Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.

A synthetic RNA oligonucleotide (15-mer) corresponding to the 3' end of the lysine tRNA primer was hybridized to single-stranded DNA containing the human immunodeficiency virus type 1 (HIV-1) primer-binding site and extended with a DNA polymerase. The resulting structures were used to study primer removal by the RNase H activity of HIV-1 reverse transcriptase. The initial cleavage event removes the RNA primer as a 14-mer and leaves a single ribonucleotide A residue bound to the 5' end of the DNA strand. This result explains the observation by several groups that HIV-1 circle junctions contain 4 bp that are not present in the integrated provirus instead of the predicted 3 bp. Subsequent cleavage events occur at other sites internal to the RNA molecule, and the ribonucleotide A residue on the end of the DNA strand is ultimately removed. Therefore, the biologically relevant cleavage that produces the 14-mer reflects the kinetics of the reaction as well as a specificity for nucleic acid sequence. When the RNA oligonucleotide alone was hybridized to the primer-binding site and tested as a substrate for HIV-1 RNase H, the cleavage pattern near the 3' end of the RNA was altered.

Moloney murine leukemia virus IN protein from disrupted virions binds and specifically cleaves its target sequence in vitro.

The integration of retroviral DNA plays an essential role in the viral life cycle. Previous studies of the Moloney murine leukemia virus (M-MuLV) have shown that viral integration is mediated by the integrase (IN) protein acting on the 13-bp inverted repeats that flank the linear viral DNA produced during reverse transcription. Prior studies have also shown that when the M-MuLV IN protein is produced in Escherichia coli it retains an ability to specifically associate with the viral inverted repeats (Krogstad and Champoux, 1990). In this study we present evidence that the IN protein present in detergent-disrupted virions is capable of specifically interacting with double-stranded oligonucleotides that correspond to the viral inverted repeats, and that this interaction may change after integration-related processing of the viral att sites. We further present evidence that, in vitro, detergent-disrupted virions are capable of specifically cleaving ds-IR oligonucleotides in an IN-dependent reaction that mimics the trimming step that precedes integration.

Expression of plasmid-encoded structural proteins permits engineering of bacteriophage T4 assembly.

A complementation system for studying bacteriophage T4 tail assembly has been developed and used to test the effects of nonviable mutations on the function of a specific T4 tail protein, gp48. The complementation system assays the assembly function of gp48 without requiring that viable phage be produced, circumventing the operational problems of maintaining nonviable mutants of this lytic bacteriophage. The protein to be tested was preexpressed from cloned genes in a host cell prior to infection with the challenge phage. Assembly activity was assayed by monitoring the conversion of one tail assembly intermediate, the baseplate lacking gp48, into baseplates containing gp48 or into tube baseplates (or sheathed tails) assembled from such baseplates. Specific incorporation of gp48 into these structures was confirmed using gp48-specific antiserum, and the same serum was used in direct immunoelectron microscopy experiments to localize gp48 to the baseplate-proximal end of the T4 tail tube, at the site where the tube and sheath bind to the baseplate. The protein gp48 has been previously shown to be a baseplate protein, as well as a tail-tube-associated protein, and was tested for a possible role as a tail-length tape-measure protein. Tests with a deleted variant of gp48 were inconclusive because the protein was inactive. A variant of gp48, 20% longer than wild-type protein due to an internal duplication, was found to be partly functional in our assembly complementation system. This abnormally elongated protein allows several assembly steps to proceed, including the assembly of normal length T4 tails, implying that it does not specify tail length. The insertion-duplication variant of gp48 appears to have a defect in its interaction with the tail sheath protein, leading to abnormal sheath contraction.

Expression and regulation of genes coding for three bacteriophage T4 tail tube-associated proteins.

The assembly and length regulation of the tail tube of bacteriophage T4 requires the function of three proteins: gp29, 48, and 54. Six copies of each protein are found in the completed tail, and the genes for these proteins are adjacent on the T4 genome. Evidence is presented here that gp54 is also a tail tube-associated protein that remains bound to the tail tube after the baseplate is removed by guanidine hydrochloride, suggesting that all three proteins interact structurally. There is a strong polar effect of translation termination mutants in gene 48 upon the expression of the adjacent gene 54, in cis-trans tests. Gene dosage experiments that assay the in vivo expression of these genes show that only gene 48 is expressed at slightly higher than stoichiometric levels during T4 infection. Genes 48 and 54 were placed under the control of a T7 promoter and the corresponding proteins identified. When a frameshift mutation was introduced into gene 48, neither gp48 nor gp54 was made. Transcriptional termination was not the explanation of this result because genes distal to 48 and 54 in the plasmid were expressed. These data suggest that expression of genes 48 and 54 is translationally coupled.

The structure of three bacteriophage T4 genes required for tail-tube assembly.

Three different protein molecules copurify with T4 tail tubes after the tubes are released from the baseplate by guanidine hydrochloride treatment. These tube-associated proteins (TAPs) are the products of genes 29, 48, and 54. To further investigate the structural roles that these proteins may play in T4 tail assembly we have cloned and sequenced the genes coding for these proteins and have deduced their predicted amino acid sequences. The sequence data reveal a region of amino acid sequence similarity between gp54 and the T4 tail-tube structural protein, gp19. We believe that this region of similarity is significant and consistent with the role gp54 may play in initiating T4 tail-tube polymerization.