Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products.

Hu, Yu-Wen; Kang, C. Yong

doi:10.1073/pnas.88.11.4596

Cited by 14 publications

(12 citation statements)

References 29 publications

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“…Hu & Kang (1991) reported that many forms of artificial HIV pol gene products, including partly cleaved forms, possessed RT activity in activity gels. However, using activity gel analysis of HIV RT, Starnes et al (1988) demonstrated that p66 was the only polypeptide expressing HIVspecific DNA polymerase activity in infected H9 cells, and interpreted this polypeptide species as being derived from both intracellular RT and budded but trapped virions.…”

Section: Discussionmentioning

confidence: 99%

Initiation of reverse transcription during cell-to-cell transmission of human immunodeficiency virus infection uses pre-existing reverse transcriptase

Stephenson

Brennan

Karageorgos

et al. 1994

Journal of General Virology

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Section: Discussionmentioning

confidence: 99%

Initiation of reverse transcription during cell-to-cell transmission of human immunodeficiency virus infection uses pre-existing reverse transcriptase

Stephenson

Brennan

Karageorgos

et al. 1994

Journal of General Virology

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“…The pJV.PR-RT,B.COLL expression vector contains a COLL mutation which replaces the Leu-Thr residues of the PR-RTcx cleavage site with the consensus cleavage site for collagenase, Pro-Leu-Gly-Pro. Symbols: 0 of human immunodeficiency virus (HIV) (22). The recombinant proteins undergoing specific proteolysis all have Gag sequences upstream of the PR domain.…”

Section: Resultsmentioning

confidence: 99%

“…Interpretation of these results is further complicated by the observations that different methods of purification (metal chelate or ion-exchange chromatography) or expression (bac-VOL. 67, I1993 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from 7594 STEWART AND VOGT teria or insect cells) can drastically affect the level of RT activity of HIV PR-RT (22,33). In the pararetrovirus cauliflower mosaic virus, RT was reported to be inactive as a PR-RT fusion (55) but active when the amino-terminal PR domain (56) was proteolytically removed (54) or genetically deleted (55).…”

Section: Discussionmentioning

confidence: 99%

Reverse transcriptase and protease activities of avian leukosis virus Gag-Pol fusion proteins expressed in insect cells

Stewart

Vogt

1993

J Virol

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Protease (PR)-defective avian leukosis virus particles display 300-fold-reduced levels of reverse transcriptase (RT) activity relative to wild-type particles. This observation suggests that during virion assembly RT is activated by proteolytic maturation of the Gag-Pol polyprotein precursor. To study the relationship between proteolytic cleavage and RT activation, we subjected PR-defective virion cores to digestion with purified viral PR and analyzed the structure of the major polypeptides produced as well as RT activity. Under conditions in which Gag precursors were fully matured, the RT domain was only incompletely released from the Gag-Pol precursor, remaining tethered to the upstream Gag domains PR or NC-PR. In the same reaction, RT activity was stimulated only three-fold, or 100-fold less than expected for a fully active RT. The poor activation suggested that the NC or PR domains could repress RT activity. To test this idea, we constructed recombinant baculoviruses expressing 19 different fusion proteins with upstream Gag or downstream Pol sequences attached to RT. Each protein was partially purified and assayed for its inherent RT activity. The results are consistent with the idea that Gag sequences can inhibit RT activity but indicate that the size of the Pol domain as well as the status of the PR domain (wild-type or mutant) also can profoundly influence activity. Several of the constructed Gag-Pol fusion proteins contained a wild-type PR domain. Some of these underwent intracellular PR-mediated processing, while others did not. All proteins in which the PR domain was preceded by upstream Gag sequences showed specific proteolysis. By contrast, all proteins initiated with a methionine placed one residue upstream of the natural N terminus of PR failed to show specific proteolysis. Aminoterminal sequencing of one such protein yielded the correct amino acid sequence and showed that the initiating methionine was not removed. One interpretation of these findings is that activation of PR requires the generation of the precise N terminus of the mature PR.

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“…feet the RT activity. 11,[13][14][15][16][17][18][19] The RT activity in these cells is between 10-and 20-fold higher than in the HT-1080 parental cell line and is stable for at least 30 cell generations, even in the absence of G418 selection. Indirect immunofluorescence of the cells revealed cytoplasmic localization of the expressed RT subunits.…”

Section: Localization Of Reverse Transcriptase Expressionmentioning

confidence: 99%

“…All other studies have depended on bacterial, yeast, or insect cell expression systems, none of which allow the longterm intracellular study of RT. [12][13][14][15][16][17][18][19][20][21][22] In this study the expression of HIV-1 RT in the presence or the absence of PR expression in the HT-1080 human fibrosarcoma cell line was investigated. The RT activity associated with the individual subunits was also examined.…”

Section: Introductionmentioning

confidence: 99%

Analysis of HIV Type 1 Reverse Transcriptase Expression in a Human Cell Line

Ansari-Lari

Gibbs²

1994

AIDS Research and Human Retroviruses

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The functional analysis of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) subunits on transient and constitutive expression, in the absence or presence of the HIV-1 protease (PR) expression, in a human cell line is described. HIV-1 RT is a heterodimer composed of a 51-kDa subunit (p51) and a 66-kDa subunit (p66). Cloning and expression of the RT region of the HIV-1 pol gene in the HT-1080 human fibrosarcoma cell line yielded p66 without any detectable p51 and a low level of RT activity could be measured. Transient expression of PR and RT in cis generated p51 and p66, but when RT and PR were expressed in trans only p66 was produced. Attempts to establish a stable cell line expressing the PR-RT region of the pol gene were hampered by an apparent intolerance of HT-1080 cells to the HIV-1 PR expression. Therefore, to generate p51 independent of PR expression, the 51-kDa subunit was cloned separately. p51 lacked detectable RT activity. Coexpression of p51 and p66 resulted in a dramatic increase in RT activity. Stable HT-1080 cells producing both p51 and p66 exhibited on average a 15-fold increase in RT activity compared to the parental cell line. Immunofluorescence revealed a diffuse cytoplasmic localization of p51 and p66. To date, this is the first example of a human cell line that is constitutively expressing HIV-1 RT in the absence of HIV-1 infection.

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Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products.

Abstract: 4596The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Cited by 14 publications

References 29 publications

Initiation of reverse transcription during cell-to-cell transmission of human immunodeficiency virus infection uses pre-existing reverse transcriptase

Initiation of reverse transcription during cell-to-cell transmission of human immunodeficiency virus infection uses pre-existing reverse transcriptase

Reverse transcriptase and protease activities of avian leukosis virus Gag-Pol fusion proteins expressed in insect cells

Analysis of HIV Type 1 Reverse Transcriptase Expression in a Human Cell Line

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