Protease Coding Sequence Research Articles

HIV-1 Reverse Transcriptase Error Rates and Transcriptional Thresholds Based on Single-strand Consensus Sequencing of Target RNA Derived From In Vitro-transcription and HIV-infected Cells

Nucleotide incorporation and lacZ-based forward mutation assays have been widely used to determine the accuracy of reverse transcriptases (RTs) in RNA-dependent DNA polymerization reactions. However, they involve quite complex and laborious procedures, and cannot provide accurate error rates. Recently, NGS-based methods using barcodes opened the possibility of detecting all errors introduced by the RT, although their widespread use is limited by cost, due to the large size of libraries to be sequenced. In this study, we describe a novel and relatively simple NGS assay based on single-strand consensus sequencing that provides robust results with a relatively small number of raw sequences (around 60 Mb). The method has been validated by determining the error rate of HIV-1 (BH10 strain) RT using the HIV-1 protease-coding sequence as target. HIV-1 reverse transcription error rates in standard conditions (37 °C/3 mM Mg2+) using an in vitro-transcribed RNA were around 7.3 × 10−5. In agreement with previous reports, an 8-fold increase in RT’s accuracy was observed after reducing Mg2+ concentration to 0.5 mM. The fidelity of HIV-1 RT was also higher at 50 °C than at 37 °C (error rate 1.5 × 10−5). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg2+ (7.4 × 10−5) were similar to those determined with the in vitro-transcribed RNA, and were reduced to 1.8 × 10−5 in the presence of 0.5 mM Mg2+. Values obtained at low magnesium concentrations were modestly higher than the transcription error rates calculated for human cells, thereby suggesting a realistic transcriptional threshold for our NGS-based error rate determinations.

Hepatitis C Virus (HCV) NS3 sequence diversity and antiviral resistance-associated variant frequency in HCV/HIV coinfection.

HIV coinfection accelerates disease progression in chronic hepatitis C and reduces sustained antiviral responses (SVR) to interferon-based therapy. New direct-acting antivirals (DAAs) promise higher SVR rates, but the selection of preexisting resistance-associated variants (RAVs) may lead to virologic breakthrough or relapse. Thus, pretreatment frequencies of RAVs are likely determinants of treatment outcome but typically are below levels at which the viral sequence can be accurately resolved. Moreover, it is not known how HIV coinfection influences RAV frequency. We adopted an accurate high-throughput sequencing strategy to compare nucleotide diversity in HCV NS3 protease-coding sequences in 20 monoinfected and 20 coinfected subjects with well-controlled HIV infection. Differences in mean pairwise nucleotide diversity (π), Tajima's D statistic, and Shannon entropy index suggested that the genetic diversity of HCV is reduced in coinfection. Among coinfected subjects, diversity correlated positively with increases in CD4(+) T cells on antiretroviral therapy, suggesting T cell responses are important determinants of diversity. At a median sequencing depth of 0.084%, preexisting RAVs were readily identified. Q80K, which negatively impacts clinical responses to simeprevir, was encoded by more than 99% of viral RNAs in 17 of the 40 subjects. RAVs other than Q80K were identified in 39 of 40 subjects, mostly at frequencies near 0.1%. RAV frequency did not differ significantly between monoinfected and coinfected subjects. We conclude that HCV genetic diversity is reduced in patients with well-controlled HIV infection, likely reflecting impaired T cell immunity. However, RAV frequency is not increased and should not adversely influence the outcome of DAA therapy.

Placement of p6pol between tandem repeat HIV-1 protease domains reduces Gag cleavage efficiency

Background HIV-1 protease (PR) is encoded by pol, which is initially translated as a Pr160 polyprotein by a ribosomal frameshift event [1]. Pr160 is incorporated into virions via interactions with assembling Pr55. The PRmediated proteolytic cleavage of Pr55 and Pr160, known as virus maturation, is essential for the acquisition of viral infectivity. Within the Gag-Pol, the p6gag is truncated and is replaced by a transframe domain referred to as p6* or p6pol. Removal of p6pol improves Gag-Pol autoprocessing, suggesting that p6pol is involved in regulation of PR activation [2]. However, overlapping of p6gag/p6pol reading frame hampers generic approach to studying p6pol biological function. To assess the p6pol contribution to PR-mediated virus maturation without affecting p6gag reading frame, we introduced an extra copy of p6pol-PR or PR coding sequence at the PR C-terminus.

Development of a 2,4-Dinitrotoluene-Responsive Synthetic Riboswitch in E. coli Cells

Riboswitches are RNA sequences that regulate expression of associated downstream genes in response to the presence or absence of specific small molecules. A novel riboswitch that activates protein translation in E. coli cells in response to 2,4-dinitrotoluene (DNT) has been engineered. A plasmid library was constructed by incorporation of 30 degenerate bases between a previously described trinitrotoluene aptamer and the ribosome binding site. Screening was performed by placing the riboswitch library upstream of the Tobacco Etch Virus (TEV) protease coding sequence in one plasmid; a second plasmid encoded a FRET-based construct linked with a peptide containing the TEV protease cleavage site. Addition of DNT to bacterial culture activated the riboswitch, initiating translation of TEV protease. In turn, the protease cleaved the linker in the FRET-based fusion protein, causing a change in fluorescence. This new riboswitch exhibited a 10-fold increase in fluorescence in the presence of 0.5 mM DNT compared to the system without target.

FRET-Based Optical Assay for Monitoring Riboswitch Activation

Riboswitches are regulatory RNAs located in the 5'-untranslated region of mRNA sequences that recognize and bind to small molecules and regulate the expression of downstream genes. Creation of synthetic riboswitches to novel ligands depends on the ability to monitor riboswitch activation in the presence of analyte. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins. The theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence. Our FRET construct was composed of eGFP and a nonfluorescent yellow fluorescent protein mutant called REACh (for resonance energy-accepting chromoprotein) connected with a peptide linker containing a TEV protease cleavage site. Addition of theophylline to the E. coli cells activates the riboswitch and initiates the translation of mRNA. Synthesized protease cleaves the linker in the FRET-based fusion protein causing a change in the fluorescence signal. By this method, we observed an 11-fold increase in cellular extract fluorescence in the presence of theophylline. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence. This leads to an improved detection of FRET via better signal-to-noise ratio, allowing us to monitor riboswitch activation in a wide range of analyte concentrations from 0.01 to 2.5 mM.

HCV NS3 Serine Protease-neutralizing Single-chain Antibodies Isolated by a Novel Genetic Screen

Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable beta-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant beta-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal) plates), while induction of NS3 expression results in loss of beta-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources.

Natural Variation in HIV-1 Protease, Gag p7 and p6, and Protease Cleavage Sites within Gag/Pol Polyproteins: Amino Acid Substitutions in the Absence of Protease Inhibitors in Mothers and Children Infected by Human Immunodeficiency Virus Type 1

Reduced sensitivity of human immunodeficiency virus type 1 (HIV-1) to protease inhibitors is associated with multiple amino acid substitutions in the virus-encoded protease. The combination of changes that contribute to drug resistance is dependent in part upon the amino acid residues comprising protease alleles prior to drug therapy. We analyzed within peripheral blood mononuclear cells from HIV-1-infected mothers and their children viralgag/polregions, which included p7, transframe p6/p6*, and protease coding sequences, as well as six protease cleavage sites. Sixty protease alleles from 12 individuals differed by at least 3 to as many as 10 amino acids from proteases encoded by molecular clones of HIV-1, indicating that there is no prototype or consensus wild-type HIV-1 protease sequence. Protease variants with a proline at position 63, a substitution associated with resistance to protease inhibitors, appeared in the absence of antiprotease therapy in 7 patients and were transmitted by 2 mothers to their infants. Gag p7 and p6 regions were significantly more variable than protease. The p6/p6* region contained length variants and amino acid repeats in both reading frames. Five protease cleavage sites (B, D′, D, E, and F) contained highly conserved amino acid sequences in individuals infected by epidemiologically distinct viruses. In contrast, C cleavage sites, localized between Gag p2 and Gag p7, displayed considerable amino acid variability, were unique among groups of infected individuals, and appeared to be related to particular protease alleles. Genetic variabilityin vivoin protease, in cleavage sites, and in proteins upstream of protease provides the potential to modulate enzyme activity and susceptibility to protease inhibitors.

Extensive Regions ofpolAre Required for Efficient Human Immunodeficiency Virus Polyprotein Processing and Particle Maturation

Human Immunodeficiency type 1 particle maturation is dependent upon proteolytic cleavage of the gag and gag-pol precursors by thepol-encoded viral protease. We have investigated the importance of domains ofpolother than the protease for particle maturation and gag proteolytic processing. Truncations of the gag-pol polyprotein precursor of HIV-1 were created by deleting segments of the reverse transcriptase coding region or by introducing stop codons in the integrase region of an HIV-1 infectious molecular clone. In these mutants, the protease coding sequence was left intact. Particles produced by all of the mutants displayed abnormal morphologies and impaired proteolytic processing of gag. The severity of particle morphology abnormalities and of gag polyprotein processing impairment appeared to be affected both by the size and by the position of the deletions inpol,suggesting that the integrity of severalpoldomains within the gag-pol precursor is required for optimal protease activation and particle maturation. Additionally, cotransfection of a deletion mutant with wild-type provirus led to a marked reduction in the titer of infectious virus, suggesting that truncated gag-pol precursors can interfere with wild-type virus assembly and maturation.

Resistance of human immunodeficiency virus type 1 to protease inhibitors: selection of resistance mutations in the presence and absence of the drug

Inhibitors of the human immunodeficiency virus (HIV) protease are a promising class of antiviral agents that dramatically reduce HIV replication both in culture and in infected patients. However, as for many other antiviral compounds, long-term efficacy of these agents is impeded by the emergence of virus variants with increased resistance to their inhibitory action, following selection of specific mutations in the protease coding sequence. We have studied HIV-1 variants that emerged at different stages of selection in the presence of the C2-symmetrical protease inhibitor ABT-77003. The selection of variants was a gradual process during which mutations accumulated at different sites in the protease, generating virus populations with increasing levels of resistance to the drug. The initially selected viruses had a low level of resistance as well as a markedly reduced replicative capacity. Further accumulation of mutations at secondary sites led to an improvement in both drug resistance and replication. In spite of their reduced infectivity, partially selected virus populations did not readily revert to wild-type when serially passaged in drug-free conditions. Instead, even in the absence of drug, secondary mutations identical to those selected in the presence of the inhibitor continued to emerge. These mutations improved both the intrinsic replicative capacity of the virus and its level of resistance to the inhibitor, suggesting that once committed to drug resistance, readaptation of the enzyme to its natural substrate leads to a reduction of its sensitivity to the inhibitor.

High level expression in Escherichia coli cells and purification of poliovirus protein 2Apro

The poliovirus protease 2Apro has been produced to high levels in Escherichia coli using the inducible system that utilizes T7 RNA polymerase. The protease coding sequences that contained an additional AUG to start translation were cloned in pET vectors. Synthesis of 2Apro was induced by IPTG or IPTG plus rifampicin, the levels of the protein made being higher when IPTG alone was used. The expression of the protein is not toxic for E. coli cells and can be readily visualized by Coomassie blue staining of total bacterial protein extracts separated in polyacrylamide gels. Centrifugation of the broken bacterial cells sediments more than 95% of the 2Apro synthesized at a 95% purity level after sarkosyl treatment. Antibodies raised against 2Apro in E. coli recognize a 16K protein in poliovirus-infected cells. In addition, 2Apro shows activity in trans as measured by the cleavage of p220 in HeLa cell extracts and by cleavage of a poliovirus protein substrate that contains the junction between the P1 and P2 polypeptides.

Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein

We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.

Expression of gag precursor protein and secretion of virus-like gag particles of HIV-2 from recombinant baculovirus-infected insect cells

A recombinant baculovirus carrying the gag gene but lacking the protease coding sequences of human immunodeficiency virus type 2 (HIV-2) has been constructed. When this recombinant baculovirus is used to infect insect cells, a high level of gag precursor protein, gag pr41, is expressed. Electron microscopy showed that the majority of gag pr41 was budding through the plasma membrane and being released into the culture medium in spherical virus-like particles with a diameter of approximately 100 nm. Metabolic labeling demonstrates that gag pr41 is myristylated. Our results demonstrated that HIV-2 gag pr41 can be assembled into virus-like particles in the absence of other HIV proteins. Rabbits immunized with purified gag pr41 particles produced high-titer antibody and Western blot analysis showed that anti- gag pr41 rabbit sera recognize p17, p24, and p55 gag proteins of HIV-1. These results show that gag pr41 particles are highly immunogenic and that gag proteins of HIV-1 and HIV-2 have similar antigenic epitopes.

Identification of HTLV-I gag protease and its sequential processing of the gag gene product.

The full-length provirus of human T-cell leukemia virus type I (HTLV-I) was isolated from MT-2, a lymphoid cell line producing HTLV-I. In transfected cells, structural proteins of HTLV-I, the gag and env products, were formed and processed in the same manner as observed in MT-2 cells. The nucleotide sequence was determined for a region between the gag and pol genes of the proviral DNA clone containing an open-reading frame. The deduced amino acid sequences show that this open-reading frame encodes a putative HTLV-I protease. The protease gene (pro) of HTLV-I was investigated using a vaccinia virus expression vector. Processing of 53k gag precursor polyprotein into mature p19, p24, and p15 gag structural proteins was detectable with a recombinant plasmid harboring the entire gag- and protease-coding sequence. We demonstrated that the protease processed the gag precursor polyprotein in a trans-action. A change in the sequence Asp(64)-Thr-Gly, the catalytic core sequence among aspartyl proteases, to Gly-Thr-Gly was shown to abolish correct processing, suggesting that HTLV-I protease may belong to the aspartyl protease group. The 76k gag-pro precursor polyprotein was identified, implying that a cis-acting function of HTLV-I protease may be necessary to trigger the initial cleavage event for its own release from a precursor protein, followed by the release of p53 gag precursor protein. The p53 gag precursor protein is then processed by the trans-action of the released protease to form p19, p24, and p15.

Characterization and autoprocessing of precursor and mature forms of human immunodeficiency virus type 1 (HIV 1) protease purified from Escherichia coli.

A recombinant plasmid encompassing the human immunodeficiency virus type 1 (HIV 1) protease coding sequence and flanking regions (Ala-13 to Gly-185 of the pol open reading frame) has been expressed in two distinct strains of Escherichia coli, AR58 and AR68. In the first strain, AR58, the primary translation product, a 25 kilodalton (kDa) precursor protein, is short-lived and rapidly processes itself to the 11 kDa mature protease in vivo. In the second strain, AR68, the 25 kDa species is only partially processed, and it, a 13 kDa intermediate, and the mature 11 kDa enzyme accumulate at a ratio of 3:4.5:2.5, respectively. The 11 kDa mature protease from AR58 and the 25 kDa precursor from AR68 have been purified to homogeneity. The yield of 11 kDa enzyme from AR58 is approximately 0.02 mg/g wet weight of E. coli cell pellet. The protease has both the expected NH2- and COOH-terminal sequences. The yield of 25 kDa enzyme from AR68 is approximately 0.1 mg/g wet weight of E. coli cell pellet. In vitro, the 25 kDa precursor enzyme rapidly (t1/2 approximately equal to 9 min) processes itself into a species with a mass of approximately 13 kDa and a species with a mass of approximately 11 kDa. Both of these latter species can be separated by RP-HPLC, have the NH2-terminal sequence expected for the mature protease, and are active. The 11 kDa enzyme from AR58 comigrates with the 11 kDa enzyme from AR68 on RP-HPLC and SDS polyacrylamide gel electrophoresis. On extended incubation at 4 degrees C at either neutral or acidic pH all species of the protein exhibit further autodegradation at defined sequences. The availability of the mature, 11 kDa enzyme and the 25 kDa precursor will allow biochemical and physical studies on this critical viral enzyme.

Processing of gag precursor polyprotein of human T-cell leukemia virus type I by virus-encoded protease

The biological activity encoded in the putative protease gene (pro) of human T-cell leukemia virus type I was investigated by using a vaccinia virus expression vector. The 53-kilodalton gag precursor polyprotein was processed into the mature p19, p24, and p15 gag proteins when the gag and protease-coding sequence was expressed under the control of a vaccinia virus promoter, suggesting that the protease may be synthesized through the mechanism of ribosomal frame shifting. The processing defect of a protease mutant could be complemented by cointroduction of a wild-type construct into the cell, demonstrating that the pro gene encodes the biologically active protease molecules which are capable of processing the gag precursor polyprotein in vivo in trans. A study involving the use of a variety of mutants constructed in vitro revealed that the protease consists of a nonessential carboxy-terminal region and a part essential for its activity, including the putative catalytic residue, aspartic acid. Furthermore, a cluster of adenine residues positioned at the overlapping region between the gag and pro genes was shown to be involved in the ribosomal frameshifting event for the synthesis of protease. To mimic the formation of the 76-kilodalton gag-pro precursor polyprotein formed by ribosomal slipping, the coding frames of the gag and pro gene were adjusted. The processing of the gag-pro precursor polyprotein depended on an intact protease gene, implying that a cis-acting function of human T-cell leukemia virus type I protease may be necessary to trigger the initial cleavage event that leads to the release of protease from the precursor protein.

Physical and genetic analysis of DNA regions encoding the immunoglobulin A proteases of different specificities produced by Haemophilus influenzae.

The structural gene for immunoglobulin A protease (iga) from Haemophilus influenzae serotype d was cloned in pBR322. The gene was used as a probe for Southern hybridization analysis of chromosomal DNA from the five other H. influenzae serotypes (a, b, c, e, and f). In most cases strains from a single serotype exhibited a distinct pattern of restriction fragment(s) homologous to the iga gene probe which was unique for that serotype. Serotype f strains were unique in that they gave two distinct patterns of homologous restriction fragments which correlated well with the production of two different protease types by members of this group. An iga mutant of H. influenzae serotype d was isolated by introducing a 4-base-pair insertion into the cloned iga gene and using the altered DNA for transformation of an H. influenzae recipient. The resulting iga- mutant produced no immunoglobulin A protease but was otherwise indistinguishable from its iga+ parent in growth characteristics. Transformation of mutant cells with chromosomal DNA isolated from either a serotype d or a serotype c strain gave rise to iga+ transformants. Those obtained with serotype d DNA produced a type 1 protease, whereas those obtained with serotype c DNA produced either a type 1 protease (characteristic of serotype d) or a type 2 protease (characteristic of serotype c). Southern analysis of the latter transformants, using the iga gene probe, indicated that the type 1 transformants had a serotype d pattern of restriction fragments whereas the type 2 transformants had either a serotype c or a novel pattern of restriction fragments. These results indicate that there is considerable homology between the iga genes of the various serotypes and that the homologous sequences identified with the serotype d probe are the immunoglobulin A protease-coding sequences in each case.