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Isolated HIV-1 core is active for reverse transcription.

Warrilow D, Stenzel D, Harrich D - Retrovirology (2007)

Bottom Line: Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial.Core-like particles were identified in this active fraction by electron microscopy.We are the first to report the detection of authentic strong-stop, first-strand transfer and full-length minus strand products in this core fraction without requirement for an uncoating activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology and Infectious Disease, Queensland Institute of Medical Research, Brisbane, Queensland, 4006, Australia. David.Warrilow@qimr.edu.au

ABSTRACT
Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial. To address this question we purified cores from a virus culture and tested for the ability to generate authentic reverse transcription products. A dense fraction (approximately 1.28 g/ml) prepared without detergent, possibly derived from disrupted virions, was found to naturally occur as a minor sub-fraction in our preparations. Core-like particles were identified in this active fraction by electron microscopy. We are the first to report the detection of authentic strong-stop, first-strand transfer and full-length minus strand products in this core fraction without requirement for an uncoating activity.

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Analysis of core fractions. (A) Endogenous reverse transcriptase activity: strong-stop (squares), first-strand transfer (diamonds) and full-length targets (triangles) are shown. (B) p24 ELISA on fractions; inset shows the density of fractions calculated from weight (fractions 3–9 only are shown). Viral proteins were detected in HIV-1NL4.3 equilibrium gradient fractions 1–9 by western analysis using (C) anti-HIV-1 polyclonal antibody, (D) colorimetric reverse transcriptase ELISA using homoploymeric template (fractions 5–9 only are shown), and (E) anti-gp41 antibody. (F) Negative staining transmission electron microscopy of dense fractions showing four representative core-like structures. 100,000× magnification; bar indicates 50 nm. Please note, the fractions shown in A and B are from a separate preparation to those in C-E and hence fraction numbers do not directly correspond [see Additional file 1 for complete methods].
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Figure 1: Analysis of core fractions. (A) Endogenous reverse transcriptase activity: strong-stop (squares), first-strand transfer (diamonds) and full-length targets (triangles) are shown. (B) p24 ELISA on fractions; inset shows the density of fractions calculated from weight (fractions 3–9 only are shown). Viral proteins were detected in HIV-1NL4.3 equilibrium gradient fractions 1–9 by western analysis using (C) anti-HIV-1 polyclonal antibody, (D) colorimetric reverse transcriptase ELISA using homoploymeric template (fractions 5–9 only are shown), and (E) anti-gp41 antibody. (F) Negative staining transmission electron microscopy of dense fractions showing four representative core-like structures. 100,000× magnification; bar indicates 50 nm. Please note, the fractions shown in A and B are from a separate preparation to those in C-E and hence fraction numbers do not directly correspond [see Additional file 1 for complete methods].

Mentions: A high titre HIVNL4.3 virus stock was grown on CD4/CXCR4-expressing HeLa cells (MAGI) and was subsequently concentrated by centrifugation on a 20% sucrose cushion. We subjected two virus samples to 20–60% Optiprep density gradient centrifugation for 20 h: one with a detergent layer and a control without a detergent layer. Fractions were obtained and assayed for capsid by p24 ELISA and the ability to generate authentic reverse transcription products (endogenous reverse transcription or ERT activity). Interestingly, with repeated attempts we were not able to detect ERT products using core fractions prepared by brief passage through the detergent layer (data not shown; Warrilow et al., manuscript under review). However, a control preparation without a detergent layer had capsid and ERT activity in fractions 7–9 (Fig 1A, B) corresponding to the reported buoyant density of core (peak fraction 1.29 g/ml). A clear peak in activity was seen, for example, fraction 8 contained 30-fold more ERT activity than fraction 5. These three peak fractions represented 6% of total ERT activity of the fractions. This core fraction was capable of first-strand transfer, and full-length minus strand synthesis was also detectable above background (Fig. 1B). However, the signal was not sufficient to determine whether products indicating second-strand transfer had been generated (data not shown). This result was repeated in three separate experiments. Hence, a naturally occurring core fraction was capable of advanced reverse transcription.


Isolated HIV-1 core is active for reverse transcription.

Warrilow D, Stenzel D, Harrich D - Retrovirology (2007)

Analysis of core fractions. (A) Endogenous reverse transcriptase activity: strong-stop (squares), first-strand transfer (diamonds) and full-length targets (triangles) are shown. (B) p24 ELISA on fractions; inset shows the density of fractions calculated from weight (fractions 3–9 only are shown). Viral proteins were detected in HIV-1NL4.3 equilibrium gradient fractions 1–9 by western analysis using (C) anti-HIV-1 polyclonal antibody, (D) colorimetric reverse transcriptase ELISA using homoploymeric template (fractions 5–9 only are shown), and (E) anti-gp41 antibody. (F) Negative staining transmission electron microscopy of dense fractions showing four representative core-like structures. 100,000× magnification; bar indicates 50 nm. Please note, the fractions shown in A and B are from a separate preparation to those in C-E and hence fraction numbers do not directly correspond [see Additional file 1 for complete methods].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2169257&req=5

Figure 1: Analysis of core fractions. (A) Endogenous reverse transcriptase activity: strong-stop (squares), first-strand transfer (diamonds) and full-length targets (triangles) are shown. (B) p24 ELISA on fractions; inset shows the density of fractions calculated from weight (fractions 3–9 only are shown). Viral proteins were detected in HIV-1NL4.3 equilibrium gradient fractions 1–9 by western analysis using (C) anti-HIV-1 polyclonal antibody, (D) colorimetric reverse transcriptase ELISA using homoploymeric template (fractions 5–9 only are shown), and (E) anti-gp41 antibody. (F) Negative staining transmission electron microscopy of dense fractions showing four representative core-like structures. 100,000× magnification; bar indicates 50 nm. Please note, the fractions shown in A and B are from a separate preparation to those in C-E and hence fraction numbers do not directly correspond [see Additional file 1 for complete methods].
Mentions: A high titre HIVNL4.3 virus stock was grown on CD4/CXCR4-expressing HeLa cells (MAGI) and was subsequently concentrated by centrifugation on a 20% sucrose cushion. We subjected two virus samples to 20–60% Optiprep density gradient centrifugation for 20 h: one with a detergent layer and a control without a detergent layer. Fractions were obtained and assayed for capsid by p24 ELISA and the ability to generate authentic reverse transcription products (endogenous reverse transcription or ERT activity). Interestingly, with repeated attempts we were not able to detect ERT products using core fractions prepared by brief passage through the detergent layer (data not shown; Warrilow et al., manuscript under review). However, a control preparation without a detergent layer had capsid and ERT activity in fractions 7–9 (Fig 1A, B) corresponding to the reported buoyant density of core (peak fraction 1.29 g/ml). A clear peak in activity was seen, for example, fraction 8 contained 30-fold more ERT activity than fraction 5. These three peak fractions represented 6% of total ERT activity of the fractions. This core fraction was capable of first-strand transfer, and full-length minus strand synthesis was also detectable above background (Fig. 1B). However, the signal was not sufficient to determine whether products indicating second-strand transfer had been generated (data not shown). This result was repeated in three separate experiments. Hence, a naturally occurring core fraction was capable of advanced reverse transcription.

Bottom Line: Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial.Core-like particles were identified in this active fraction by electron microscopy.We are the first to report the detection of authentic strong-stop, first-strand transfer and full-length minus strand products in this core fraction without requirement for an uncoating activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology and Infectious Disease, Queensland Institute of Medical Research, Brisbane, Queensland, 4006, Australia. David.Warrilow@qimr.edu.au

ABSTRACT
Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial. To address this question we purified cores from a virus culture and tested for the ability to generate authentic reverse transcription products. A dense fraction (approximately 1.28 g/ml) prepared without detergent, possibly derived from disrupted virions, was found to naturally occur as a minor sub-fraction in our preparations. Core-like particles were identified in this active fraction by electron microscopy. We are the first to report the detection of authentic strong-stop, first-strand transfer and full-length minus strand products in this core fraction without requirement for an uncoating activity.

Show MeSH
Related in: MedlinePlus