Active human immunodeficiency virus protease is required for viral infectivity.

Retroviral proteins are synthesized as polyprotein precursors that undergo proteolytic cleavages to yield the mature viral proteins. The role of the human immunodeficiency virus (HIV) protease in the viral replication cycle was examined by use of a site-directed mutation in the protease gene. The HIV protease gene product was expressed in Escherichia coli and observed to cleave HIV gag p55 to gag p24 and gag p17 in vitro. Substitution of aspartic acid residue 25 (Asp-25) of this protein with an asparagine residue did not affect the expression of the protein, but it eliminated detectable in vitro proteolytic activity against HIV gag p55. A mutant HIV provirus was constructed that contained the Asn-25 mutation within the protease gene. SW480 human colon carcinoma cells transfected with the Asn-25 mutant proviral DNA produced virions that contained gag p55 but not gag p24, whereas virions from cells transfected with the wild-type DNA contained both gag p55 and gag p24. The mutant virions were not able to infect MT-4 lymphoid cells. In contrast, these cells were highly sensitive to infection by the wild-type virions. These results demonstrate that the HIV protease is an essential viral enzyme and, consequently, an attractive target for anti-HIV drugs.     

Mechanisms associated with the generation of biologically active human immunodeficiency virus type 1 particles from defective proviruses.

The human immunodeficiency virus (HIV) is the etiological agent of acquired immunodeficiency syndrome (AIDS). HIV exhibits extensive genetic diversity and it is apparent that an infected individual contains different populations of distinct viral strains, a large proportion of which has been found surprisingly to be defective for replication. A similar phenomenon has also been observed with some cell lines that are known to produce infectious viral particles but harbor defective proviral genomes. Here, we investigated the molecular basis of this phenomenon by cloning proviral genomes of HIV from a cell line that was capable of producing high titers of biologically active HIV particles that readily induced syncytia with CD4+ cell lines and peripheral blood lymphocytes. This cell line was found to contain five proviral genomes, all of which, when tested individually, failed to produce replication-competent viruses upon transfection into human cells. However, when a specific combination of two proviral genomes was used in such transfection studies, it was possible to obtain biologically active, replication-competent viral particles that infected and replicated in CD4+ cell lines and induced syncytia characteristic of HIV. Such a result may be due to homologous recombination between proviral DNAs occurring in cells after transfection and/or complementation of replication-defective proviral DNAs. The diploid nature of the viral RNA genome present in the viral particle may enable the persistence of defective HIV genomes.     

Dynamics of HIV-1 Gag Processing as Revealed by Fluorescence Lifetime Imaging Microscopy and Single Virus Tracking

The viral polyprotein Gag plays a central role for HIV-1 assembly, release and maturation. Proteolytic processing of Gag by the viral protease is essential for the structural rearrangements that mark the transition from immature to mature, infectious viruses. The timing and kinetics of Gag processing are not fully understood. Here, fluorescence lifetime imaging microscopy and single virus tracking are used to follow Gag processing in nascent HIV-1 particles in situ. Using a Gag polyprotein labelled internally with eCFP, we show that proteolytic release of the fluorophore from Gag is accompanied by an increase in its fluorescence lifetime. By tracking nascent virus particles in situ and analyzing the intensity and fluorescence lifetime of individual traces, we detect proteolytic cleavage of eCFP from Gag in a subset (6.5%) of viral particles. This suggests that for the majority of VLPs, Gag processing occurs with a delay after particle assembly.     

Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease

The virally encoded protease of human immunodeficiency virus is responsible for the processing of the gag and gag-pol polyprotein precursors to their mature polypeptides. Since correct processing of the viral polypeptides is essential for the production of infectious virus, HIV protease represents a potential target for therapeutic agents that may prove beneficial in the treatment of AIDS. In this study, full-length gag polyprotein has been synthesized in vitro to serve as a substrate for bacterially expressed HIV-1 protease. Expression of the protease in E. coli from the lac promoter was enhanced approximately five-fold by deletion of a potential hairpin loop upstream from the codon determining the amino terminus of mature protease. Extracts of induced cultures of E. coli harboring a protease-containing plasmid served as the source of protease activity. The gag polyprotein synthesized in vitro was cleaved by such lysates, producing fragments corresponding in size to p17 plus p24 and mature p24. Immunoprecipitations with monoclonal antibodies to p17 and p24 polypeptides suggest that initial cleavage of gag polyprotein occurs near the p24-p15 junction. The proteolysis was inhibited by pepstatin with an IC50 of 0.15 mM for cleavage at the p24-p15 junction and 0.02 mM for cleavage at the p17-p24 junction.     

Engineering human immunodeficiency virus 1 protease heterodimers as macromolecular inhibitors of viral maturation.

Dimerization of human immunodeficiency virus type 1 protease (HIV-1 PR) monomers is an essential prerequisite for viral proteolytic activity and the subsequent generation of infectious virus particles. Disruption of the dimer interface inhibits this activity as does formation of heterodimers between wild-type and defective monomers. A structure-based approach was used to identify amino acid substitutions at the dimer interface of HIV-1 PR that facilitate preferential association of heterodimers and inhibit self-association of the defective monomers. Expression of the designed PR monomers inhibits activity of wild-type HIV-1 PR and viral infectivity when assayed in an ex vivo model system. These results show that it is possible to design PR monomers as macromolecular inhibitors that may provide an alternative to small molecule inhibitors for the treatment of HIV infection.     

Maturation of human immunodeficiency virus particles assembled from the gag precursor protein requires in situ processing by gag-pol protease

The vaccinia virus expression system was used to determine the role of human immunodeficiency virus type 1 (HIV-1) protease in viral morphogenesis and maturation. The unprocessed p55 gag precursor polyprotein alone was assembled to form HIV-1 particles which budded from cells. The particles were spherical and immature, containing an electron-dense shell in the particle submembrane; there was no evidence of core formation. Expression of both gag and pol proteins from a recombinant containing the complete gag-pol coding sequences resulted in intracellular processing of gag-pol proteins and the production of mature particles with electron-dense cores characteristic of wild-type HIV virions. To ascertain the role of protein processing in particle maturation, the pol ORF in the gag-pol recombinant was truncated to limit expression of the pol gene to the protease domain. With this recombinant expressing p55 gag and protease, intracellular processing was observed. Some of the resultant particles were partially mature and contained processed gag protein subunits. In contrast, particle maturation was not observed when the HIV-1 protease and p55 gag were coexpressed from separate recombinants, despite evidence of intracellular gag processing. These findings suggest that HIV-1 protease must be an integral component of the full-length gag-pol precursor for optimal processing and virion maturation.     

Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6(gag), a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6(gag). Consistent with this, viruses with mutations in PR or p6(gag) were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.     

Abelson murine leukemia virus: molecular cloning of infectious integrated proviral DNA.

The integrated proviral genome of Abelson murine leukemia virus (A-MuLV) was cloned in lambda gtWES . lambda B bacteriophage after EcoRI endonuclease digestion and enrichment of proviral sequences by sequential RPC-5 column chromatography and agarose gel electrophoresis. Recombinant DNA clones containing a 7.8-kilobase-pair EcoRI insert were shown to have the entire integrated A-MuLV genome with both 5' and 3' ends flanked by mink cellular DNA sequences. This DNA fragment was shown to induce focus transformation upon transfection of NIH/3T3 mouse cells. Moreover, focus-forming virus could be rescued from transformed nonproducer cells upon superinfection with a type C helper virus. A polyprotein of molecular weight 120,000 (p120) containing murine leukemia virus gag gene determinants was invariably deteced by immunoprecipitation analysis of individual transformants induced by the 7.8-kilobase-pair DNA. Molecularly cloned integrated A-MuLV in its infectious form should be of use in elucidating the mechanisms involved in transformation by this virus.     

Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes.

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having alpha,beta-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.     

Plasmoviruses: nonviral/viral vectors for gene therapy.

We have generated a chimeric gene transfer vector that combines the simplicity of plasmids with the infectivity and long-term expression of retroviruses. We replaced the env gene of a Moloney murine leukemia virus-derived provirus by a foreign gene, generating a plasmid that upon transfer to tumor cells generates noninfectious retroviral particles carrying the transgene. We added to this plasmid an independent expression cassette comprising a cytomegalovirus promoter, an amphotropic retroviral envelope, and a polyadenylylation signal from simian virus 40. These constructs were designed to minimize the risk of recombination generating replication-competent retroviruses. Their only region of homology is a 157-bp sequence with 53% identity. We show that the sole transfection of this plasmid in various cell lines generates infectious but defective retroviral particles capable of efficiently infecting and expressing the transgene. The formation of infectious particles allows the transgene propagation in vitro. Eight days after transfection in vitro, the proportion of cells expressing the transgene is increased by 10-60 times. There was no evidence of replication-competent retrovirus generation in these experiments. The intratumoral injection of this plasmid, but not of the control vector lacking the env gene, led to foci of transgene-expressing cells, suggesting that the transgene had propagated in situ. Altogether, these "plasmoviruses" combine advantages of viral and non-viral vectors. They should be easy to produce in large quantity as clinical grade materials and should allow efficient and safe in situ targeting of tumor cells.     

Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors.

The observed in vitro and in vivo benefit of combination treatment with anti-human immunodeficiency virus (HIV) agents prompted us to examine the potential of resistance development when two protease inhibitors are used concurrently. Recombinant HIV-1 (NL4-3) proteases containing combined resistance mutations associated with BMS-186318 and A-77003 (or saquinavir) were either inactive or had impaired enzyme activity. Subsequent construction of HIV-1 (NL4-3) proviral clones containing the same mutations yielded viruses that were severely impaired in growth or nonviable, confirming that combination therapy may be advantageous. However, passage of BMS-186318-resistant HIV-1 (RF) in the presence of either saquinavir or SC52151, which represented sequential drug treatment, produced viable viruses resistant to both BMS-186318 and the second compound. The predominant breakthrough virus contained the G48V/A71T/V82A protease mutations. The clone-purified RF (G48V/A71T/V82A) virus, unlike the corresponding defective NL4-3 triple mutant, grew well and displayed cross-resistance to four distinct protease inhibitors. Chimeric virus and in vitro mutagenesis studies indicated that the RF-specific protease sequence, specifically the Ile at residue 10, enabled the NL4-3 strain with the triple mutant to grow. Our results clearly indicate that viral genetic background will play a key role in determining whether cross-resistance variants will arise.     

Isolation of defective mutant of avian sarcoma virus

A colony of transformed cells was isolated from chick-embryo cells infected with a stock of nondefective Schmidt-Ruppin strain of Rous sarcoma virus. The virus recovered from this colony was a stable defective mutant very similar to the Bryan strain of Rous sarcoma virus in the following characteristics: (i) noninfectiousness of virus particles released from transformed cells that lack helper factor; (ii) formation of infectious pseudotypes by coinfection with avian leukosis virus or by interaction with endogenous-helper factor in chicken cells; (iii) ability of the noninfectious form of virus to transform chick-embryo cells in the presence of ultraviolet light-inactivated Sendai virus; (iv) absence of glycoprotein in the noninfectious form; (v) failure to produce nondefective virus by recombination with avian leukosis virus; and (vi) segregation of polymerase-negative virus.The morphology of transformed cells is characteristic of those infected by the Schmidt-Ruppin strain. The demonstration of segregation of such a defective virus from nondefective sarcoma virus and failure to detect revertants of this mutant suggest that the deletion of some genes may be involved in this mutation.     

De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells.

We have investigated the block to expression of Moloney murine leukemia virus in murine embryonal carcinoma (EC) cells. Infected EC cells were found to contain up to 100 integrated proviral genomes. However, expression of virus as measured by XC plaque and virus-specific RNA synthesis did not occur at significant levels, in contrast to productively infected differentiated cells. Analysis of the DNA in the infected EC cells revealed that the proviral genomes were highly methylated, as shown by their resistance to cleavage by Sma I. Integrated proviral genomes in infected differentiated cells were readily cut by Sma I and thus were not methylated at these sites. Transfection of DNA from infected EC cells to cells permissive for virus expression failed to induce virus expression. The proviral genomes, however, were potentially infectious because they induced XC plaques when the recipient cells for transfection were treated with 5-azacytidine. This drug is believed to interfere with DNA methylation. We conclude that expression of proviral genomes introduced into EC cells is suppressed and that this inactivation can be correlated with the de novo methylation of the viral DNA. De novo methylation activity thus may be a characteristic of early embryonic cells.     

A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC(50) value of 20 microM, which was improved to 600 nM via synthetic optimization. The resulting compound, GRL0617, inhibited SARS-CoV viral replication in Vero E6 cells with an EC(50) of 15 microM and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs.     

Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor.

The mature gag and pol proteins of human immunodeficiency virus (HIV) and all retroviruses derive from large gag and gag-pol polyprotein precursors by posttranslational cleavage. A highly specific, virally encoded protease is required for this essential proteolytic processing. In this study, the HIV protease gene product was expressed in Escherichia coli and shown to autocatalyze its maturation from a larger precursor. In addition, this bacterially produced HIV protease specifically processed an HIV p55 gag polyprotein precursor when coexpressed in E. coli. This system will allow detailed structure-function analysis of the HIV protease and provides a simple assay for the development of potential therapeutic agents directed against this critical viral enzyme.     

DNA methylation and gene expression: endogenous retroviral genome becomes infectious after molecular cloning.

The Mov-3 substrain of mice carries Moloney murine leukemia virus as a Mendelian gene in its germ line. All mice segregating the Mov-3 locus activate virus and develop viremia and leukemia. The integrated provirus (i.e., Mov-3 locus) was molecularly cloned from Mov-3 liver DNA as a 16.8 kilobase long EcoRI fragment. Comparison of the cloned and genomic Mov-3 specific EcoRI fragment by restriction enzyme analysis showed no differences in the size of the fragments, indicating that no major sequence rearrangements occurred during cloning. The genomic and cloned Mov-3 DNAs were compared for methylation and infectivity. Analysis with Hha I showed that the genomic proviral and the flanking mouse sequences were methylated at cytosine residues, in contrast to the cloned Mov-3 locus. The cloned Mov-3 locus, however, was highly infectious in a transfection assay (1 x 10(-3) plaque-forming unit per viral genome) in contrast to the genomic Mov-3 DNA (less than 10(-7) per viral genome). Our results suggest that genes containing 5-methylcytosine are not expressed after transfection into susceptible cells and that removal of the methyl groups by molecular cloning in prokaryotes leads to expression generating infectious proviral DNA. If gene expression of transfected DNA is controlled by mechanisms that are relevant for gene expression in the animal, this suggests that DNA methylation may play a causative role in eukaryotic gene regulation.