Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.23.16 (HIV-1 protease)
 2,107 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structural proteins of the retroviral capsid are translated as a polyprotein (the Gag precursor) that is cleaved by a virally encoded protease. Processing of the human immunodeficiency virus type 1 Gag precursor Pr55 was analyzed through a combination of pulse-chase labeling, cell fractionation, and immunoprecipitation. We observed a membrane-associated processing pathway for the Gag precursor that gives rise to virions. In addition, we found that a significant amount of processing occurs in the cytoplasm of infected cells resulting in the intracellular accumulation of appropriately processed viral proteins. This observation suggests the viral protease is active in the cytoplasmic compartment of the cell. Processing of the Gag protein was blocked in both compartments by the addition of a viral protease inhibitor. A comparison of the amount of cytoplasmic processing seen in lytically infected cells with that seen in chronically infected cells showed that cytoplasmic processing was associated with the lytic infection. These observations raise the possibility that activation of the human immunodeficiency virus type 1 protease in the cytoplasm of lytically infected cells might result in the cleavage of cellular proteins and thus contribute to cytotoxicity.
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PMID:Human immunodeficiency virus type 1 Gag proteins are processed in two cellular compartments. 203 93

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.
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PMID:The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions. 796 91

Protease inhibitors are currently the most effective antiviral agents against human immunodeficiency virus type 1 (HIV-1). In this study we determined the effect of four HIV-1 protease inhibitors on human T cell leukemia virus type 1 (HTLV-I). Rhesus monkey cells infected with HTLV-I were treated with different concentrations of indinavir, saquinavir, ritonavir, or nelfinavir. The effect of these inhibitors was monitored through their effect on the processing efficiency of the viral Gag protein in cells, the natural substrate for the viral protease. These inhibitors failed to block processing of HTLV-I Gag. To confirm these findings, human cells were cotransfected with plasmids encoding infectious copies of HIV-1 and HTLV-I, and the cells were subsequently treated with these same HIV-1 protease inhibitors. At concentrations between 5 and 50 times the IC50 for inhibition of HIV-1 replication, inhibition of HIV-1 Gag cleavage was apparent. In contrast, no effect on HTLV-I Gag processing was seen. At higher concentrations, HIV-1 Gag processing was essentially completely inhibited whereas HTLV-I Gag cleavage was still unaffected. Thus, these inhibitors are not effective inhibitors of HTLV-I Gag processing. Sequence alignments of the HIV-1 and HTLV-I viral proteases and processing sites suggest that the active site of the HTLV-I protease may have subtle differences in substrate recognition compared with the HIV-1 protease.
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PMID:HIV type 1 protease inhibitors fail to inhibit HTLV-I Gag processing in infected cells. 968 47

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4(+) T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4(+) T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.
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PMID:A bipartite membrane-binding signal in the human immunodeficiency virus type 1 matrix protein is required for the proteolytic processing of Gag precursors in a cell type-dependent manner. 976 51

A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cell-mediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1(SF2) p55(Gag) were modified extensively. First, the viral codons were changed to conform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55(Gag) protein expression to levels that ranged from 322- to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and these showed high-level Rev-independent expression of p55(Gag) and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag and gag protease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gag showed Gag-specific antibody and CD8(+) cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with plasmids containing the native gag gene. Most importantly, four of four rhesus monkeys that received two or three immunizations with modified gag plasmid DNA demonstrated substantial Gag-specific CTL responses. These results highlight the useful application of modified gag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.
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PMID:Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene. 1068 77

Despite the effectiveness of currently available human immunodeficiency virus type 1 (HIV-1) therapies, a continuing need exists for new drugs to treat HIV-1 infection. We investigated the mechanism by which 3-O-[3',3'-dimethylsuccinyl]-betulinic acid (DSB) inhibits HIV-1 replication. DSB functions at a late stage of the virus life cycle but does not inhibit the HIV-1 protease in vitro or interfere with virus assembly or release. DSB specifically delays the cleavage of Gag between the capsid (CA) and p2, resulting in delayed formation of the mature viral core and reduced HIV-1 infectivity. Replication of simian immunodeficiency virus (SIV) was resistant to DSB; however, a chimeric SIV carrying CA-p2 sequences from HIV-1 was inhibited by the drug, indicating that susceptibility to DSB maps to the CA-p2 region of the HIV-1 Gag protein. A single point mutation at the CA-p2 cleavage site of HIV-1 conferred strong resistance to DSB, confirming the target of the drug. HIV-1 strains that are resistant to a variety of protease inhibitors were sensitive to DSB. These findings indicate that DSB specifically protects the CA-p2 cleavage site from processing by the viral protease during virion maturation, thereby revealing a novel mechanism for pharmacologic inhibition of HIV-1 replication.
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PMID:Small-molecule inhibition of human immunodeficiency virus type 1 replication by specific targeting of the final step of virion maturation. 1469 23

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles.
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PMID:Protease cleavage leads to formation of mature trimer interface in HIV-1 capsid. 2292 21

Viruses can incorporate foreign glycoproteins to form infectious particles through a process known as pseudotyping. However, not all glycoproteins are compatible with all viruses. Despite the fact that viral pseudotyping is widely used, what makes a virus/glycoprotein pair compatible is poorly understood. To study this, we chose to analyze a gammaretroviral glycoprotein (Env) whose compatibility with different viruses could be modulated through small changes in its cytoplasmic tail (CT). One form of this glycoprotein is compatible with murine leukemia virus (MLV) particles but incompatible with human immunodeficiency virus type 1 (HIV-1) particles, while the second is compatible with HIV-1 particles but not with MLV particles. To decipher the factors affecting virus-specific Env incompatibility, we characterized Env incorporation, maturation, cell-to-cell fusogenicity, and virus-to-cell fusogenicity of each Env. The HIV-1 particle incompatibility correlated with less efficient cleavage of the R peptide by HIV-1 protease. However, the MLV particle incompatibility was more nuanced. MLV incompatibility appeared to be caused by lack of incorporation into particles, yet incorporation could be restored by further truncating the CT or by using a chimeric MLV Gag protein containing the HIV-1 MA without fully restoring infectivity. The MLV particle incompatibility appeared to be caused in part by fusogenic repression in MLV particles through an unknown mechanism. This study demonstrates that the Env CT can dictate functionality of Env within particles in a virus-specific manner.IMPORTANCE Viruses utilize viral glycoproteins to efficiently enter target cells during infection. How viruses acquire viral glycoproteins has been studied to understand the pathogenesis of viruses and develop safer and more efficient viral vectors for gene therapies. The CTs of viral glycoproteins have been shown to regulate various stages of glycoprotein biogenesis, but a gap still remains in understanding the molecular mechanism of glycoprotein acquisition and functionality regarding the CT. Here, we studied the mechanism of how specific mutations in the CT of a gammaretroviral envelope glycoprotein distinctly affect infectivity of two different viruses. Different mutations caused failure of glycoproteins to function in a virus-specific manner due to distinct fusion defects, suggesting that there are virus-specific characteristics affecting glycoprotein functionality.
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PMID:Sequence Determinants in Gammaretroviral Env Cytoplasmic Tails Dictate Virus-Specific Pseudotyping Compatibility. 3089 64