Inhibition of murine leukaemia virus retrotranscription by the intracellular expression of a phage-derived anti-reverse transcriptase antibody fragment.
The intracellular targeting of recombinant antibodies is an experimental strategy to interfere with the function of selected molecules that is being utilized in a variety of different systems for research and medical applications. Since recombinant antibodies are increasingly being derived from phage display libraries, we have exploited phage technology to isolate, from a large combinatorial library, human antibody fragments directed against human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). We describe in this paper the in vitro and in vivo properties of a neutralizing anti-RT antibody fragment. We demonstrate that the heavy chain domain (VH-CH1) of the phage-derived antibody is able to inhibit the retroviral enzyme, in that it neutralizes the RNA-dependent DNA polymerase activity of HIV-1 RT. The VH-CH1 antibody fragment also neutralizes the activity of RT of drug-resistant HIV-1 mutants as well as that of murine retrovirus RT. To confirm the broad reactivity of the synthetic antibody fragment, we have assessed the ability of the intracellularly expressed VH-CH1 protein to interfere with murine retroviral infection. To this end, we developed an in vivo selection procedure based on the antibody-mediated resistance to a cytotoxic retrovirus and used this selection procedure to rescue, from a heterogeneous population, cells expressing the VH-CH1 antibody fragment. We finally demonstrate that the intracellular expression of the recombinant heavy chain antibody fragment leads to an efficient inhibition of viral retrotranscription by murine-based retrovirus.
Gargano
N
N
MRC Laboratory of Molecular Biology, Cambridge, UK.
Cattaneo
A
A
D016475:3T3 Cells; D000818:Animals; D000918:Antibody Specificity; D001435:Bacteriophages; D003600:Cytosol / Q000276:immunology / Q000821:virology; D004279:DNA, Viral / Q000096:biosynthesis*; D015967:Gene Expression Regulation, Viral; D015483:HIV Antibodies / Q000235:genetics / Q000276:immunology*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000276:immunology*; D051379:Mice; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D012194:RNA-Directed DNA Polymerase / Q000276:immunology*; D018894:Reverse Transcriptase Inhibitors; D013045:Species Specificity
D004279:DNA, Viral; D015483:HIV Antibodies; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase