Dynamic copy choice: steady state between murine leukemia virus polymerase and polymerase-dependent RNase H activity determines frequency of in vivo template switching

Abstract

We recently proposed a dynamic copy-choice model for retroviral recombination in which a steady state between the rates of polymerization and RNA degradation determines the frequency of reverse transcriptase (RT) template switching. The relative contributions of polymerase-dependent and polymerase-independent RNase H activities during reverse transcription and template switching in vivo have not been determined. We developed an in vivo trans-complementation assay in which direct repeat deletion through template switching reconstitutes a functional green fluorescent protein gene in a retroviral vector. Complementation in trans between murine leukemia virus Gag-Pol proteins lacking polymerase and RNase H activities restored viral replication. Because only polymerase-independent RNase H activity is present in this cell line, the relative roles of polymerase-dependent and -independent RNase H activities in template switching could be determined. We also analyzed double mutants possessing polymerase and RNase H mutations that increased and decreased template switching, respectively. The double mutants exhibited low template switching frequency, indicating that the RNase H mutations were dominant. Trans-complementation of the double mutants with polymerase-independent RNase H did not restore the high template switching frequency, indicating that polymerase-dependent RNase H activity was essential for the increased frequency of template switching. Additionally, trans-complementation of RNase H mutants in the presence and absence of hydroxyurea, which slows the rate of reverse transcription, showed that hydroxyurea increased template switching only when polymerase-dependent RNase H activity was present. This is, to our knowledge, the first demonstration of polymerase-dependent RNase H activity in vivo. These results provide strong evidence for a dynamic association between the rates of DNA polymerization and polymerase-dependent RNase H activity, which determines the frequency of in vivo template switching.

Figure 1

Dynamic copy-choice model for RT template switching. Shaded boxes represent direct repeats in an RNA template. Horizontal arrows represent nascent DNA. The thickness of these arrows indicates relative polymerization rate: the thicker the arrow, the faster the polymerization rate. Small boxes represent degraded RNA by the RNase H domain. In the case of slow RNase H activity, the degraded RNA fragments are shown as larger boxes. Hydrogen bonds between the RNA template and nascent DNA are designated by vertical marks. Vertical arrows of various thicknesses indicate the relative efficiency of template switching.

Figure 2

Structure of MLV-based direct repeat vector and trans-complementation assay for determining which RNase H mode is important for template switching. (A) The pES-GFFP vector contains MLV-derived cis-acting elements including LTRs. The GFFP and neo genes are transcribed from the LTR promoter, and neo is expressed by the internal ribosomal entry site. During reverse transcription, deletion of one of the “F” repeats (shaded) results in the reconstitution of functional GFP. (B) Experimental protocol. A D150E A3–4 cell line stably expressing the pol⁻ RNase H⁺ mutant D150E, pES-GFFP, and pSV-A-MLV-env was constructed. Wild-type and mutant pRMBNB constructs were separately cotransfected (Tf) with pSV-hygro into the A3–4 cell line. Virus harvested from the A3–4 cell line was used to infect D17 target cells and placed under G418 selection. After selection, G418-resistant clones were pooled and analyzed by flow cytometry to determine the frequency of direct repeat deletion.

Figure 3

Trans-complementation and direct repeat deletion assay. The frequency of GFP reconstitution is shown on the left axis and represented by gray bars, whereas the titers are shown as a log scale on the right axis and represented by white bars. The error bars represent the SEM of at least two independent experiments. Squares represent the polymerase domain, and circles represent the RNase H domain of RT. White squares and circles represent wild type; black squares and circles represent replication-defective mutants. Mutants separated by a “/” represent trans-complementation; mutants with a “+” represent mutations contained within the same RT.

Figure 4

Trans-complementation of polymerase, RNase H, and polymerase + RNase H double mutants. Error bars represent the SEM of a least three independent experiments. Symbols are the same as in Fig. 3. Squares and circles containing an “X” represent mutants that are able to undergo viral replication.

Figure 5

Effect of HU on the frequency of direct repeat deletion during trans-complementation. Gray bars represent experiments performed without HU, whereas black bars represent experiments performed with HU. Error bars represent the SEM of at least three independent experiments. Symbols are the same as in Fig. 3.

Figure 6

Trans-complementation of RNase H mutants. Wild-type and RNase H mutants were trans-complemented with D150E to determine the effects of providing polymerase-independent RNase H in trans. Error bars represent the SEM of at least three independent experiments. Circles containing an “X” represent RNase H mutants that are able to undergo viral replication. Symbols are the same as in Fig. 3.

Figure 7

Model for in vivo template switching. (A) Wild type, (B) wild type + HU, and (C) D150E trans-complemented with D524N in the presence and absence of HU. Shaded boxes represent direct repeats within the RNA template, with the top box signifying the acceptor template and the bottom box the donor template. Horizontal lines between the shaded boxes represent nascent DNA. Small shaded boxes represent RNA that has been degraded by the RNase H domain. Vertical lines represent hydrogen bonds between the nascent DNA and RNA. RT symbols are the same as in Fig. 3. RTs annealed to the bottom of the shaded box represent polymerase-independent RNase H activity.