Biomedical subjects

M Tisdale

Publications and source records attributed to M Tisdale.

At least 37 records · Page 2Linked to original sources

PubMed1996

Human immunodeficiency virus. Mutations in the viral protease that confer resistance to saquinavir increase the dissociation rate constant of the protease-saquinavir complex.

Mutations in the human immunodeficiency virus (HIV) protease (L90M, G48V, and L90M/G48V) arise when HIV is passaged in the presence of the HIV protease inhibitor saquinavir. These mutations yield a virus with less sensitivity to the drug (L90M > G48V >> L90M/G48V). L90M, G48V, and L90M/G48V proteases have 1/20, 1/160, and 1/1000 the affinity for saquinavir compared to WT protease, respectively. Therefore, the affinity of mutant protease for saquinavir decreased as the sensitivity of the virus to saquinavir decreased. Association rate constants for WT and mutant proteases with saquinavir were similar, ranging from 2 to 4 x 10(7) M-1 s-1. In contrast, the dissociation rate constants for WT, L90M, G48V, and L90M/G48V proteases complexed with saquinavir were 0.0014, 0.019, 0.128, and 0. 54 s-1, respectively. This indicated that the reduced affinity for mutant proteases and saquinavir is primarily the result of larger dissociation rate constants. The increased dissociation rate constants may be the result of a decrease in the internal equilibrium between the bound inhibitor with the protease flaps up and the bound inhibitor with the flaps down. Interestingly, the affinity of these mutant proteases for VX-478, ABT-538, AG-1343, or L-735,524 was not reduced as much as that for saquinavir. Finally, the catalytic constants of WT and mutant proteases were determined for eight small peptide substrates that mimic the viral cleavage sites in vivo. WT and L90M proteases had similar catalytic constants for these substrates. In contrast, G48V and L90M/G48V proteases had catalytic efficiency (kcat/Km) values with TLNF-PISP, RKIL-FLDG, and AETF-YVDG that were 1/10 to 1/20 the value of WT protease. The decreased catalytic efficiencies were primarily the result of increased Km values. Thus, mutations in the protease decrease the affinity of the enzyme for saquinavir and the catalytic efficiency with peptide substrates.

Antiviral Agents↗

PubMed1996

Characterization of a cancer cachectic factor.

Cancer cachexia is a syndrome of progressive wasting which has been suggested to be mediated by tumour-necrosis factor-alpha, interleukins 1 and 6, interferon-gamma and leukaemia-inhibitory factor. It has proved difficult to correlate levels of tumour-necrosis factor-alpha and interleukin-6 with cancer cachexia, and the weight loss induced by leukaemia-inhibitory factor may be due to toxicity. In the murine adenocarcinoma MAC16, cachexia is mediated by circulatory catabolic factors, which we have now isolated using an antibody cloned from splenocytes of mice transplanted with the MAC16 tumour, with a delayed cachexia. The material is a proteoglycan of relative molecular mass 24K which produces cachexia in vivo by inducing catabolism of skeletal muscle. The 24K material was also present in urine of cachectic cancer patients, but was absent from normal subjects, patients with weight loss due to trauma, and cancer patients with little or no weight loss. This suggests that cachexia in mice and humans may be produced by the same material.

Adenocarcinoma↗

PubMed1996

In vitro antiviral activity of 141W94 (VX-478) in combination with other antiretroviral agents.

141W94 (VX-478) is a novel HIV-1 protease inhibitor with an IC50 of 0.08 microM against HIV-1 (strain IIIB) and a mean IC50 of 0.012 microM against six HIV clinical isolates. 141W94 was synergistic on the basis of isobologram analysis with each of the following reverse transcriptase inhibitors: AZT, 935U83, 524W91, 1592U89 and ddl, 141W94 was also synergistic with saquinavir and additive with either indinavir or ritonavir. Resistance to 141W94 has been reported in vitro passage experiments. The binding of 141W94 to human alpha 1-acid glycoprotein was relatively weak (Kd = 4 microM) and the off-rate for the drug is very fast (> or = 100 s-1). Only a 2-fold reduction of in vitro antiviral activity was observed in the presence of 45% human plasma. No serious drug associated adverse experiences were reported in a Phase I placebo-controlled, single-dose escalation, pharmacokinetic and safety study. The average concentration of 141W94 at 8 and 12 h after single doses of 900 and 1200 mg, respectively, was in excess of 10 times the IC50. As 141W94 is synergistic with a variety of anti-HIV-1 agents and exhibits a unique cross resistance profile compared to other protease inhibitors, 141W94 is considered a good candidate for combination therapy.

Carbamates↗

PubMed1995

Inhibition of influenza virus transcription by 2'-deoxy-2'-fluoroguanosine.

The nucleoside analog 2'-deoxy-2'-fluoroguanosine (2'-fluorodGuo) is phosphorylated by cellular enzymes and reversibly inhibits influenza virus replication in chick embryo cells within the first 4 h of infection. RNA hybridization studies revealed that primary and secondary transcription of influenza virus RNA were blocked at a compound concentration of 10 microM, but no inhibition of cell protein synthesis was seen even at high compound concentrations (200 microM). In vitro, the triphosphate of 2'-fluorodGuo is a competitive inhibitor of influenza virus transcriptase activity from disrupted virus, with a Ki of 1.0 microM. The cellular polymerases DNA polymerase alpha and RNA polymerase II were only weakly inhibited or were insusceptible to 2'-fluorodGTP. In kinetic studies with the influenza virus transcriptase, 2'-fluorodGTP, in the absence of GTP, blocked elongation of the virus RNA chain. Similarly, by using purified ribonucleoprotein complexes it was found that the addition of a single nucleotide of 2'-fluorodGTP to the virus RNA caused chain termination, which resulted in the blockage of further virus transcription. Furthermore, the specificity for influenza virus transcriptase was confirmed when the transcriptase from partially resistant virus was found to be 10-fold less susceptible to 2'-fluorodGTP (Ki = 13.1 microM).

Antiviral Agents↗

PubMed1995

Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors.

Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy.

Amino Acid Sequence↗

PubMed1995

Antiviral, metabolic, and pharmacokinetic properties of the isomeric dideoxynucleoside 4(S)-(6-amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol.

4(S)-(6-Amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol (IsoddA) is the most antivirally active member of a novel class of optically active isomeric dideoxynucleosides in which the base has been transposed from the natural 1' position to the 2' position and the absolute configuration is (S,S). IsoddA was active against human immunodeficiency virus type 1 (HIV-1) (strain IIIB), HIV-2 (strain ZY), and HIV-1 clinical isolates. Combinations of the compound with zidovudine (3'-azido-3'-deoxythymidine), 2',3'-dideoxyinosine, or 5-fluoro-2'-deoxy-3'-thiacytidine showed synergistic inhibition of HIV. A moderate reduction of activity was observed with clinical isolates resistant to zidovudine. An IsoddA-resistant virus (eightfold-increased 50% inhibitory concentration) was selected in vitro by repeated passage of HIV-1 (HXB2) in the presence of increasing concentrations of IsoddA. The reverse transcriptase-coding region of the mutant virus contained a single base change resulting in a change at codon 184 from Met to Val. IsoddA was also active against hepatitis B virus (HBV) in vitro; however, it lacked substantial selective activity in an in vivo HBV model. IsoddA was inefficiently phosphorylated in CEM cells; however, the half-life of the triphosphate was 9.4 h, and IsoddATP was a potent inhibitor of HIV-1 reverse transcriptase, with a Ki of 16 nM. The cytotoxicity 50% inhibitory concentrations of IsoddA were greater than 100 microM for CEM, MOLT-4, IM9, and the HepG2-derived HBV-infected 2.2.15 (subclone P5A) cell lines but were 12 and 11 microM for human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Deaminase↗

PubMed1995

In vitro selection and characterization of human immunodeficiency virus type 1 (HIV-1) isolates with reduced sensitivity to hydroxyethylamino sulfonamide inhibitors of HIV-1 aspartyl protease.

Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observation in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the inhibitors than the parent enzyme (< or = 80-fold). The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val shows an even greater decrease in inhibitor binding (< or = 270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially.

Amino Acid Sequence↗

PubMed1994

Influence of stereochemistry on antiviral activities and resistance profiles of dideoxycytidine nucleosides.

beta-L-2',3'-Dideoxycytidine (beta-L-ddC) and beta-L-5-fluoro-2',3'-dideoxycytidine (5-F-beta-L-ddC) were prepared and shown to have potent activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). These compounds were compared with beta-D-2',3'-dideoxycytidine (beta-D-ddC) and two beta-L-oxathiolane nucleosides (beta-L-3'-thio-2',3'-dideoxycytidine and beta-L-5-fluoro-3'-thio-2',3'-dideoxycytidine) in terms of anti-HIV and anti-HBV activity, cytotoxicity, and development of HIV-1 resistance. Compared with beta-D-ddC, the beta-L-dideoxycytidine nucleosides had similar anti-HIV-1 activities, significantly greater anti-HBV activities, and decreased toxicities to a B-cell line, T-cell lines, and human bone marrow progenitor cells. HIV-1 strains resistant to beta-D-ddC were susceptible to the beta-L-ddC analogs. Compared with the beta-L-oxathiolane nucleosides, beta-L-ddC and 5-F-beta-L-ddC had similar anti-HIV-1 activities, decreased anti-HBV activities, and greater toxicities to B- and T-cell lines and bone marrow progenitor cells. There were similarities between the beta-L-ddC and beta-L-oxathiolane nucleosides in the rate of development and pattern of resistant HIV-1 selection. While the in vitro activity and cytotoxicity profiles of the beta-L-ddC nucleosides differed from those of the beta-D-ddC and beta-L-oxathiolane nucleosides, the data presented herein suggest that the sugar configuration of a dideoxynucleoside analog may play a major role in the rate of development and the pattern of HIV-1 resistance.

Animals↗

PubMed1994

Efficacy of 2'-deoxy-2'-fluororibosides against influenza A and B viruses in ferrets.

Single-dose treatments (5 to 40 mg/kg of body weight given intraperitoneally) of ferrets with 2'-deoxy-2'-fluoroguanosine or its prodrug, 2,6-diamino-purine-2'-fluororiboside, 1 h after infection with influenza A virus significantly inhibited replication of virus in the upper respiratory tract, resulting in amelioration of fever and nasal inflammation. Replication of virus in the lower respiratory tract was also reduced > 100-fold, but three doses were required to prevent replication in the lungs. In ferrets infected with influenza B virus, single-dose treatment (40 mg/kg given intraperitoneally) produced a similar but reduced response in comparison with that in ferrets infected with influenza A virus, indicating that dosing was not optimal for this virus.

Adenosine↗

PubMed1993

Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase.

Resistant variants of human immunodeficiency virus type 1 (HIV-1) have been selected by limited passage in MT4 cells of both wild-type and 3'-azido-3'-deoxythymidine (AZT, zidovudine)-resistant strains with the nucleoside analogues (-)-2'-deoxy-3'-thiacytidine (3TC) and (-)-2'-deoxy-5-fluoro-3'-thiacytidine (FTC). Virus variants selected independently were crossresistant to both inhibitors. This rapid in vitro selection of resistant virus has not previously been seen with nucleoside analogues but is reminiscent of that observed with the nonnucleoside reverse transcriptase inhibitors. However, passage of wild-type virus with a combination of AZT and FTC appreciably delayed emergence of FTC-resistant virus. DNA sequence analysis of the reverse transcriptase coding region from FTC-resistant virus revealed changes at codon 184 in the highly conserved Tyr, Met, Asp, Asp (YMDD) region. When the mutation Met184-->Val was introduced into the infectious clone HXB2, this change alone accounted for the resistance (> 1000-fold) seen with both 3TC and FTC, and for a 5- to 15-fold reduction in sensitivity to their (+) enantiomers. It had no effect on susceptibility to AZT or nevirapine and minimal effect on susceptibility to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine. To determine the influence of this mutation in a background of mutations conferring resistance to AZT and nonnucleoside reverse transcriptase inhibitors, a series of HIV-1 variants were created by site-directed mutagenesis. All mutants with Met184-->Val were cross-resistant to 3TC and FTC. The Met184-->Val mutation did not influence nevirapine resistance, but resistance to AZT was suppressed. Similar suppression of AZT resistance was seen with Tyr181-->Cys. Interestingly, when both Met184-->Val and Tyr181-->Cys substitutions were present, highly resistant virus reverted to complete AZT sensitivity. Assessment of the interactive effects of multiple drug-resistance mutations may help to establish a rationale for using these drugs in the future therapy of HIV disease.

Amino Acid Sequence↗

PubMed1993

Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleases H.

The reverse transcriptase (RT) of HIV-1 has been mutagenized within the carboxyl-terminal domain which harbors the RNase H. Two amino acids highly conserved among all 14 known RT sequences but not in the bacterial RNase H have been mutagenized resulting in the mutant proteins N494D and Q475E. They were expressed as recombinant proteins, purified, and analyzed for their in vitro properties in comparison to the p66 homodimeric wild-type and a previously described H539N mutant. The N494D mutant closely resembles the wild-type RNase H, exhibits an endonuclease activity and a processive RNase H activity, gives rise to small RNA hydrolysis products, and acts in concert with the RT. The Q475E mutant is more defective and resembles the H539N mutant, exhibits a retarded endonuclease activity and an impaired 3'-->5' processive RNA cleavage activity, gives rise to predominantly larger RNA hydrolysis products, is less processive in the presence of competitor substrate, and is defective in its ability to hydrolyze the polypurine tract and homopolymeric hybrids. Short homopolymeric stretches cause a pausing of the RT of wild-type and mutants which results in a coordinated action of the RNase H. Pausing of the RT correlates with RNase H cleavages about 20 nucleotides behind the point of synthesis. The defects of the mutant enzymes can be interpreted on the basis of the known crystallography data.

Amino Acid Sequence↗

PubMed1993

Purine 2'-deoxy-2'-fluororibosides as antiinfluenza virus agents.

Twenty purine 2'-deoxy-2'-fluororibosides were synthesized by enzymic pentosyl transfer from 2'-deoxy-2'-fluorouridine. Each nucleoside analogue was assayed for cytotoxicity in uninfected Madin-Darby canine kidney cells and for their ability to suppress influenza A virus infections in these cells. The most potent antivirial activity was observed with analogues having an amino group in the 2-position of the purine moiety. All 2-unsubstituted analogues were less potent than their 2-amino counterparts. Furthermore, 2-methyl,2-methoxy, or 2-fluoro substitution obliterated antivirial activity. The most cytotoxic member of the series was the 2-fluoro-6-amino analogue (IC50 = 120 microM). 2'-Deoxy-2'-fluoroguanosine and those congeners readily converted to it by adenosine deaminase showed the most potent antivirial activity (IC50 = 15-23 microM). Little cytotoxicity was observed with this subgroup of analogues which renders them worthy of further investigation as potential antiinfluenza agents.

Animals↗

PubMed1993

Isolation and characterization of monoclonal antibodies raised against the reverse transcriptase of human immunodeficiency virus type 2 and cross-reactivity with that of type 1.

Monoclonal antibodies to human immunodeficiency virus (HIV)-2 reverse transcriptase have been raised with the ultimate goal of generating Fab fragments for future co-crystallization studies. A number of mouse monoclonal antibodies to recombinant HIV-2 reverse transcriptase have been obtained and characterized in terms of the possible epitopes they recognise together with cross-reactivity with a related reverse transcriptase. The antibodies were shown to fall into three groups that recognize different regions of the reverse transcriptase enzyme. One antibody, which recognizes part of the RNase H domain, demonstrated cross-reactivity between the HIV-1 and HIV-2 reverse transcriptase.

Amino Acid Sequence↗

PubMed1991

Rapid purification and characterisation of HIV-1 reverse transcriptase and RNaseH engineered to incorporate a C-terminal tripeptide alpha-tubulin epitope.

The C-termini of p66 and p51 forms of HIV-1 reverse transcriptase have been engineered to contain a Glu-Glu-Phe sequence recognized by a monoclonal antibody to alpha-tubulin, YL1/2. Mutated RTs were purified in a single step using peptide elution from columns of immobilized YL1/2. The known sequence requirements of the YL1/2 epitope are consistent with protein eluting from the column with an intact C-terminus. Kinetic parameters of these mutated RTs are essentially unchanged from wild-type enzyme. The p15 RNaseH domain has been purified using this method and shown to have low enzyme activity compared to the parental p66 subunit.

Amino Acid Sequence↗

PubMed1991

Monoclonal antibodies define linear and conformational epitopes of HIV-1 pol gene products.

Purified recombinant reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) was used to raise 21 monoclonal antibodies with anti-RT specificities. The antibodies were characterized using Western blotting against native virus and recognized either the p66 or p66, p51 components of RT. Further immunoblotting using either cyanogen bromide fragmented RT or truncated mutants of RT along with cross-competition studies enabled the location of various immunogenic regions of RT to be identified. Three antibodies recognized a linear epitope in the N-terminal region (amino acids 128-176). Also, a neutralizing RT antibody recognized a conformational epitope in this region. Three monoclonals had epitopes mapped to linear sequences in the RNase H region at the C-terminus of the RT. Another neutralizing antibody, also requiring folding of the RT protein had its epitope more centrally located (231-353). Of the remaining 13 monoclonals, 7 were roughly located in the C-terminal region and required folding of the protein for epitope recognition and only three of the remaining six could be mapped to conformational epitopes in N-terminal and central regions of the RT. None of the antibodies tested recognized HIV-2 RT products p68 and p55 in Western blot.

Animals↗

PubMed1991

Mutations within the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase abolish virus infectivity.

The C-terminal region of human immunodeficiency virus (HIV) reverse transcriptase (RT) contains the domain responsible for RNase H activity. To determine the importance of this RNase H domain, specific changes in the C-terminal region of a recombinant RT expressed in Escherichia coli were introduced by amino acid substitutions and specific deletions. The enzyme activities of purified wild-type and mutant RT/RNase H proteins, standardized for protein content, were compared by filter assays and thermal inactivation kinetics. A point mutation of His 539----Asn produced an enzyme with a marked thermolabile RNase H function (nine-fold increase in inactivation), whereas RT function was only marginally more labile than that of the wild-type (two-fold). A second mutation, His 539----Asp, impaired both enzyme activities to a similar degree (four- to five-fold). A C-terminal deletion of 19 amino acids (aa) (aa 540 to 558) and a C-terminal truncation of 21 aa (aa 540 to 560) reduced RT as well as RNase H activity. A 130 aa deletion enzyme exhibited no RNase H activity and insufficient RT activity to allow inactivation studies. Two mutants, the 19 aa deletion and His----Asn, were introduced into proviral HIV-1 DNA clones to determine whether changes in enzyme activity, particularly RNase H activity, affected virus infectivity. Both mutants were non-infectious, indicating that the C-terminal 19 to 21 amino acids and His 539 of the RT/RNase H protein are essential for HIV replication. These results are consistent with the assumption that RNase H is essential for the infectivity of HIV-1.

Amino Acid Sequence↗

PubMed1989

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors.

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme.

Animals↗

PubMed1988

HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis.

Bacterially expressed recombinant HIV-1 reverse transcriptase is active as both a homodimer of Mr 66,000 subunits and a heterodimer of Mr 66,000 and 51,000 subunits. The heterodimer is formed by cleavage of a C-terminal fragment from one Mr 66,000 polypeptide, which occurs during purification and crystallization of reverse transcriptase. Thus, crystals obtained from purified Mr 66,000 polypeptide preparations consisted of an apparently equimolar mixture of Mr 66,000 and 51,000 polypeptides, which were apparently analogous to the Mr 66,000 and 51,000 polypeptides detected in HIV-infected cells and in virions. Limited proteolysis of the homodimer with alpha-chymotrypsin also resulted in cleavage to a stable Mr 66,000/51,000 mixture, and proteolysis with trypsin resulted in the transient formation of some Mr 51,000 polypeptide. These results are consistent with the reverse transcriptase molecule having a protease-sensitive linker region following a structured domain of Mr 51,000. Further digestion with trypsin resulted in cleavage of the Mr 51,000 polypeptide after residue 223, yielding peptides of apparent Mr 29,000 and 30,000. A minor peptide of Mr 40,000 was also produced by cleavage of the Mr 66,000 polypeptide after residue 223. About half the original Mr 66,000 polypeptides remained resistant to proteolysis and existed in complex with the above peptides in solution. During both chymotrypsin and trypsin digestion there was an increase in the reverse transcriptase activity caused by a doubling of Vmax with little change in Km for dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗