Role of divalent ion complex formation in pyran--inhibition of nucleic acid biosynthesis.

The degree of inhibition of mammalian DNA-dependent RNA polymerases I and II and Moloney leukemia virus RNA-dependent DNA polymerase by pyran copolymer was dependent on the concentration of the divalent cation cofactor in the reaction mixture. Inhibition was completely blocked by an excess of divalent cations. It was concluded that pyran inhibited these enzymes by complexing with the essential divalent cation cofactor.

Effects of Poly(1-vinyluracil) and Poly(9-vinyladenine) on viral RNA-directed DNA polymerase.

The effects of poly(1-vinyluracil) [poly(vU)] and poly(9-vinyladenine) [poly(vA)] on the RNA-dependent DNA polymerase activity of murine leukemia virus (Moloney strain) were studied. Vinyl polymers themselves cannot act as templates for the polymerase. However, if a vinyl polymer is added to a polymerase reaction mixture in which a complementary polynucleotide serves as the template, the reaction is inhibited: thus with polyribocytidylic acid as template and oligodeoxyguanylic acid as primer, neither poly(vU) nor poly(vA) had a significant effect; when polyribouridylic acid was used as template and oligodeoxyadenylic acid as primer, poly(vA) inhibited polymerase activity while poly(vU) had little effect; when polyriboadenylic acid was a template and oligodeoxy thymidylic acid was a primer, poly(vU) was an inhibitor. Complex effects were noted with the latter system and poly(vA); either stimulation or inhibition of the reaction was observed, depending on the concentration of poly(vA). The stimulation brings about a decrease in the amount of lower-molecular-weight materials in the product and is caused by the interaction of poly(vA) with the template-primer. Thus vinyl polymers differ from polynucleotides in their mechanism of inhibition of viral polymerase, since the latter inhibit the enzyme by binding to it.

Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain.

Reverse transcriptase (RT) serves as the replicative polymerase for retroviruses by using RNA and DNA-directed DNA polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain detailed structural information about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 A resolution of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the fingers and palm domains from Moloney murine leukemia virus reverse transcriptase. We have also determined the crystal structure at 3.0 A resolution of the fragment complexed to DNA with a single-stranded template overhang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA atoms involved in the interactions include the 3'-OH group from the primer strand and minor groove base atoms and sugar atoms from the n-2 and n-3 positions of the template strand, where n is the template base that would pair with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residues in the beta4-beta5 loop (beta3-beta4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to HIV-1 RT but related in the types of interactions formed between protein and DNA. In addition, the DNA in all of these complexes is bound in the same cleft of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and have characterized the resulting enzymes. We now propose that nucleic acid binding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process.

Differentiation of the seven serotypes of foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction.

A strategy for reverse transcriptase polymerase chain reaction (RT-PCR) using multiple primers was developed to detect and to differentiate the seven serotypes of foot-and-mouth disease virus (FMDV) simultaneously, quickly and accurately. The development of the test was carried out on virus isolates grown in tissue culture prior to cDNA synthesis and PCR using various sets of primers. Primers P33 and P32 were used for the consensus PCR to detect FMDV regardless of the serotype. Positive cDNA was assayed in two multi-primer PCR mixes containing type-specific primers capable of distinguishing between the seven serotypes. The serotype-specific primers were selected to correspond to regions of the genome coding for parts of the VP1 polypeptide that is responsible for the antigenic diversity of the virus group. Multi-primer mix P33-P(A-C-O-ASIA 1) gave products of 732, 596, 402, 292 bp for the A,C,O and ASIA 1 serotypes, respectively, and no target products for South African Territories serotypes (SAT 1, SAT 2 and SAT 3). The multi-primer mix P33-P(A-C-O-ASIA 1) was also capable of detecting a mixture of two different serotypes. Multi-primer mix P1-P(SAT 1-3-2) gave products of 246, 201 and 75 bp for the SAT 1, SAT 3 and SAT 2 serotypes, respectively, and no specific products for serotypes A, C, O and ASIA 1. This is the first PCR assay to be described that differentiates between the SAT serotypes of FMDV. The method has been applied to 25 cell-culture-derived isolates of the SAT serotypes of FMDV and the results were totally compatible with the standard techniques for FMDV detection and serotyping.

Inhibition of reverse transcriptase by polyvinyl sulfate (PVS).

The effect of the ribonuclease inhibitor polyvinyl sulfate on the activity of retroviral DNA polymerase and terminal c transferase was examined. This substance was found to be a potent inhibitor of these enzymes by virtue of competition between the sulfated sidechains of the molecule and the template primer for a site on the enzyme. The significance of these findings in relation to searching for reverse transcriptase is discussed.

In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution containing dithiothreitol in addition to 0.1% Nonidet P-40 allowed the efficient processing of P40 to p30 and a band migrating with p10. Immunoprecipitation with monospecific sera indicated that P40 contained p30 and p10, whereas P25 contained p15 and pp12 determinants. P40 and P25 are similar in size and antigenic properties to Pr40gag and Pr25gag observed in infected cells (Naso et al, J. Virol. 32:187-198, 1979).

Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled.

The functional interaction between the RNA-dependent DNA polymerase and the RNase H activities of reverse transcriptases (RTs) were examined using a 272 nucleotide long plasmid-derived RNA transcript primed in a specific location. Properties of the avian myeloblastosis virus (AMV) RT, the human immunodeficiency virus RT and the Moloney murine leukemia virus RT were examined. All three enzymes formed stable complexes with the primer-template with half-lives ranging from about 16 to 41 s. Each enzyme synthesized full-length primer extension products and cleaved the RNA template at least once during DNA synthesis. Polymerization was then assayed in the presence of challenger RNA that effectively sequestered RTs after one round of processive DNA synthesis. This assay allowed measurement of the number of endonucleolytic cleavages catalyzed by the RT during one encounter with the primer-template. Results indicated that each of the three RTs cut the transcript before dissociating from the primer-template, whether or not deoxynucleoside triphosphates were present to allow synthesis. During synthesis, the extent of RNA degradation differed among the RTs, with AMV-RT generating mostly large segments of RNA-DNA hybrid, and virtually no small RNA cleavage products. Human immunodeficiency virus and Moloney murine leukemia virus-RT generated more small degradation products than AMV-RT, but still left much of the potentially degradable hybrid undigested. Results demonstrate that the RNase H function is much less active than the polymerization function during processive DNA synthesis and that the activities are not strictly coupled.

An oligonucleotide affinity column for RNA-dependent DNA polymerase from RNA tumor viruses.

Columns of (dT)(12-18)-cellulose provide a one-step enrichment procedure for RNA-dependent DNA polymerase. The enzyme of the virus from RD-114 cells, as well as that from Rauscher murine leukemia virus, have been purified in this way. The preference of viral as compared to cellular DNA polymerases for (dT)(12-18) as a primer is reflected in the fact that the DNA polymerases of uninfected cells do not bind to this column. Viral enzymes have been purified and identified from crude cellular extracts.