PR with an H69E mutation (PR H69E ) folds into active enzymes, and it does so with an apparent K d (dissociation constant) significantly higher than that of the wild-type protease, corroborating the marked retardation of the in vitro N-terminal autocatalytic processing of TFR-PR H69E and suggesting a folding defect in the precursor.

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“…Unlike the flanking C-terminal residues (reverse transcriptase region), which do not appear to affect the catalytic activity or the K d in fusion with the protease (9,22,23), the native transframe region, which flanks the N terminus of the protease in the Gag-Pol precursor, drastically influences the dimer stability of the protease indicated by the very low catalytic activity of the protease prior to its maturation at its N terminus ( Fig. 1) (10, 11).…”

mentioning

confidence: 99%

Mutational and Structural Studies Aimed at Characterizing the Monomer of HIV-1 Protease and Its Precursor

Ishima

Torchia

Louis

2007

Journal of Biological Chemistry

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An experimental protocol for folding the mature human immunodeficiency virus-1 (HIV-1) protease is presented that facilitates NMR studies at a low protein concentration of ϳ20 M. Under these conditions, NMR spectra show that the mature protease lacking its terminal ␤-sheet residues 1-4 and 96 -99 (PR 5-95 ) exhibits a stable monomer fold spanning the region 10 -90 that is similar to that of the single subunit of the wildtype dimer and the dimer bearing a D25N mutation (PR D25N ). Urea-induced unfolding monitored both by changes in 1 H-15 N heteronuclear single quantum correlation spectra and by protein fluorescence indicates that although PR 5-95 monomer displays a transition profile similar to that of the PR D25N dimer (50% unfolded (U 50 ) ‫؍‬ ϳ1.9 M), extending the protease with 4 residues (SFNF) of its N-terminally flanking sequence in the Gag-Pol precursor ( SFNF PR D25N ) decreases the stability of the fold (U 50 ‫؍‬ ϳ1.5 M). Assigned backbone chemical shifts were used to elucidate differences in the stability of the PR T26A (U 50 ‫؍‬ 2.5 M) and SFNF PR D25N monomers and compared with PR D25N/T26A monomer. Discernible differences in the backbone chemical shifts were observed for N-terminal protease residues 3-6 of SFNF PR D25N that may relate to the increase in the equilibrium dissociation constant (K d ) and the very low catalytic activity of the protease prior to its autoprocessing at its N terminus from the Gag-Pol precursor.

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Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins 1 1Edited by J. Karn

Cited by 42 publications

References 59 publications

Virion Instability of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Mutated in the Protease Cleavage Site between RT p51 and the RT RNase H Domain

Virion Instability of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Mutated in the Protease Cleavage Site between RT p51 and the RT RNase H Domain

Modulation of Human Immunodeficiency Virus Type 1 Protease Autoprocessing by Charge Properties of Surface Residue 69

Mutational and Structural Studies Aimed at Characterizing the Monomer of HIV-1 Protease and Its Precursor

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