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Labeling of multiple HIV-1 proteins with the biarsenical-tetracysteine system.

Pereira CF, Ellenberg PC, Jones KL, Fernandez TL, Smyth RP, Hawkes DJ, Hijnen M, Vivet-Boudou V, Marquet R, Johnson I, Mak J - PLoS ONE (2011)

Bottom Line: This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus.We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein.Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Virology, Burnet Institute, Melbourne, Victoria, Australia.

ABSTRACT
Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.

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Related in: MedlinePlus

Genetic labeling of HIV-1 proteins and its impact on virus infectivity.(A) Diagram of the HIV-1 gag (red), pol (brown) and env (blue) open reading frames and the positions where the tetracysteine (TC) tag motif was inserted. The majority of the TC motifs were inserted five amino acids downstream (-N tags) or upstream (-C tags) of the protease cleavage site and the first or last five amino acids of the viral protein coding sequence were repeated to prevent alterations in the minimal recognition site for the viral protease. The left insert shows the crystal structure of the RT enzyme and the positions in the “thumb” region where the TC tag was inserted are shown in blue. The right insert shows the crystal structure of the Env gp120 and the positions in the V1/V2 region where the TC tag was inserted are shown in blue. (B) Virion protein processing profiles of 293T cell-derived HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRT-C, HIVRN-N, HIVRN-C and HIVIN-N (lanes 1-12, respectively). HIV-1 proteins were detected by Western Blot using pooled HIV-positive patient sera. Data are representative of 2 independent experiments. (C) The infectivity of HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRN-N, HIVRN-C and HIVIN-N was assessed by comparing their replication kinetics in peripheral blood mononuclear cells with HIVwt. Samples were collected at 3, 7, 11 and 14 days post infection, and the levels of virus replication were monitored using an in vitro reverse transcriptase assay that is specific for HIV-1 enzymatic activity. Data are representative of 2-3 independent donors. Error bars represent technical replicates. (D) The capacity of several HIVTC to enter target cells was assessed by measuring β-lactamase activity in MT-2 cells that have been infected with HIVTC containing a β-lactamase-Vpr fusion protein. Error bars, s.d. are based on the averages of 2-3 independent experiments. (E) The capacity of HIVTC to infect target cells was assessed by measuring luciferase activity in the indicator TZM-bl cells that have been infected with the indicated viruses. Error bars, s.d. are based on the averages of 2-3 independent experiments.
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pone-0017016-g001: Genetic labeling of HIV-1 proteins and its impact on virus infectivity.(A) Diagram of the HIV-1 gag (red), pol (brown) and env (blue) open reading frames and the positions where the tetracysteine (TC) tag motif was inserted. The majority of the TC motifs were inserted five amino acids downstream (-N tags) or upstream (-C tags) of the protease cleavage site and the first or last five amino acids of the viral protein coding sequence were repeated to prevent alterations in the minimal recognition site for the viral protease. The left insert shows the crystal structure of the RT enzyme and the positions in the “thumb” region where the TC tag was inserted are shown in blue. The right insert shows the crystal structure of the Env gp120 and the positions in the V1/V2 region where the TC tag was inserted are shown in blue. (B) Virion protein processing profiles of 293T cell-derived HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRT-C, HIVRN-N, HIVRN-C and HIVIN-N (lanes 1-12, respectively). HIV-1 proteins were detected by Western Blot using pooled HIV-positive patient sera. Data are representative of 2 independent experiments. (C) The infectivity of HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRN-N, HIVRN-C and HIVIN-N was assessed by comparing their replication kinetics in peripheral blood mononuclear cells with HIVwt. Samples were collected at 3, 7, 11 and 14 days post infection, and the levels of virus replication were monitored using an in vitro reverse transcriptase assay that is specific for HIV-1 enzymatic activity. Data are representative of 2-3 independent donors. Error bars represent technical replicates. (D) The capacity of several HIVTC to enter target cells was assessed by measuring β-lactamase activity in MT-2 cells that have been infected with HIVTC containing a β-lactamase-Vpr fusion protein. Error bars, s.d. are based on the averages of 2-3 independent experiments. (E) The capacity of HIVTC to infect target cells was assessed by measuring luciferase activity in the indicator TZM-bl cells that have been infected with the indicated viruses. Error bars, s.d. are based on the averages of 2-3 independent experiments.

Mentions: In the first labeling strategy we inserted the TC tag within the N-terminus or C-terminus of the major mature Gag and Pol proteins. The natural proteolytic cleavage sites were preserved by duplicating the first or last five amino acids of the viral protein coding sequences of the corresponding mature proteins (Figure 1A and Table 1). Using this strategy the following HIVTC were generated: HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRT-C, HIVRN-N, HIVRN-C and HIVIN-N. The protein processing profiles of these HIVTC were very similar from HIVwt with a few exceptions. HIVRT-N, HIVRT-C, HIVRN-N, HIVRN-C and HIVIN-N have reduced levels or completely lack the p66-RT subunit (Figure 1B). The MA protein in HIVMA-C showed a slower electrophoretic mobility, which is likely due to the additional six amino acids TC tag, as described previously [11], [12]. Similarly, the CA protein in HIVCA-N and HIVCA-C also showed a slower electrophoretic mobility and appeared as a double band in HIVCA-N. The appearance of the CA doublet may result from non-specific protease cleavage at the N-terminus of CA in HIVCA-N. To assess the effect of these modifications on the viral life-cycle, the replication kinetics of HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRN-N, HIVRN-C and HIVIN-N were assessed in peripheral blood mononuclear cells (PBMCs), which are the natural target cells of HIV-1 (Figure 1C). HIVMA-C replication levels and kinetics were very similar to HIVwt. HIVNC-C and HIVPR-C were 54% and 50% less infectious than HIVwt, respectively. The replication kinetics of HIVNC-N was delayed (maximum RT activity at day 11). The remaining viruses were between 79-91% less infectious than HIVwt.


Labeling of multiple HIV-1 proteins with the biarsenical-tetracysteine system.

Pereira CF, Ellenberg PC, Jones KL, Fernandez TL, Smyth RP, Hawkes DJ, Hijnen M, Vivet-Boudou V, Marquet R, Johnson I, Mak J - PLoS ONE (2011)

Genetic labeling of HIV-1 proteins and its impact on virus infectivity.(A) Diagram of the HIV-1 gag (red), pol (brown) and env (blue) open reading frames and the positions where the tetracysteine (TC) tag motif was inserted. The majority of the TC motifs were inserted five amino acids downstream (-N tags) or upstream (-C tags) of the protease cleavage site and the first or last five amino acids of the viral protein coding sequence were repeated to prevent alterations in the minimal recognition site for the viral protease. The left insert shows the crystal structure of the RT enzyme and the positions in the “thumb” region where the TC tag was inserted are shown in blue. The right insert shows the crystal structure of the Env gp120 and the positions in the V1/V2 region where the TC tag was inserted are shown in blue. (B) Virion protein processing profiles of 293T cell-derived HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRT-C, HIVRN-N, HIVRN-C and HIVIN-N (lanes 1-12, respectively). HIV-1 proteins were detected by Western Blot using pooled HIV-positive patient sera. Data are representative of 2 independent experiments. (C) The infectivity of HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRN-N, HIVRN-C and HIVIN-N was assessed by comparing their replication kinetics in peripheral blood mononuclear cells with HIVwt. Samples were collected at 3, 7, 11 and 14 days post infection, and the levels of virus replication were monitored using an in vitro reverse transcriptase assay that is specific for HIV-1 enzymatic activity. Data are representative of 2-3 independent donors. Error bars represent technical replicates. (D) The capacity of several HIVTC to enter target cells was assessed by measuring β-lactamase activity in MT-2 cells that have been infected with HIVTC containing a β-lactamase-Vpr fusion protein. Error bars, s.d. are based on the averages of 2-3 independent experiments. (E) The capacity of HIVTC to infect target cells was assessed by measuring luciferase activity in the indicator TZM-bl cells that have been infected with the indicated viruses. Error bars, s.d. are based on the averages of 2-3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037950&req=5

pone-0017016-g001: Genetic labeling of HIV-1 proteins and its impact on virus infectivity.(A) Diagram of the HIV-1 gag (red), pol (brown) and env (blue) open reading frames and the positions where the tetracysteine (TC) tag motif was inserted. The majority of the TC motifs were inserted five amino acids downstream (-N tags) or upstream (-C tags) of the protease cleavage site and the first or last five amino acids of the viral protein coding sequence were repeated to prevent alterations in the minimal recognition site for the viral protease. The left insert shows the crystal structure of the RT enzyme and the positions in the “thumb” region where the TC tag was inserted are shown in blue. The right insert shows the crystal structure of the Env gp120 and the positions in the V1/V2 region where the TC tag was inserted are shown in blue. (B) Virion protein processing profiles of 293T cell-derived HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRT-C, HIVRN-N, HIVRN-C and HIVIN-N (lanes 1-12, respectively). HIV-1 proteins were detected by Western Blot using pooled HIV-positive patient sera. Data are representative of 2 independent experiments. (C) The infectivity of HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRN-N, HIVRN-C and HIVIN-N was assessed by comparing their replication kinetics in peripheral blood mononuclear cells with HIVwt. Samples were collected at 3, 7, 11 and 14 days post infection, and the levels of virus replication were monitored using an in vitro reverse transcriptase assay that is specific for HIV-1 enzymatic activity. Data are representative of 2-3 independent donors. Error bars represent technical replicates. (D) The capacity of several HIVTC to enter target cells was assessed by measuring β-lactamase activity in MT-2 cells that have been infected with HIVTC containing a β-lactamase-Vpr fusion protein. Error bars, s.d. are based on the averages of 2-3 independent experiments. (E) The capacity of HIVTC to infect target cells was assessed by measuring luciferase activity in the indicator TZM-bl cells that have been infected with the indicated viruses. Error bars, s.d. are based on the averages of 2-3 independent experiments.
Mentions: In the first labeling strategy we inserted the TC tag within the N-terminus or C-terminus of the major mature Gag and Pol proteins. The natural proteolytic cleavage sites were preserved by duplicating the first or last five amino acids of the viral protein coding sequences of the corresponding mature proteins (Figure 1A and Table 1). Using this strategy the following HIVTC were generated: HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRT-C, HIVRN-N, HIVRN-C and HIVIN-N. The protein processing profiles of these HIVTC were very similar from HIVwt with a few exceptions. HIVRT-N, HIVRT-C, HIVRN-N, HIVRN-C and HIVIN-N have reduced levels or completely lack the p66-RT subunit (Figure 1B). The MA protein in HIVMA-C showed a slower electrophoretic mobility, which is likely due to the additional six amino acids TC tag, as described previously [11], [12]. Similarly, the CA protein in HIVCA-N and HIVCA-C also showed a slower electrophoretic mobility and appeared as a double band in HIVCA-N. The appearance of the CA doublet may result from non-specific protease cleavage at the N-terminus of CA in HIVCA-N. To assess the effect of these modifications on the viral life-cycle, the replication kinetics of HIVMA-C, HIVCA-N, HIVCA-C, HIVNC-N, HIVNC-C, HIVPR-C, HIVRT-N, HIVRN-N, HIVRN-C and HIVIN-N were assessed in peripheral blood mononuclear cells (PBMCs), which are the natural target cells of HIV-1 (Figure 1C). HIVMA-C replication levels and kinetics were very similar to HIVwt. HIVNC-C and HIVPR-C were 54% and 50% less infectious than HIVwt, respectively. The replication kinetics of HIVNC-N was delayed (maximum RT activity at day 11). The remaining viruses were between 79-91% less infectious than HIVwt.

Bottom Line: This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus.We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein.Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Virology, Burnet Institute, Melbourne, Victoria, Australia.

ABSTRACT
Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.

Show MeSH
Related in: MedlinePlus