Replication defect of moloney murine leukemia virus with a mutant reverse transcriptase that can incorporate ribonucleotides and deoxyribonucleotides

Abstract

Reverse transcriptase (RT) plays a critical role in retrovirus replication, directing the synthesis of a double- stranded DNA copy of the viral RNA genome. We have previously described a mutant RT of the Moloney murine leukemia virus in which F155 was replaced by valine, and we demonstrated that this substitution allowed the enzyme to incorporate ribonucleotides to form RNA while still retaining its normal ability to incorporate deoxyribonucleotides to form DNA. When introduced into the viral genome, this mutation rendered the virus incapable of replication. Characterization of the mutant virus revealed that the enzyme was still active and able to synthesize minus-strand strong stop DNA and some longer products but failed to make full-length minus-strand DNA. We propose that the failure of the enzyme to complete DNA synthesis in vivo resulted from its ability to incorporate ribonucleotides into the products, which served as inhibitors for DNA synthesis. We also tested seven other amino acid residues for their abilities to substitute for F155 in virus replication; of these, only tyrosine could support virus replication. In an attempt to select for second-site suppressor mutations, the F155V mutant was subjected to random mutagenesis and was used as a parent for the isolation of revertant viruses. Two independent revertants were found to have changed the valine residue at position 155 back to the wild- type phenylalanine. These results suggest that an aromatic ring at this position is important for virus replication.

FIG. 1

Test of replication of viral RT mutants. Proviral DNAs encoding RTs with various amino acid substitutions of Y155 were analyzed for replication ability by DEAE dextran-mediated transient transfection of NIH 3T3 cells. Mock, mock transfection; WT, wild-type DNA; F, phenylalanine; A, alanine; M, methionine; I, isoleucine; H, histidine; L, leucine; W, tryptophan; V, valine; and Y, tyrosine. Revertant 1 and 2, revertants isolated from the F155V parent. Culture supernatants were harvested at 2, 5, and 10 days after transfection and analyzed for RT activity by the homopolymer RT assay as a measure of viral spread.

FIG. 2

Comparison of mutant and wild-type viruses for virion-associated RT protein and RT activities. 293T cells were transiently transfected with the indicated proviral DNAs on a plasmid containing the SV40 origin of replication. Mock, no DNA; pNCS-WT, wild-type viral DNA; pNCS-WT-H, RNase H-deficient mutant; pNCS-F155V, the substitution mutant; and pNCS-F155V-H, the double mutant. Culture supernatants were harvested 48 h after transfection, and various amounts of the supernatant, as indicated, were analyzed for RT activity by homopolymer RT assay (upper panel). The virions were pelleted through a 25% sucrose cushion and then analyzed by Western blotting for RT levels with a polyclonal anti-RT antiserum (middle panel). Relative specific activities for each enzyme were calculated (lower panel).

FIG. 3

Detection of viral DNA in wild-type and mutant virus-infected NIH 3T3 cells by Southern blotting. Supernatants harvested from transfected 293T cells were used to acutely infect NIH 3T3 cells. Twelve hours after infection, low-molecular-weight DNA was isolated, and resolved by electrophoresis, and viral DNA was detected by Southern blotting with a viral DNA probe. MMLV, virus collected from a stable NIH 3T3 cell line producing wild-type M-MuLV. Note that two forms of the plasmid DNAs used to transfect the 293T cells were carried over in the viral supernatants to the NIH 3T3 cells and detected in the analysis. The migration positions of the double-stranded viral DNA (8.8 kb) and plasmid DNA are indicated.

FIG. 4

Endogenous RT assay of products generated in wild-type and mutant virions collected from transfected 293T cells. Either [α-³²P]dTTP (lanes 1 to 5) or [α-³²P]rUTP (lanes 6 to 10) was included with all four dNTP substrates. The migration position of minus-strand strong stop DNA covalently linked to the tRNA primer is indicated.

FIG. 5

Endogenous assay to detect full-length minus-strand DNA in wild-type and mutant virions. DNA products were labeled by [α-³²P]dTTP and resolved on a 0.8% agarose gel, followed by autoradiography. The migration position of full-length minus-strand DNA is indicated.

FIG. 6

Processivity analysis of RT-WT and mutant RTs. ³²P-end-labeled primer was extended by purified recombinant RT-F155V (lanes 2 to 4) or RT- WT with a 320-nt-long RNA as the template. No dNTPs, no dNTP substrates added to the reaction; dNTPs only, 500 μM each dNTP added to the reaction; dNTPs + comp., dNTPs substrates, together with excess template-primer competitor, poly(rA) · oligo(dT)_12–18, added to the reaction to trap enzymes that dissociate from the elongation complex. The migration position of labeled primer is indicated.

FIG. 7

Inhibition of the enzymatic activity of RT-F155V by ribonucleotide rUTP. (A) Supernatants from transfected 293T cells were analyzed for RT activity in the homopolymer RT assay with poly(rA) · oligo(dT) as the template-primer, in the presence of various concentrations of rUTP as inhibitors as indicated. Aliquots were removed at the indicated time points and spotted on DEAE paper. The paper was then washed and exposed to either X-ray film for autoradiography (upper panel) or PhosphoImager analysis for quantitation. The relative enzymatic activities of RT-WT and mutant RTs were plotted and normalized to the wild-type enzyme without inhibitor (lower panel). Gray bars, RT-F155V; black bars, RT-WT. (B) Culture supernatants containing either wild-type or F155V virions were analyzed for RT activity on poly(rA) · oligo(dT)_12–18 as the template-primer, with labeled dTTP as the substrate, in the presence of different unlabeled NTPs as indicated. Reactions were performed with dTTP at 10 μM and with each inhibitor at 100 μM.

FIG. 8

Enzymatic activities of mutant RT and RT-WT enzymes with deoxyribonucleotides or ribonucleotides as substrates. Recombinant enzymes were partially purified and quantitated by Western blotting so that the same amount of RT protein was used in each reaction. Reactions were performed for 5 min with poly(rA) · oligo(dT) as the template-primer with substrates as indicated.

FIG. 9

Sequence comparison of the revertants, RT-WT and the original F155V mutant. The restriction sites used in the random mutagenesis are shown on the schematic sequences of RT (open bar) and part of integrase (closed bar). The AflII restriction site, which was generated by silent mutations during the construction of RT-F155V, is underlined.