Proteolytic events of HIV-1 replication as targets for therapeutic intervention.

Acquired immunodeficiency syndrome (AIDS) is a worldwide epidemic caused by infection with HIV, a human retrovirus. Proteolysis occurs at many points of the retroviral life-cycle, and these events can be considered as targets for chemotherapy. The most well-known proteolytic action in the retroviral life-cycle is the processing of the Gag and Gag-Pro-Pol polyproteins with the virally encoded protease at the late phase of viral infection. Protease inhibitors, together with reverse transcriptase inhibitors, are important components of the drug combinations currently used to treat HIV patients. The current combination therapy substantially reduced morbidity and mortality in HIV-infected patients. However, these drugs do not allow viral eradication, therefore their long-term use is required, allowing the development of resistance in a large portion of patients. Furthermore, several adverse metabolic side effects have been observed associated with the therapy. Thus, new approaches are required to eradicate HIV infection, which may include targeting of the potential early-phase function of the viral protease, and other crucial proteolytic events of the viral replication, such as the ubiquitin-dependent proteolytic degradation of the unfolded viral proteins as well as the inhibition of envelope protein processing.

Comparison of the substrate specificity of the human T-cell leukemia virus and human immunodeficiency virus proteinases.

Human T-cell leukemia virus type-1 (HTLV-1) is associated with a number of human diseases. Based on the therapeutic success of human immunodeficiency virus type 1 (HIV-1) PR inhibitors, the proteinase (PR) of HTLV-1 is a potential target for chemotherapy. To facilitate the design of potent inhibitors, the subsite specificity of HTLV-1 PR was characterized and compared to that of HIV-1 PR. Two sets of substrates were used that contained single amino-acid substitutions in peptides representing naturally occurring cleavage sites in HIV-1 and HTLV-1. The original HIV-1 matrix/capsid cleavage site substrate and most of its substituted peptides were not hydrolyzed by the HTLV-1 enzyme, except for those with hydrophobic residues at the P4 and P2 positions. On the other hand, most of the peptides representing the HTLV-1 capsid/nucleocapsid cleavage site were substrates of both enzymes. A large difference in the specificity of HTLV-1 and HIV-1 proteinases was demonstrated by kinetic measurements, particularly with regard to the S4 and S2 subsites, whereas the S1 subsite appeared to be more conserved. A molecular model of the HTLV-1 PR in complex with this substrate was built, based on the crystal structure of the S9 mutant of Rous sarcoma virus PR, in order to understand the molecular basis of the enzyme specificity. Based on the kinetics of shortened analogs of the HTLV-1 substrate and on analysis of the modeled complex of HTLV-1 PR with substrate, the substrate binding site of the HTLV-1 PR appeared to be more extended than that of HIV-1 PR. Kinetic results also suggested that the cleavage site between the capsid and nucleocapsid protein of HTLV-1 is evolutionarily optimized for rapid hydrolysis.

Cloning of the bovine leukemia virus proteinase in Escherichia coli and comparison of its specificity to that of human T-cell leukemia virus proteinase.

The proteinase of bovine leukemia virus (BLV) was cloned into pMal-c2 vector with N-terminal or with N- as well as C-terminal flanking sequences, and expressed in fusion with maltose binding protein. The proteinase self-processed itself from the fusion protein during expression and formed inclusion bodies. The enzyme was purified from inclusion bodies by cation-exchange chromatography followed by gel filtration. Specificity of the enzyme was compared to that of human T-cell leukemia proteinase type 1. Although the two viruses belong to the same subfamily of retroviruses, the differences in their proteinase specificity, based on kinetics with oligopeptide substrates representing naturally occurring cleavage sites as well as on inhibition pattern, appear to be pronounced.

Comparison of the effect of FK506 and cyclosporin A on virus production in H9 cells chronically and newly infected by HIV-1.

The presence of FK506-binding protein-12 was demonstrated in virions of HIV-1, although its concentration was lower than that of cyclophilin A. The effect of two inhibitors of the peptidyl-prolyl cis-trans isomerases FK506 and cyclosporin A (CsA) was studied in H9 cells that were chronically infected by HIV-1. Both drugs inhibited virus production in the infected cells in a concentration-dependent manner, by decreasing the number of the producing cells. FK506 did not have an effect on Gag processing, based on the p24 antigen content of virions produced in the presence of this drug. Furthermore, FK506 treatment of uninfected H9 cells did not diminish their susceptibility toward HIV-1 infection, whereas CsA treatment decreased the degree of HIV-1 infection with an IC(50) of 1-2 microg/ml. Also, pretreatment of the virus with CsA decreased its infectivity in HeLaCD4-LTR/beta-gal cells; in contrast, at concentrations up to 10 microg/ml, FK506 did not have an effect. Our findings on the antiviral activity of FK506 and CsA suggest that FK506 is effective only in chronically infected cells, by selectively inhibiting the growth of HIV-1 infected cells, whereas CsA has a specific effect on virus replication.

Effect of substrate residues on the P2' preference of retroviral proteinases.

The substrate sequence requirements for preference toward P2' Glu residue by human immunodeficiency virus type 1 (HIV-1) proteinase were studied in both the matrix protein/ capsid protein (MA/CA) and CA/p2 cleavage site sequence contexts. These sequences represent typical type 1 (-aromatic*Pro-) and type 2 (-hydrophobic* hydrophobic-) cleavage site sequences, respectively. While in the type 1 sequence context, the preference for P2' Glu over Ile or Gln was found to be strongly dependent on the ionic strength and the residues being outside the P2-P2' region of the substrate, it remained preferable in the type 2 substrates when typical type 1 substrate sequence residues were substituted into the outside regions. The pH profile of the specificity constants suggested a lower pH optimum for substrates having P2' Glu in contrast to those having uncharged residues, in both sequence contexts. The very low frequency of P2' Glu in naturally occurring retroviral cleavage sites of various retroviruses including equine infectious anemia virus (EIAV) and murine leukemia virus (MuLV) suggests that such a residue may not have a general regulatory role in the retroviral life cycle. In fact, unlike HIV-1 and HIV-2, EIAV and MuLV proteinases do not favor P2' Glu in either the MA/CA or CA/p2 sequence contexts.

Effect of serine and tyrosine phosphorylation on retroviral proteinase substrates.

Vimentin, a cellular substrate of HIV type 1 (HIV-1) proteinase, contains a protein kinase C (PKC) phosphorylation site at one of its cleavage sites. Peptides representing this site were synthesized in P2 Ser-phosphorylated and nonphosphorylated forms. While the nonphosphorylated peptide was a fairly good substrate of the enzyme, phosphorylation prevented hydrolysis. Phosphorylation of human recombinant vimentin by PKC prevented its processing within the head domain, where the phosphorylation occurred. Oligopeptides representing naturally occurring cleavage sites at the C-terminus of the Rous sarcoma virus integrase were assayed as substrates of the avian proteinase. Unlike the nonphosphorylated peptides, a Ser-phosphorylated peptide was not hydrolyzed by the enzyme at the Ser-Pro bond, suggesting the role of previously established phosphorylation in processing at this site. Ser-phosphorylated and Tyr-phosphorylated forms of model substrates were also tested as substrates of the HIV-1 and the avian retroviral proteinases. In contrast to the moderate effect of P4 Ser phosphorylation, phosphorylation of P1 Tyr prevented substrate hydrolysis by HIV-1 proteinase. Substrate phosphorylation had substantially smaller effects on the hydrolysis by the avian retroviral proteinase. As the active retroviral proteinase as well as various protein kinases are incorporated into mature virions, substrate phosphorylation resulting in attenuation or prevention of proteolytic processing may have important consequences in the regulation of the retroviral life cycle as well as in virus-host cell interactions.

Transfer RNA modification status influences retroviral ribosomal frameshifting.

The possibility of whether tRNAs with and without a highly modified base in their anticodon loop may influence the level of retroviral ribosomal frameshifting was examined. Rabbit reticulocyte lysates were programmed with mRNA encoding UUU or AAC at the frameshift site and the corresponding Phe tRNA with or without the highly modified wyebutoxine (Y) base on the 3' side of its anticodon or Asn tRNA with or without the highly modified queuine (Q) base in the wobble position of its anticodon added. Phe and Asn tRNAs without the Y or Q base, respectively, stimulated the level of frameshifting, suggesting that the frameshift event is influenced by tRNA modification status. In addition, when AAU occurred immediately upstream of UUU as the penultimate frameshift site codon, addition of tRNAAsn without the Q base reduced the stimulatory effect of tRNAPhe without the Y base, whereas addition of tRNAAsn with the Q base did not alter the stimulatory effect. The addition of tRNAAsn without the Q base and tRNAPhe with the Y base inhibited frameshifting. The latter studies suggest an interplay between the tRNAs decoded at the penulimate frameshift and frameshift site codons that is also influenced by tRNA modification status. These data may be intrepreted as indicating that a hypomodified isoacceptor modulates frameshifting in an upward manner when utilized at the frameshift site codon, but modulates frameshifting in a downward manner when utilized at the penultimate frameshift site codon.

Stabilization from autoproteolysis and kinetic characterization of the human T-cell leukemia virus type 1 proteinase.

We have developed a system for expression and purification of wild-type human T-cell leukemia virus type 1 (HTLV-1) proteinase to attain sufficient quantities for structural, kinetic, and biophysical investigations. However, similar to the human immunodeficiency virus type 1 (HIV-1) proteinase, HTLV-1 proteinase also undergoes autoproteolysis rapidly upon renaturation to produce two products. The site of this autoproteolytic cleavage was mapped, and a resistant HTLV-1 proteinase construct (L40I) as well as another construct, wherein the two cysteine residues were exchanged to alanines, were expressed and purified. Oligopeptide substrates representing the naturally occurring cleavage sites in HTLV-1 were good substrates of the HTLV-1 proteinase. The kinetic parameters kcat and Km were nearly identical for all the three enzymes. Although three of four peptides representing HTLV-1 proteinase cleavage sites were fairly good substrates of HIV-1 proteinase, only two of nine peptides representing HIV-1 proteinase cleavage sites were hydrolyzed by the HTLV-1 proteinase, suggesting substantial differences in the specificity of the two enzymes. The large difference in the specificity of the two enzymes was also demonstrated by inhibition studies. Of the several inhibitors of HIV-1 or other retroviral proteinases that were tested on HTLV-1 proteinase, only two inhibit the enzyme with a Ki lower than 100 nM.

Expression and characterization of human foamy virus proteinase.

The human foamy virus proteinase was expressed in fusion with maltose binding protein in Escherichia coli and purified. The specific activity of the fusion protein was similar to that of the processed enzyme. The kinetic constants on foamy virus cleavage site substrates were very low but comparable to those obtained with the gag-encoded avian proteinase on its own substrates. The proteinase showed preference for high ionic strength and a pH optimum of 6.6. None of the tested retroviral cleavage site peptides were substrates, however, some peptides representing cleavage sites in retrotransposons were properly processed by the enzyme.

Comparison of the specificity of homo- and heterodimeric linked HIV-1 and HIV-2 proteinase dimers.

The specificity of linked homo- and heterodimeric HIV-1 and HIV-2 proteinases was characterized by using oligopeptide substrates. For two substrates the k(cat)/Km values for the heterodimers were the mean values for those of the homodimers, suggesting that these substrates could productively bind into the heterodimers in both directions. However, for two other substrates the k(cat)/Km values for the heterodimers were higher than those of the homodimers, suggesting that these substrates could productively bind into the enzymes in a preferable direction. However, the mode of binding does not seem to depend on the sequential position of the subunits. The studied linked homo- and heterodimers may represent intermediate stages in the evolution of bilobal aspartic proteinases. As divergence in sequence of the two halves of such a proteinase increases, the possibility of bidirectional binding is likely lost at the expense of the optimized side-chain subsite interactions. The differences in observed and calculated k(cat)/Km values revealed dependence of the substrate specificity at one subsite of the enzyme from the next residue in sequence of substrate. These findings were also supported by molecular modeling studies.

Ubiquitin is covalently attached to the p6Gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12Gag protein of Moloney murine leukemia virus.

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.

Studies on the symmetry and sequence context dependence of the HIV-1 proteinase specificity.

Two major types of cleavage sites with different sequence preferences have been proposed for the human immunodeficiency virus type 1 (HIV-1) proteinase. To understand the nature of these sequence preferences better, single and multiple amino acid substitutions were introduced into a type 1 cleavage site peptide, thus changing it to a naturally occurring type 2 cleavage site sequence. Our results indicated that the previous classification of the retroviral cleavage sites may not be generally valid and that the preference for a residue at a particular position in the substrate depends strongly on the neighboring residues, including both those at the same side and at the opposite side of the peptide backbone of the substrate. Based on these results, pseudosymmetric (palindromic) substrates were designed. The retroviral proteinases are symmetrical dimers of two identical subunits; however, the residues of naturally occurring cleavage sites do not show symmetrical arrangements, and no obvious symmetrical substrate preference has been observed for the specificity of HIV proteinase. To examine the role of the asymmetry created by the peptide bonds on the specificity of the respective primed and nonprimed halves of the binding site, amino acid substitutions were introduced into a palindromic sequence. In general, the results suggested that the asymmetry does not result in substantial differences in specificity of the S3 and S3' subsites, whereas its effect is more pronounced for the S2 and S2' subsites. Although it was possible to design several good palindromic substrates, asymmetrical arrangements may be preferred by the HIV proteinase.

Protein composition and morphology of human foamy virus intracellular cores and extracellular particles.

Characterization of human foamy virus (HFV) gag-encoded precursors and the search for a Gag-Pol polyprotein and mature proteins derived from proteolytic processing were carried out in HFV-infected cells and with purified preassembled cores and extracellular virus by Western blotting and radioimmunoprecipitation using antisera against synthetic peptides corresponding to putative Gag and protease proteins. Precursor proteins, Pr78gag/74gag and Pr135pol, were found in the nucleus of epithelial and fibroblast cells 3-4 days after HFV infection. Kinetic analysis of HFV Pr78gag and Pr74gag indicated that Pr78gag is a precursor to Pr74gag. South-Western blot analysis indicated that Pr78gag and Pr74gag have properties associated with nucleic acid binding protein although they lack the typical zinc-finger motifs found in retroviral nucleocapsid proteins. Western blot analyses of preassembled HFV cores isolated from the cytoplasm of infected cells and purified by sucrose gradient centrifugation demonstrated the presence of Pr78gag/74gag and Pr135pol, but no proteolytically processed Gag proteins were observed. The majority of extracellular HFV particles were found to have pentagon-shaped cores, as observed intracellularly, and are believed to be the immature extracellular form of the virus. The highest concentration of extracellular particles, estimated by EM, Western blot, and reverse transcriptase assays were found in sucrose gradient fractions having a density of 1.21-1.24 g/cm3. Western blot analysis revealed that Pr78gag/74gag and Pr135pol were the major viral proteins associated with these extracellular particles, as only small amounts of putative proteolytically cleaved capsid (p32) were observed. Our results support the notion that Pol is translated independent of Gag in HFV-infected cells.

Activity of tethered human immunodeficiency virus 1 protease containing mutations in the flap region of one subunit.

The tethered-dimer protease of human immunodeficiency virus 1 (HIV-1) [Cheng Y.-S. E., Yin, F.H., Foundling, S., Blomstrom, D. & Kettner, C. A. (1990) Proc. Natl Acad. Sci. USA 87, 9660-9664] and its mutants containing amino acid substitutions or deletions or both in only one flap region were expressed in Escherichia coli. These mutant enzymes showed various degrees of self-processing and significantly reduced catalytic activity toward oligopeptide substrates compared with the wild type. Kinetic parameters determined for one of the oligopeptide substrates showed a dramatic increase in K(m) and decrease in Kcat values. Unexpectedly, the substrate cleavage was more efficient in low salt concentration for a mutant containing a shortened hydrophilic flap. Assays with oligopeptides representing naturally occurring cleavage sites or oligopeptides containing single amino acid substitutions at the P2 and P2' substrate positions showed only moderate changes in the substrate specificity of the mutant proteases. Predicted structures for the mutants were constructed by molecular modeling and used to interpret the results of kinetic measurements. In general, the data suggest that the mutated part of the flaps does not have a major role in determining substrate specificity; rather, it provides the hydrophobic environment and hydrogen-bond interactions with the conserved water that are necessary for efficient substrate binding and catalysis.

Effect of cyclosporin A on the replication cycle of human immunodeficiency virus type 1 derived from H9 and Molt-4 producer cells.

The effect of cyclosporin A (CsA) on the replication of human immunodeficiency virus type 1 (HIV-1) was studied. CsA treatment inhibited virus production in chronically infected H9 and Molt-4 cells. CsA treatment of HeLaCD4-LTR/beta-gal cells or extracellular viruses also inhibited infection (IC50 1 microg/ml). The intracellular CsA-binding molecule cyclophilin A was detected in HIV-1 derived from chronically infected H9 cells, but it was present at a substantially lower level in HIV-1 derived from chronically infected Molt-4 cells. The low level of cyclophilin A in viral particles derived from Molt-4 cells correlated well with their substantially lower infectivity as assayed on HeLaCD4-LTR beta-gal cells. CsA treatment of infected cells showed a dose-dependent reduction of cyclophilin A incorporation into virions; the amount of cyclophilin A incorporation was found to be dependent on the producer cell type.

Activity of linked HIV-1 proteinase dimers containing mutations in the active site region.

Mutations were introduced into the active site triplet (Asp-Thr-Gly) of one or both subunits of a linked dimer of human immunodeficiency virus type 1 proteinase. Mutation of Thr to Ser in one or both subunits did not alter the activity of the enzyme substantially, whereas its mutation to Asn in one subunit caused a dramatic decrease in catalytic efficiency. Mutation of Gly to Val in one subunit also yielded an enzyme with very low activity. The enzymes containing Thr-->Asn and Gly-->Val mutations in both subunits resulted in inactive enzymes, based on their inability to self-process and on assay with an oligopeptide substrate. The dramatic decrease in enzyme efficiency of the mutants was interpreted using molecular models of the enzymes.

Comparative studies on the substrate specificity of avian myeloblastosis virus proteinase and lentiviral proteinases.

The retroviral proteinase (PR) seems to play crucial roles in the viral life cycle, therefore it is an attractive target for chemotherapy. Previously we studied the specificity of human immunodeficiency virus (HIV) type 1 and type 2 as well as equine infectious anemia virus PRs using oligopeptide substrates. Here a similar approach is used to characterize the specificity of avian myeloblastosis virus (AMV) PR and to compare it with those of the previously characterized lentiviral PRs. All peptides representing naturally occurring Gag and Gag-Pol cleavage sites were substrates of the AMV PR. Only half of these peptides were substrates of HIV-1 PR. The Km values for AMV PR were in a micromolar range previously found for the lentiviral PRs; however, the kcat values were in a 10 30-fold lower range. A series of peptides containing single amino acid substitutions in a sequence representing a naturally occurring HIV cleavage site was used to characterize the seven substrate binding subsites of the AMV PR. The largest differences were found at the P4 and P2 positions of the substrate. Detailed analysis of the results by molecular modeling and comparison with previously reported data revealed the common characteristics of the specificity of the retroviral PRs as well as its strong dependence on the sequence context of the substrate.

Mutational analysis of the substrate binding pocket of murine leukemia virus protease and comparison with human immunodeficiency virus proteases.

The differences in substrate specificity between Moloney murine leukemia virus protease (MuLV PR) and human immunodeficiency virus (HIV) PR were investigated by site-directed mutagenesis. Various amino acids, which are predicted to form the substrate binding site of MuLV PR, were replaced by the equivalent ones in HIV-1 and HIV-2 PRs. The expressed mutants were assayed with the substrate Val-Ser-Gln-Asn-Tyr decreases Pro-Ile-Val-Gln-NH2 (decreases indicates the cleavage site) and a series of analogs containing single amino acid substitutions in positions P4(Ser) to P3'(Val). Mutations at the predicted S2/S2' subsites of MuLV PR have a strong influence on the substrate specificity of this enzyme, as observed with mutants H37D, V39I, V54I, A57I, and L92I. On the other hand, substitutions at the flap region of MuLV PR often rendered enzymes with low activity (e.g. W53I/Q55G). Three amino acids (His-37, Val-39, and Ala-57) were identified as the major determinants of the differences in substrate specificity between MuLV and HIV PRs.

Binding of human prothymosin alpha to the leucine-motif/activation domains of HTLV-I Rex and HIV-1 Rev.

Rex of human T-cell leukemia virus type I (HTLV-I) and Rev of human immunodeficiency virus 1 (HIV-1) are post-transcriptional regulators of viral gene expression. By means of affinity chromatography, we purified an 18-kDa cellular protein that bound to the conserved leucine-motif/activation domain of HTLV-I Rex or HIV-1 Rev. The protein that was purified through a Rev-affinity column was found to bind to Rex immunoprecipitated with anti-Rex IgG from an HTLV-I-producing cell line. We analyzed the purified approximately 18-kDa protein biochemically and identified it as prothymosin alpha. The binding activity of prothymosin alpha to Rev or Rex was completely abolished when the epsilon-amino groups of its lysine residues were chemically modified by N-succinimidyl-3-(4-hydroxy-3,5-diodo- phenyl)propionate. The functional relationship between the nuclear protein prothymosin alpha and Rex-Rev is discussed.

Intracellular transport of the murine leukemia virus during acute infection of NIH 3T3 cells: nuclear import of nucleocapsid protein and integrase.

The entry and intracellular transport of Moloney-murine leukemia virions inside mouse NIH 3T3 cells have been followed by electron microscopy techniques. Five viral proteins--matrix (MA, p15), capsid (CA, p30), nucleocapsid (NC, p10), integrase (IN), and the envelope glycoprotein (SU, gp70)--were located by immunolabeling using gold probes. After entering the cells, viral particles were frequently detected inside cytoplasmic vesicles of variable size. Their viral envelope was apparently lost during intracytoplasmic transport. When the unenveloped viral cores reached the nuclear membrane or its vicinity, they were disrupted. Two of the immunolabeled proteins, NC and IN, were detected entering the nucleus of non-dividing cells, where both were targeted to the nucleolus. However, MA and CA were found only in the cytoplasm. NC is a nucleic acid-binding protein which contains potential nuclear localization signals. We suggest that NC could enter the nucleus as part of a nucleoprotein complex, associated with IN, and possibly, also with viral DNA.