Wild-type and mutant HIV type 1 nucleocapsid proteins increase the proportion of long cDNA transcripts by viral reverse transcriptase.
HIV-1 nucleocapsid, p7, contains two retroviral zinc fingers, which are both necessary for efficient packaging of genomic RNA and infectivity. The nucleocapsid protein is bound tightly to genomic RNA in the mature virion. In this study, the effect of p7 on polymerization of nascent cDNA by viral reverse transcriptase (RT) was examined. An 874-base RNA of HIV-1 was synthesized and used as a template in RT assays with varying concentrations of intact p7, mutants of p7 that have transposed or repeated zinc fingers, and several different peptides that represent various structural regions of p7. Results indicate that at greater than or equal to 50% saturation of p7-binding sites, with p7, there is up to a 90% reduction in total cDNA synthesis, as measured by nucleotide incorporation. However, the cDNA products that are made are almost exclusively full length. Three zinc finger mutants exhibited effects similar to those of wild-type p7. N-terminal and C-terminal halves of p7 inhibited total nucleotide incorporation, but also inhibited synthesis of long cDNA products by RT. In the absence of p7 an array of short transcripts (< 200 bases) was produced by RT. These studies show that full-length p7 is necessary to increase the proportion of long cDNA transcripts produced by RT. The relative position of the two zinc fingers is not critical for this effect.
Drummond
J E
JE
NCI-Frederick Cancer Research and Development Center, SAIC Frederick, Maryland 21702, USA.
Mounts
P
P
Gorelick
R J
RJ
Casas-Finet
J R
JR
Bosche
W J
WJ
Henderson
L E
LE
Waters
D J
DJ
Arthur
L O
LO
D000595:Amino Acid Sequence; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D004251:DNA Transposable Elements; D018076:DNA, Complementary / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D015683:Gene Products, gag / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000032:analysis; D016335:Zinc Fingers / Q000235:genetics; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D004251:DNA Transposable Elements; D018076:DNA, Complementary; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D012367:RNA, Viral; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D054303:HIV Reverse Transcriptase