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Chapter 25 : Retroviral DNA Integration

Author: Robert Craigie
Content Type: Monograph
Publication Year: 2002

Category: Microbial Genetics and Molecular Biology

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Abstract:

This chapter provides an overview of the current understanding of the molecular mechanism of retroviral DNA integration and points to some of the issues that are not yet well understood. The mechanism of retroviral DNA integration is closely related to the mechanism by which many transposons and retrotransposons move from one location to another in the genome of the host cell. Retroviral integrase is encoded by the 3' part of the gene and is assembled into virus particles as the Gag-Pol polyprotein precursor. Retroviral integrases share a common domain structure, and the biochemical activities of integrase proteins from different retroviruses are fundamentally similar. Integrase has a nonspecific nuclease activity that is most easily monitored by observing nicking of closed circular DNA. Indeed, this nonspecific nuclease activity was the first biochemical activity detected for a retroviral integrase. This activity may reflect an inefficient 3' processing reaction acting on an aberrant DNA substrate. Complementation experiments demonstrate that HIV-1 integrase functions as a multimer. Individual proteins lacking either the N-terminal or the C-terminal domain are inactive both for 3' end processing and DNA strand transfer. Mu-mediated PCR footprinting has been used to probe the nucleoprotein organization within preintegration complexes (PICs). Several cellular proteins have been implicated to play a role in retroviral DNA integration. Further studies of retroviral DNA integration will be needed to address these and other questions for which there are only partial answers at this time.

Citation: Craigie R. 2002. Retroviral DNA Integration, p 613-630. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch25
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Retroviral DNA Integration
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Citation: Craigie R. 2002. Retroviral DNA Integration, p 613-630. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch25
/content/10.1128/9781555817954.chap25.fig25-1
Figure 1

DNA cutting and joining steps in retroviral integration. (A) The viral DNA (thick lines) made by reverse transcription is linear and blunt ended. (B) In the first step of the integration process, 3′end processing, two or three nucleotides are cleaved from each 3′end of the viral DNA. (C) In the next step, DNA strand transfer, the hydroxyl groups at the 3′ends of the processed viral DNA attack a pair of phosphodiester bonds in the target DNA (thin lines). The spacing between the sites of attack on each target DNA strand is fixed and characteristic for each retrovirus. (D) The resulting integration intermediate is redrawn to clarify the connections between viral and target DNA. Integrase is responsible for both the 3processing and DNA strand transfer reactions that give rise to the integration intermediate. Completion of DNA integration requires removal of the two unpaired nucleotides at the 5′ends of the viral DNA, filling in the single-strand gaps between host and viral DNA by a DNA polymerase, and finally ligation. These steps are likely to be performed by cellular enzymes (8, 154).

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Figure 1

DNA cutting and joining steps in retroviral integration. (A) The viral DNA (thick lines) made by reverse transcription is linear and blunt ended. (B) In the first step of the integration process, 3′end processing, two or three nucleotides are cleaved from each 3′end of the viral DNA. (C) In the next step, DNA strand transfer, the hydroxyl groups at the 3′ends of the processed viral DNA attack a pair of phosphodiester bonds in the target DNA (thin lines). The spacing between the sites of attack on each target DNA strand is fixed and characteristic for each retrovirus. (D) The resulting integration intermediate is redrawn to clarify the connections between viral and target DNA. Integrase is responsible for both the 3processing and DNA strand transfer reactions that give rise to the integration intermediate. Completion of DNA integration requires removal of the two unpaired nucleotides at the 5′ends of the viral DNA, filling in the single-strand gaps between host and viral DNA by a DNA polymerase, and finally ligation. These steps are likely to be performed by cellular enzymes (8, 154).

Citation: Craigie R. 2002. Retroviral DNA Integration, p 613-630. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch25
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Figure 2

Domain structure of retroviral integrases. Retroviral integrases are composed of three structural domains. The central core domain contains the D,D-35-E motif, the triad of acidic residues that play a key role in catalysis. This domain is structurally highly conserved among many mobile genetic elements, including some prokaryotic transposons. The N-terminal (N-term) domain includes the HHCC motif that binds zinc. This domain is conserved among retroviruses and retrotransposons, but a related domain has not been seen in nonretroviral elements. The primary amino acid sequence of the C-terminal (C-term) domain is the most variable among retroviral integrases. The isolated C-terminal domain of HIV-1 integrase has a nonspecific DNA-binding activity.

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Image of Figure 2
Figure 2

Domain structure of retroviral integrases. Retroviral integrases are composed of three structural domains. The central core domain contains the D,D-35-E motif, the triad of acidic residues that play a key role in catalysis. This domain is structurally highly conserved among many mobile genetic elements, including some prokaryotic transposons. The N-terminal (N-term) domain includes the HHCC motif that binds zinc. This domain is conserved among retroviruses and retrotransposons, but a related domain has not been seen in nonretroviral elements. The primary amino acid sequence of the C-terminal (C-term) domain is the most variable among retroviral integrases. The isolated C-terminal domain of HIV-1 integrase has a nonspecific DNA-binding activity.

Citation: Craigie R. 2002. Retroviral DNA Integration, p 613-630. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch25
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Figure 3

Activities of retroviral integrases. (A) 3′end processing. In the presence of a divalent metal ion integrase cleaves the two terminal nucleotides from the 3′ends of a DNA substrate that mimics a viral DNA end. (B) DNA strand transfer. Integrase also inserts a processed 3′end into another DNA molecule by a one-step transesterification reaction. (C) Disintegration. Integrase can also "resolve" a DNA substrate that mimics the strand transfer product into its component viral and target DNA segments. Viral DNA substrate is shown as thick lines, and target DNA substrate is shown as thin lines.

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10.1128/9781555817954/fig25-3.gif
Image of Figure 3
Figure 3

Activities of retroviral integrases. (A) 3′end processing. In the presence of a divalent metal ion integrase cleaves the two terminal nucleotides from the 3′ends of a DNA substrate that mimics a viral DNA end. (B) DNA strand transfer. Integrase also inserts a processed 3′end into another DNA molecule by a one-step transesterification reaction. (C) Disintegration. Integrase can also "resolve" a DNA substrate that mimics the strand transfer product into its component viral and target DNA segments. Viral DNA substrate is shown as thick lines, and target DNA substrate is shown as thin lines.

Citation: Craigie R. 2002. Retroviral DNA Integration, p 613-630. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch25
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Figure 4

Chirality of the scissile phosphorothioate is inverted during both DNA strand transfer and 3′end processing. (A) DNA strand transfer. Phosphorothioate of one chirality (Rp) is incorporated into target DNA. In the product of DNA strand transfer its chirality is inverted to the Sp form. (B and C) 3′end processing. Rp form phosphorothioate is substituted for the scissile phosphate at the end of the viral DNA substrate. When the attacking nucleophile is from water (B), the product is a simple dinucleotide and chirality is lost. However, in an alternative 3′processing pathway (C), the nucleophile is the 3′-hydroxyl at the end of the viral DNA. The cleavage product is therefore a cyclic dinucleotide. The chirality of the phosphorothioate in this cyclic dinucleotide is inverted to the Sp form.

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10.1128/9781555817954/fig25-4.gif
Image of Figure 4
Figure 4

Chirality of the scissile phosphorothioate is inverted during both DNA strand transfer and 3′end processing. (A) DNA strand transfer. Phosphorothioate of one chirality (Rp) is incorporated into target DNA. In the product of DNA strand transfer its chirality is inverted to the Sp form. (B and C) 3′end processing. Rp form phosphorothioate is substituted for the scissile phosphate at the end of the viral DNA substrate. When the attacking nucleophile is from water (B), the product is a simple dinucleotide and chirality is lost. However, in an alternative 3′processing pathway (C), the nucleophile is the 3′-hydroxyl at the end of the viral DNA. The cleavage product is therefore a cyclic dinucleotide. The chirality of the phosphorothioate in this cyclic dinucleotide is inverted to the Sp form.

Citation: Craigie R. 2002. Retroviral DNA Integration, p 613-630. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch25
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