Modeling, analysis, and validation of a novel HIV integrase structure provide insights into the binding modes of potent integrase inhibitors.

It has been shown that L-731988, a potent integrase inhibitor, targets a conformation of the integrase enzyme formed when complexed to viral DNA, with the 3'-end dinucleotide already cleaved. It has also been shown that diketo acid inhibitors bind to the strand transfer complex of integrase and are competitive with the host target DNA. However, published X-ray structures of HIV integrase do not include the DNA; thus, there is a need to develop a model representing the strand transfer complex. In this study, we have constructed an active-site model of the HIV-1 integrase complexed with viral DNA using the crystal structure of DNA-bound transposase and have identified a binding mode for inhibitors. This proposed binding mechanism for integrase inhibitors involves interaction with a specific Mg(2+) in the active site, accentuated by a hydrophobic interaction in a cavity formed by a flexible loop upon DNA binding. We further validated the integrase active-site model by selectively mutating key residues predicted to play an important role in the binding of inhibitors. Thus, we have a binding model that is applicable to a wide range of potent integrase inhibitors and is consistent with the available resistant mutation data.

Combination therapy with lamivudine and famciclovir for chronic hepatitis B-infected Chinese patients: a viral dynamics study.

In vitro studies have shown that lamivudine and penciclovir (the active metabolite of famciclovir) act synergistically to inhibit hepatitis B virus (HBV) replication. We compared the effectiveness of HBV viral suppression by lamivudine monotherapy versus lamivudine plus famciclovir combination therapy in Chinese patients with chronic HBV infection. Twenty-one Chinese hepatitis B e antigen (HBeAg)-positive patients, with detectable HBV DNA (Digene Hybrid Capture II), were randomized to receive either lamivudine 150 mg/d orally (group 1, 9 patients) or lamivudine 150 mg/d plus famciclovir 500 mg 3 times a day orally (group 2, 12 patients) for 12 weeks, with a follow-up period of at least 16 weeks. Serial serum HBV-DNA levels were determined and a mathematical model with provision for incomplete inhibition of virus production during therapy was applied to analyze the dynamics of viral clearance. The mean antiviral efficacy was significantly greater in group 2 than in group 1 (0.988 +/- 0.012 vs. 0.94 +/- 0.03, P =.0012). HBV DNA returned to pretreatment level within 16 weeks after the end of initial treatment in 4 patients (66.7%) in group 1 and none in group 2 (P =.08), who remained HBeAg positive and received no further treatment after week 12. Hence, in Chinese chronic HBeAg-positive patients, combination therapy using lamivudine and famciclovir was superior to lamivudine monotherapy in inhibiting HBV replication. Further studies of longer duration are needed to define whether combination therapy will increase the HBeAg seroconversion rate and decrease the rate of emergence of lamivudine-resistant variants.

Biphasic clearance kinetics of hepatitis B virus from patients during adefovir dipivoxil therapy.

In a recent phase II clinical study, 13 chronic hepatitis B-infected patients treated daily with 30 mg adefovir dipivoxil for 12 weeks displayed a median 4.1-log10 decrease in plasma hepatitis B virus (HBV)-DNA levels. The decline of viral load during therapy displayed a biphasic kinetic profile that was modeled to determine the efficacy of inhibition of viral production, as well as kinetic constants for the clearance of free virus and the loss of infected cells. Viral production was suppressed with an efficacy of 0.993 +/- 0.008, indicating that only 0.7% of viral production persisted during therapy. The initial, faster phase of viral load decline reflects the clearance of HBV particles from plasma with a half-life of 1.1 +/- 0.3 days, translating to a 48% daily turnover of the free virus. The second, slower phase of viral load decline closely mirrors the rate-limiting process of infected cell loss, with a half-life of 18 +/- 7 days. The duration of therapy required to completely eliminate the virus from plasma or suppress it to levels sufficient to induce seroconversion is a function of the half-life of the free virus, the half-life of infected cells, and the efficacy of inhibition of virus production from infected cells. These quantitative analyses provide a more detailed picture of the dynamics of HBV infection and therapy, and can be used to compare the efficacy of various doses and inhibitors of HBV replication for the treatment of HBV infections.

Allosteric effects of a monoclonal antibody against thrombin exosite II.

We previously isolated a monoclonal antithrombin IgG from a patient with multiple myeloma [Colwell et al. (1997) Br. J. Haematol. 97, 219-226]. Using a panel of 55 surface mutants of recombinant thrombin, we now show that the epitope for the IgG most likely includes Arg-101, Arg-233, and Lys-236 in exosite II. The IgG affects the rate at which thrombin cleaves various peptide p-nitroanilide substrates with arginine in the P1 position, increasing the kcat for substrates with a P2 glycine residue but generally decreasing the kcat for substrates with a P2 proline. The allosteric effect of the IgG is altered by deletion of Pro-60b, Pro-60c, and Trp-60d from the 60-loop of thrombin, which lies between exosite II and the catalytic triad. The effect of the IgG, however, does not depend on the presence or absence of sodium ions, a known allosteric regulator of thrombin. The IgG does not affect the conformation of thrombin exosite I as determined by binding of a fluorescent derivative of hirudin54-65. These results provide evidence for a direct allosteric linkage between exosite II and the catalytic site of thrombin.

Functional requirements for inhibition of thrombin by antithrombin III in the presence and absence of heparin.

Mutation of 79 highly exposed amino acids that comprise approximately 62% of the solvent accessible surface of thrombin identified residues that modulate the inhibition of thrombin by antithrombin III, the principal physiological inhibitor of thrombin. Mutations that decreased the susceptibility of thrombin to inhibition by antithrombin III in the presence and absence of heparin (W50A, E229A, and R233A) also decreased hydrolysis of a small tripeptidyl substrate. These residues were clustered around the active site cleft of thrombin and were predicted to interact directly with the "substrate loop" of antithrombin III. Despite the large size of antithrombin III (58 kDa), no residues outside of the active cleft were identified that interact directly with antithrombin III. Mutations that decreased the susceptibility of thrombin to inhibition by antithrombin III in the presence but not in the absence of heparin (R89A/R93A/E94A, R98A, R245A, K248A, K252A/D255A/Q256A) in general did not also affect hydrolysis of the tripeptidyl substrate. These residues were clustered among a patch of basic residues on a surface of thrombin perpendicular to the face containing the active site cleft and were predicted to interact directly with heparin. Three mutations (E25A, R178A/R180A/D183A, and E202A) caused a slight enhancement of inhibition by antithrombin III.

Protein engineering thrombin for optimal specificity and potency of anticoagulant activity in vivo.

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

Conversion of thrombin into an anticoagulant by protein engineering.

At sites of vascular injury, thrombin interacts with multiple procoagulant substrates, to mediate both fibrin clotting and platelet aggregation. But upon binding to thrombomodulin on the vascular endothelium, thrombin instead activates protein C, thereby functioning as an anticoagulant and attenuating clot formation. Upon infusion in vivo, both the procoagulant and anticoagulant effects of thrombin were observed. Preliminary studies indicating that thrombin's protein C activating and fibrinogen clotting activities could be dissociated by mutagenesis suggested to us that a thrombin variant that lacked procoagulant activity while retaining anticoagulant function might be an attractive antithrombotic agent. Using protein engineering, we introduced a single substitution, E229A, that substantially shifted thrombin's specificity in favour of the anticoagulant substrate, protein C. In monkeys, this modified thrombin functioned as an endogenous protein C activator demonstrating dose-dependent, reversible anticoagulation without any indication of procoagulant activity. Notably, template bleeding times were not prolonged, suggesting a reduced potential for bleeding complications.

Selection of a suppressor mutation that restores affinity of an oligonucleotide inhibitor for thrombin using in vitro genetics.

The thrombin aptamer is a single-stranded DNA of 15 nucleotides that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. This prototype aptamer of thrombin has a unique double G-tetrad structure capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite I. Substitution of arginine 70 in thrombin exosite I with glutamic acid effectively eliminated binding of the prototype thrombin aptamer. In contrast, aptamers selected against R70E thrombin were able to bind and inhibit both wild-type and R70E thrombins, and displayed potassium-independent inhibition. Aptamers selected against R70-E thrombin bound to sites identical or overlapping with that of the prototype thrombin aptamer. These aptamers retained the potential to form double G-tetrad structures; however, these structures would be destabilized by a T-->A substitution, disrupting the T4-T13 base pairing found in the prototype. This destabilization appeared to be partially compensated by newly recruited structural elements. Thus, selection against R70E thrombin did not lead to aptamers that bound to alternative sites, but instead to ssDNA structures with a suppressor mutation that accommodated the mutation in thrombin within a double G-tetrad context. These results provide insight into the aptamer-thrombin interaction and suggest that the binding site for the prototype is the dominant aptamorigenic site on thrombin.

Functional mapping of the surface residues of human thrombin.

Utilizing site-directed mutagenesis, 77 charged and polar residues that are highly exposed on the surface of human thrombin were systematically substituted with alanine. Functional assays using thrombin mutants identified residues that were required for the recognition and cleavage of the procoagulant substrate fibrinogen (Lys21, Trp50, Lys52, Asn53 + Thr55, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Asp193 + Lys196, Glu202, Glu229, Arg233, Asp234) and the anticoagulant substrate protein C (Lys21, Trp50, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Glu229, Arg233), interactions with the cofactor thrombomodulin (Gln24, Arg70) and inhibition by the thrombin aptamer, an oligonucleotide-based thrombin inhibitor (Lys65, His66, Arg70, Tyr71, Arg73). Although there is considerable overlap between the functional epitopes, distinct and specific residues with unique functions were identified. When the functional residues were mapped on the surface of thrombin, they were located on a single hemisphere of thrombin that included both the active site cleft and the highly basic exosite 1. No functional residues were located on the opposite face of thrombin. Residues with procoagulant or anticoagulant functions were not spatially separated but interdigitated with residues of opposite or shared function. Thus thrombin utilizes the same general surface for substrate recognition regardless of substrate function although the critical contact residues may vary.

Structure-function relationships of the thrombin-thrombomodulin interaction.

Thrombomodulin is an anticoagulant protein cofactor that modulates the substrate specificity of thrombin and promotes the cleavage of protein C. The structure-function relationships of the thrombin-thrombomodulin interaction have been explored by recombinant DNA and protein chemistry methods. Thrombomodulin binds to thrombin at an anion-binding exosite on the carboxyl-terminal side of the substrate binding cleft. This interaction interferes with the recognition and cleavage of fibrinogen, factor V, and the platelet thrombin receptor. Binding to thrombomodulin also protects thrombin from inhibition by heparin cofactor II. The major thrombin binding site on thrombomodulin consists of EGF-like domains 5 and 6. In addition, EGF-like domain 4 is required for thrombomodulin to accelerate the activation of protein C. Some thrombomodulin molecules contain a chondroitin sulfate moiety attached to a Ser/Thr-rich domain adjacent to the cell membrane. This modification is not required for the cofactor activity of thrombomodulin, but appears to contribute to 'direct anticoagulant' activity--the ability of thrombomodulin to inhibit fibrinogen clotting, factor V activation, and platelet activation. The chondroitin sulfate moiety of thrombomodulin also can affect the rate of thrombin inhibition by antithrombin III, possibly by competing with heparin for the heparin binding site on thrombin. Detailed understanding of these interactions could lead to new strategies for the treatment of bleeding or thrombotic disorders.

Localization of the single-stranded DNA binding site in the thrombin anion-binding exosite.

Single-stranded DNA molecules containing a 15-nucleotide consensus sequence have been reported to inhibit thrombin activity. The mechanism of the inhibition was studied using a consensus 15-mer oligonucleotide and two recombinant mutant thrombins: the anion-binding exosite mutant thrombin R70E, and thrombin K154A, in which the mutation was located in a surface loop outside of the exosite. The consensus 15-mer oligonucleotide inhibited both fibrinogen-clotting and platelet-activation activities of plasma-derived thrombin, recombinant wild type thrombin, and mutant thrombin K154A in a sequence-specific and dose-dependent manner, whereas it did not inhibit either activity of mutant thrombin R70E. The 15-mer oligonucleotide also inhibited thrombomodulin-dependent protein C activation by plasma-derived thrombin. In competition equilibrium binding experiments, binding of 125I-labeled diisopropyl phosphoryl-thrombin to thrombomodulin was completely inhibited by the consensus 15-mer oligonucleotide with a Kd value of 2.68 +/- 0.16 nM. These results suggest that Arg-70 in the anion-binding exosite of thrombin is a key determinant for interaction with specific single-stranded DNA molecules, and that binding of single-stranded DNA molecules to the exosite prevents the interaction of thrombin with fibrinogen, the platelet thrombin receptor, and thrombomodulin.

Ligand specificity of human thrombomodulin. Equilibrium binding of human thrombin, meizothrombin, and factor Xa to recombinant thrombomodulin.

Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.

Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity.

Thrombomodulin is an endothelial cell thrombin receptor that serves as a cofactor for thrombin-catalyzed activation of protein C. Structural requirements for thrombin binding and cofactor activity were studied by mutagenesis of recombinant human thrombomodulin expressed on COS-7 and CV-1 cells. Deletion of the fourth epidermal growth factor (EGF)-like domain abolished cofactor activity but did not affect thrombin binding. Deletion of either the fifth or the sixth EGF-like domain markedly reduced both thrombin binding affinity and cofactor activity. Thrombin binding sequences were also localized by assaying the ability of synthetic peptides derived from thrombomodulin to compete with diisopropyl fluorophosphate-inactivated 125I-thrombin binding to thrombomodulin. The two most active peptides corresponded to (a) the entire third loop of the fifth EGF-like domain (Kp = 85 +/- 6 microM) and (b) parts of the second and third loops of the sixth EGF-like domain (Kp = 117 +/- 9 microM). These data suggest that thrombin interacts with two discrete elements in thrombomodulin. Deletion of the Ser/Thr-rich domain dramatically decreased both thrombin binding affinity and cofactor activity and also prevented the formation of a high molecular weight thrombomodulin species containing chondroitin sulfate. Substitutions of this domain with polypeptide segments of decreasing length and devoid of glycosylation sites progressively decreased both cofactor activity and thrombin binding affinity. This correlation suggests that increased proximity of the membrane surface to the thrombin binding site may hinder efficient thrombin binding and the subsequent activation of protein C. Membrane-bound thrombomodulin therefore requires the Ser/Thr-rich domain as an important spacer, in addition to EGF-like domains 4-6, for efficient protein C activation.

Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin.

Thrombin is a serine protease that acts as a procoagulant by clotting fibrinogen and activating platelets and as an anticoagulant by activating protein C in a thrombomodulin-dependent reaction. Fibrinogen and thrombomodulin bind competitively to an anion-binding exosite on thrombin. We prepared recombinant normal human thrombin and mutant thrombins with single amino acid substitutions in order to localize and distinguish the fibrinogen- and thrombomodulin-binding sites. Normal and mutant thrombins had similar amidolytic activity. Thrombin K52E had approximately 2.5-fold increased protein C-activating activity but only approximately 17% of normal fibrinogen-clotting activity. Thrombin R70E had normal fibrinogen-clotting activity but only approximately 7% of normal protein C-activating activity. Thrombin R68E had markedly reduced activity in both assays. Decreased activation of protein C correlated with decreased binding affinity for thrombomodulin, and ability to activate platelets correlated directly with fibrinogen-clotting activity. These results demonstrate that thrombins with predominantly anticoagulant or procoagulant activity can be created by mutagenesis and that thrombomodulin- and fibrinogen-binding sites on thrombin may overlap but are not identical.

Regulation of thrombomodulin by tumor necrosis factor-alpha: comparison of transcriptional and posttranscriptional mechanisms.

The procoagulant properties of cultured vascular endothelial cells are enhanced in response to inflammatory cytokines such as tumor necrosis factor-alpha (TNF). A major component of this response is a reduction in expression of thrombomodulin, a cell surface cofactor for the activation of protein C. Regulation of thrombomodulin expression by TNF has been reported to occur through multiple mechanisms. To determine the relative roles of transcriptional and posttranscriptional regulation, the effect of TNF on the turnover of thrombomodulin protein and mRNA was examined in human and bovine endothelial cells. Quantitative nuclease S1 protection assays showed a 70% to 90% reduction in thrombomodulin mRNA within 4 hours of the addition of 1.0 nmol/L TNF to the culture medium. The decrease in thrombomodulin mRNA resulted from inhibition of transcription, followed by rapid degradation of thrombomodulin transcripts (t1/2 less than or equal to 3 hours). In pulse-chase incubations, thrombomodulin synthesis decreased parallel with mRNA, but the rate of degradation of radiolabeled thrombomodulin was not significantly altered by TNF. Human thrombomodulin was degraded with a t1/2 of 8.2 +/- 2.4 hours (SD) or 7.5 +/- 1.3 hours (SD) in the absence or presence of TNF, respectively. We conclude that TNF acts primarily to inhibit thrombomodulin transcription. The subsequent decrease in activity results from the inherent instability of thrombomodulin mRNA and protein in these cells, and not from the regulation of thrombomodulin degradation.

Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues.

Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr approximately 100,000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.

Effects of 5'-terminal modifications on the biological activity of defective interfering RNAs of Sindbis virus.

We have been studying defective interfering (DI) genomes of the RNA enveloped virus Sindbis virus. Deletion mapping of a DI cDNA demonstrated that only sequences at the 3' and 5' termini of the genome are required for the DI RNA to be biologically active. We constructed a series of cDNAs that transcribe DI RNAs differing only in 5'-terminal sequences. Two of the 5' termini identical to ones found in naturally occurring DI RNAs are the 5' terminus of the virion RNA (DI-549) and the first 142 nucleotides from the 5' terminus of the subgenomic 26S mRNA attached to the 5' terminus of the virion RNA (DI-15). The latter has a 42-nucleotide deletion from nucleotides 25 to 66 in the 26S RNA sequence. These DI RNA transcripts were biologically active, but one (DI-526) which did not have the 42-nucleotide deletion of DI-15 was not replicated. The DI RNA isolated after the presumed amplification of the DI-526 transcript had deleted the first 54 nucleotides of the 26S RNA sequences. The 5' terminus of Sindbis virion RNA contains a stem and loop region that is conserved among alphaviruses. An 11-nucleotide deletion in DI-549 that disrupted this stem and loop rendered this DI RNA inactive. In contrast, this same deletion in DI-15 and one that removed an additional 100 nucleotides of the virion 5' terminus did not prevent its amplification. We did not detect by computer analysis any common secondary structures among the biologically active DI RNAs that distinguished them from those RNAs that were not amplified. Our results support the conclusion that tertiary structure or the ability of the RNA to adapt its structure upon interaction with protein is important in the recognition process.

Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging.

Defective-interfering (DI) genomes of a virus contain sequence information essential for their replication and packaging. They need not contain any coding information and therefore are a valuable tool for identifying cis-acting, regulatory sequences in a viral genome. To identify these sequences in a DI genome of Sindbis virus, we cloned a cDNA copy of a complete DI genome directly downstream of the promoter for the SP6 bacteriophage DNA dependent RNA polymerase. The cDNA was transcribed into RNA, which was transfected into chicken embryo fibroblasts in the presence of helper Sindbis virus. After one to two passages the DI RNA became the major viral RNA species in infected cells. Data from a series of deletions covering the entire DI genome show that only sequences in the 162 nucleotide region at the 5' terminus and in the 19 nucleotide region at the 3' terminus are specifically required for replication and packaging of these genomes.

Studies of defective interfering RNAs of Sindbis virus with and without tRNAAsp sequences at their 5' termini.

Three of six independently derived defective interfering (DI) particles of Sindbis virus generated by high-multiplicity passaging in cultured cells have tRNAAsp sequences at the 5' terminus of their RNAs (Monroe and Schlesinger, J. Virol. 49:865-872, 1984). In the present work, we found that the 5'-terminal sequences of the three tRNAAsp-negative DI RNAs were all derived from viral genomic RNA. One DI RNA sample had the same 5'-terminal sequence as the standard genome. The DI RNAs from another DI particle preparation were heterogeneous at the 5' terminus, with the sequence being either that of the standard 5' end or rearrangements of regions near the 5' end. The sequence of the 5' terminus of the third DI RNA sample consisted of the 5' terminus of the subgenomic 26S mRNA with a deletion from nucleotides 24 to 67 of the 26S RNA sequence. These data showed that the 5'-terminal nucleotides can undergo extensive variations and that the RNA is still replicated by virus-specific enzymes. DI RNAs of Sindbis virus evolve from larger to smaller species. In the two cases in which we followed the evolution of DI RNAs, the appearance of tRNAAsp-positive molecules occurred at the same time as did the emergence of the smaller species of DI RNAs. In pairwise competition experiments, one of the tRNAAsp-positive DI RNAs proved to be the most effective DI RNA, but under identical conditions, a second tRNAAsp-positive DI RNA was unable to compete with the tRNAAsp-negative DIs. Therefore, the tRNAAsp sequence at the 5' terminus of a Sindbis DI RNA is not the primary factor in determining which DI RNA becomes the predominant species in a population of DI RNA molecules.