Spacer Peptide Research Articles

Effects of an HIV-1 maturation inhibitor on the structure and dynamics of CA-SP1 junction helices in virus-like particles

HIV-1 maturation involves conversion of the immature Gag polyprotein lattice, which lines the inner surface of the viral membrane, to the mature capsid protein (CA) lattice, which encloses the viral RNA. Maturation inhibitors such as bevirimat (BVM) bind within six-helix bundles, formed by a segment that spans the junction between the CA and spacer peptide 1 (SP1) subunits of Gag, and interfere with cleavage between CA and SP1 catalyzed by the HIV-1 protease (PR). We report solid-state NMR (ssNMR) measurements on spherical virus-like particles (VLPs), facilitated by segmental isotopic labeling, that provide information about effects of BVM on the structure and dynamics of CA-SP1 junction helices in the immature lattice. Although BVM strongly blocks PR-catalyzed CA-SP1 cleavage in VLPs and blocks conversion of VLPs to tubular CA assemblies, 15N and 13C ssNMR chemical shifts of segmentally labeled VLPs with and without BVM are very similar, indicating that interaction with BVM does not alter the six-helix bundle structure appreciably. Only the 15N chemical shift of A280 (the first residue of SP1) changes significantly, consistent with BVM binding to an internal ring of hydrophobic side chains of L279 residues. Measurements of transverse 15N spin relaxation rates reveal a reduction in the amplitudes and/or timescales of backbone N-H bond motions, corresponding to a rigidification of the six-helix bundles. Overall, our data show that inhibition of HIV-1 maturation by BVM involves changes in structure and dynamics that are surprisingly subtle, but still sufficient to produce a large effect on CA-SP1 cleavage.

Nucleic Acid-Induced Dimerization of HIV-1 Gag Protein

HIV-1 Gag is a highly flexible multi-domain protein that forms the protein lattice of the immature HIV-1 virion. In vitro it reversibly dimerizes, but in the presence of nucleic acids (NA) it spontaneously assembles into virus-like particles (VLP). While high-resolution structures have revealed intricate detail of the interactions of the capsid domain (CA) of Gag and the flanking spacer peptide SP1 that stabilize VLPs, much less is known about the assembly pathway, and the interactions of the highly flexible NA-binding nucleocapsid (NC) domain.

Incoming HIV Virion-Derived Gag Spacer Peptide 2 (p1) is a Target of Effective CD8+ T Cell Antiviral Responses

Persistence of HIV through integration into host DNA in CD4+ T cells presents a major barrier to virus eradication. Viral integration may be curtailed when CD8+ T cells are triggered to kill infected CD4+ T cells through recognition of HLA class I-bound peptides derived from incoming virions. However, this has been reported only in individuals with ‘beneficial’ HLA alleles that are associated with superior HIV control. Through interrogation of the pre-integration immunopeptidome, we obtained proof of early presentation of a virion-derived HLA-A*02:01-restricted epitope, FLGKIWPSH (FH9), located in Gag spacer peptide 2 (SP2). FH9-specific CD8+ T cell responses were detectable in individuals with primary HIV infection and eliminated HIV-infected CD4+ T cells prior to virus production in vitro. Our data show that non-beneficial HLA class I alleles can elicit an effective antiviral response through early presentation of HIV virion-derived epitopes and also demonstrate the importance of SP2 as an immune target.

Nucleic acid–induced dimerization of HIV-1 Gag protein

HIV-1 Gag is a highly flexible multidomain protein that forms the protein lattice of the immature HIV-1 virion. In vitro, it reversibly dimerizes, but in the presence of nucleic acids (NAs), it spontaneously assembles into virus-like particles (VLPs). High-resolution structures have revealed intricate details of the interactions of the capsid (CA) domain of Gag and the flanking spacer peptide SP1 that stabilize VLPs, but much less is known about the assembly pathway and the interactions of the highly flexible NA-binding nucleocapsid (NC) domain. Here, using a novel hybrid fluorescence proximity/sedimentation velocity method in combination with calorimetric analyses, we studied initial binding events by monitoring the sizes and conformations of complexes of Gag with very short oligonucleotides. We observed that high-affinity binding of oligonucleotides induces conformational changes in Gag accompanied by the formation of complexes with a 2:1 Gag/NA stoichiometry. This NA-liganded dimerization mode is distinct from the widely studied dimer interface in the CA domain and from protein interactions arising in the SP1 region and may be mediated by protein-protein interactions localized in the NC domain. The formation of the liganded dimer is strongly enthalpically driven, resulting in higher dimerization affinity than the CA-domain dimer. Both detailed energetic and conformational analyses of different Gag constructs revealed modulatory contributions to NA-induced dimerization from both matrix and CA domains. We hypothesize that allosterically controlled self-association represents the first step of VLP assembly and, in concert with scaffolding along the NA, can seed the formation of two-dimensional arrays near the NA.

Fluorescent Protein Inserts in between NC and SP2 are Tolerated for Assembly, Release and Maturation of HIV with Limited Infectivity.

We report the design of a fluorescent HIV construct that is labeled by insertion of fluorescent protein between the nucleocapsid (NC) and spacer peptide 2 (SP2) domains of Gag and further show that the fluorescent protein is released from its confines within Gag during maturation. This fluorescent HIV is capable of budding and maturation with similar efficiency to the parental virus. Virions generated using this design within the R8 HIV backbone pseudotyped with VSV-G were capable of delivering small RNA genomes encoding GFP to the target cells; however, the same design within the NL4-3 backbone has limited HIV infectivity. The virions generated by these constructs are approximately 165 ± 35 nm in size, which is significantly larger than wild type HIV. We suggest that this design has the potential to be a vehicle for protein and small guide RNA delivery.

New Phosphorus Analogs of Bevirimat: Synthesis, Evaluation of Anti-HIV-1 Activity and Molecular Docking Study.

Since the beginning of the human immunodeficiency virus (HIV) epidemic, many groups of drugs characterized by diverse mechanisms of action have been developed, which can suppress HIV viremia. 3-O-(3′,3′-Dimethylsuccinyl) betulinic acid, known as bevirimat (BVM), was the first compound in the class of HIV maturation inhibitors. In the present work, phosphate and phosphonate derivatives of 3-carboxyacylbetulinic acid were synthesized and evaluated for anti-HIV-1 activity. In vitro studies showed that 30-diethylphosphonate analog of BVM (compound 14a) has comparable effects to BVM (half maximal inhibitory concentrations (IC50) equal to 0.02 μM and 0.03 μM, respectively) and is also more selective (selectivity indices: 3450 and 967, respectively). To investigate the possible mechanism of antiviral effect of 14a, molecular docking was carried out on the C-terminal domain (CTD) of HIV-1 capsid (CA)–spacer peptide 1 (SP1) fragment of Gag protein, designated as CTD-SP1, which was described as a molecular target for maturation inhibitors. Compared with interactions between BVM and the protein, an increased number of strong interactions between ligand 14a and protein, generated by the phosphonate group, was observed.

Expression, purification, and characterization of the Degludec precursor DesB30

Diabetes is a chronic metabolic disease, for which recombinant human insulin is the most effective and mainstream treatment. DesB30 is an insulin analogue in which the B chain lacks amino acid 30 (Thr) compared with human insulin. This analogue can be used to produce the long-acting insulin Degludec or Detemir. In this study, a spacer peptide was added before the sequence of DesB30 and the C-peptide was replaced with AAK, a short connecting peptide. The target gene was cloned under the control of AOX1 and expressed as a secretory protein in Pichia pastoris. A high-yield recombination strain was selected by screening for resistance to G418. The basal salts medium was optimized and a lower salt concentration medium was found to show the best effects. Both media were used to compare the yield of high-density fermentation. The maximum yield reached 4.51 g/L in 1/2 basal salt medium, which is the highest reported yield to date. The insulin precursor, which is single-stranded, was purified by weak cation exchange chromatograph and preparative reversed-phase high-performance liquid chromatography (RP-HPLC), from which 73.39% of product was recovered at over 95% purity. The double-stranded protein (DesB30) was obtained by digesting the insulin precursor with trypsin. Using preparative RP-HPLC, the product was obtained with over 95% purity. Finally, the structure of DesB30 was confirmed.

The Use of α-MF Signal Peptide Without Spacer for Producing Insulin Aspart Precursor in Pichia pastoris KM71

A spacer peptide (Glu-Ala repeats) connects N-terminal of a polypeptide precursor to the signal peptide of α-mating factor (α-MF) from Saccharomyces cerevisiae. During the polypeptide precursor secretion, the signal peptide will be first cleaved by signal peptidase in the endoplasmic reticulum, followed by Kex2 endopeptidase and finally the spacer peptide is removed by dipeptidyl aminopeptidase in the Golgi apparatus. In the case of insulin precursor, unfortunately, mixture forms of the insulin precursor containing a spacer peptide and the correct insulin precursor is observed in Pichia pastoris. To overcome this problem, a gene encoding insulin Aspart precursor without the spacer peptide has been constructed and its expression has been investigated in this study. The gene of insulin Aspart precursor has been successfully expressed extracellularly in P. pastoris KM71 with molecular mass of 6306.8 Da as determined by LC-ESI-MS. The expression level of insulin Aspart precursor is affected by aeration, cell density and methanol as an inducer. In shake flask production, the insulin Aspart precursor was optimally produced by 3% methanol induction every 24 h for two days, at initial cell density (OD600) of 60 and aeration (ratio of culture volume to flask volume) of 1:25. Based on Western blot analysis, there is no intracellular insulin Aspart precursor detected. Therefore, the spacer peptide is not essential for insulin Aspart precursor secretion in P. pastoris and removal of spacer resulted in insulin Aspart precursor homogeneity.

Resistance to Second-Generation HIV-1 Maturation Inhibitors.

A betulinic acid-based compound, bevirimat (BVM), inhibits HIV-1 maturation by blocking a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. Previous studies showed that mutations conferring resistance to BVM cluster around the CA-SP1 cleavage site. Single amino acid polymorphisms in the SP1 region of Gag and the C terminus of CA reduced HIV-1 susceptibility to BVM, leading to the discontinuation of BVM's clinical development. We recently reported a series of "second-generation" BVM analogs that display markedly improved potency and breadth of activity relative to the parent molecule. Here, we demonstrate that viral clones bearing BVM resistance mutations near the C terminus of CA are potently inhibited by second-generation BVM analogs. We performed de novo selection experiments to identify mutations that confer resistance to these novel compounds. Selection experiments with subtype B HIV-1 identified an Ala-to-Val mutation at SP1 residue 1 and a Pro-to-Ala mutation at CA residue 157 within the major homology region (MHR). In selection experiments with subtype C HIV-1, we identified mutations at CA residue 230 (CA-V230M) and SP1 residue 1 (SP1-A1V), residue 5 (SP1-S5N), and residue 10 (SP1-G10R). The positions at which resistance mutations arose are highly conserved across multiple subtypes of HIV-1. We demonstrate that the mutations confer modest to high-level maturation inhibitor resistance. In most cases, resistance was not associated with a detectable increase in the kinetics of CA-SP1 processing. These results identify mutations that confer resistance to second-generation maturation inhibitors and provide novel insights into the mechanism of resistance.IMPORTANCE HIV-1 maturation inhibitors are a class of small-molecule compounds that block a late step in the viral protease-mediated processing of the Gag polyprotein precursor, the viral protein responsible for the formation of virus particles. The first-in-class HIV-1 maturation inhibitor bevirimat was highly effective in blocking HIV-1 replication, but its activity was compromised by naturally occurring sequence polymorphisms within Gag. Recently developed bevirimat analogs, referred to as "second-generation" maturation inhibitors, overcome this issue. To understand more about how these second-generation compounds block HIV-1 maturation, here we selected for HIV-1 mutants that are resistant to these compounds. Selections were performed in the context of two different subtypes of HIV-1. We identified a small set of mutations at highly conserved positions within the capsid and spacer peptide 1 domains of Gag that confer resistance. Identification and analysis of these maturation inhibitor-resistant mutants provide insights into the mechanisms of resistance to these compounds.

Repeatedly Applied Peptide Film Kills Bacteria on Dental Implants

The rising use of titanium dental implants has increased the prevalence of peri-implant disease that shortens their useful life. A growing view of peri-implant disease suggests that plaque accumulation and microbiome dysbiogenesis trigger a host immune inflammatory response that destroys soft and hard tissues supporting the implant. The incidence of peri-implant disease is difficult to estimate, but with over 3 million implants placed in the USA alone, and the market growing by 500,000 implants/year, such extensive use demands additional interceptive approaches. We report a water-based, nonsurgical approach to address peri-implant disease using a bifunctional peptide film, which can be applied during initial implant placement and later reapplied to existing implants to reduce bacterial growth. Bifunctional peptides are based upon a titanium binding peptide (TiBP) optimally linked by a spacer peptide to an antimicrobial peptide (AMP). We show herein that dental implant surfaces covered with a bifunctional peptide film kill bacteria. Further, using a simple protocol for cleaning implant surfaces fouled by bacteria, the surface can be effectively recoated with TiBP-AMP to regain an antimicrobial state. Fouling, cleansing, and rebinding was confirmed for up to four cycles with minimal loss of binding efficacy. After fouling, rebinding with a water-based peptide film extends control over the oral microbiome composition, providing a novel nonsurgical treatment for dental implants.

New Insights into Amino-Terminal Translocation as Revealed by the Use of YidC and Sec Depletion Strains

Different attributes of membrane protein substrates have been proposed and characterized as translocation-pathway determinants. However, several gaps in our understanding of the mechanism of targeting, insertion, and assembly of inner-membrane proteins exist. Specifically, the role played by hydrophilic N-terminal tails in pathway selection is unclear. In this study, we have evaluated length and charge density as translocase determinants using model proteins. Strikingly, the 36-residue N-tail of 2Pf3–Lep translocates independent of YidC–Sec. This is the longest known substrate of this pathway. We confirmed this using a newly constructed YidC–Sec double-depletion strain. Increasing its N-tail length with uncharged spacer peptides led to YidC dependence and eventually YidC–Sec dependence, hence establishing that length has a linear effect on translocase dependence. Tails longer than 60 residues were not inserted; however, an MBP–2Pf3–Lep fusion protein could be ranslocated. This suggests that longer N-tails can be translocated if it can engage SecA. In addition, we have examined how the positioning of charges within the translocated N-tail affects the insertion pathway. Additional charges can be translocated by the Lep TM when the charges are distributed across a longer N-tail. We tested charge density as a translocase determinant and confirmed that the addition of positive or negatives charges led to a greater dependence on YidC–Sec when they were placed close to each other than away. Findings from this work make an important advance in our existing knowledge about the different insertion mechanisms of membrane proteins in Escherichia coli.

MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat

HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.

Maturation inhibitors facilitate virus assembly and release of HIV-1 capsid P224 mutant

HIV-1 Maturation inhibitors (MIs) bind to the C-terminal domain of capsid protein (CA-CTD) and spacer peptide 1 (SP1) in HIV-1 Gag and inhibit the CA-SP1 cleavage by stabilizing the immature Gag. The β-turn motif, GVGGP in the HIV CA-CTD (residues 220–224) is one of the key determinants of HIV Gag assembly. In the present study, we mutated each residue of HIV-1 β-turn motif to alanine and observed complete inhibition of virus release of all mutants. This defect in virus release was rescued in the presence of maturation inhibitors; BVM and PF-46396 for P224A mutant. To our knowledge, this is the first report of identification of BVM and PF-46396-dependent capsid mutant. Our results highlight the importance of the core β-turn motif residues in immature virus assembly and suggest that the presence of MIs enhances Gag membrane binding and multimerization thereby restoring virus release of HIV Gag P224 mutant.

Examining Length and Charge Distribution of the Periplasmic N-tail of Bitopic Model Proteins as Determinants of its YidC and Sec Requirement in E.coli

Membrane proteins catalyse essential functions within the cell making them attractive drug target candidates. However, the general principles of membrane protein targeting, insertion and folding are less understood. Universally conserved membrane protein YidC is a key player in this pathway; it performs the dual function of assisting the clearance of Sec dependent proteins and inserting a subset of membrane proteins independently. In this study, we tested the involvement of the translocated substrate periplasmic N-tail in specifying its membrane protein translocase requirement using a bitopic model protein Pf3-Lep which does not require YidC or Sec for its translocation. On systematically increasing the length of its N-tail with neutral spacer peptides, we found that there is a certain N-tail length requirement (∼35 resides) beyond which YidC translocase is required. N-tails longer than 50 residues required both YidC and Sec for its translocation, however translocation was severely impaired beyond 60 residues N-tail length. N-tail translocation of larger periplasmic domains were studied using Maltose-Binding Protein and Alkaline Phosphatase fusions to the N-tail of Pf3-Lep. Surprisingly, these bulky N-terminal regions could be translocated by YidC-Sec even in the absence of their respective signal sequences in the N-terminal direction. These results hint at the involvement of chaperons and other membrane-associated components like SecA in keeping the periplasmic region in an unfolded state and facilitating the translocation of larger N-tails. Finally, we tested the role of charges on Pf3-Lep N-tail as translocase determinants and found that crowding of positive or negative charges led to YidC-Sec dependence. This study provides fundamental information regarding the translocation capacity of YidC and YidC-Sec mode of insertion of single-spanning membrane proteins in E.coli.

In Vitro Exploration of the Anti-HCV Potential of the Synthetic Spacer Peptides Derived from Human, Bovine, and Camel Lactoferrins.

Chronic liver disease is often associated with the infection by hepatitis C virus (HCV), which is an enveloped RNA virus belonging to the Flaviviridae family. Many studies found that milk proteins, such as lactoferrin, might have profound antiviral activity against HCV. Various secretory fluids ranging from milk, to tears, saliva, and nasal secretion, and to bile and pancreatic juice, as well as neutrophils, mucosal surfaces, and blood contain a widely spread multifunctional glycoprotein, lactoferrin (Lf), structure of which can be depicted as two homologous domains connected by the short spacer peptide. This study aimed to understand the effectiveness of the synthetic peptides cLfsp, bLfsp, hLfsp1, and hLfsp2 corresponding to the spacer peptides of camel, bovine, and human Lfs, respectively, against HCV in in vitro settings. We used RT-nested PCR to evaluate the antiviral activity of the synthesized spacer peptides against HCV infectivity in PBMC and HepG2 cells looking at their neutralization, protection, and intracellular treatment potentials. We show that direct interaction of hLfsp1, hLfsp2, and bLfsp with viral particles is able to neutralize the HCV entry into HepG2 cells (with hLfsp2 being more potent neutralizer than hLfsp1 and bLfsp), whereas cLfsp does not show any neutralizing potential. Therefore, our analysis revealed that different spacer peptides are characterized by different antiviral potentials and use different mechanisms for antiviral protection.

Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography-Mass Spectrometry.

Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC-MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that "spacer peptides" can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC-tandem MS methods.

Immature HIV-1 lattice assembly dynamics are regulated by scaffolding from nucleic acid and the plasma membrane

The packaging and budding of Gag polyprotein and viral RNA is a critical step in the HIV-1 life cycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from subnanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA-SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while overexpression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.

In vitro assembly of the Rous Sarcoma Virus capsid protein into hexamer tubes at physiological temperature

During a proteolytically-driven maturation process, the orthoretroviral capsid protein (CA) assembles to form the convex shell that surrounds the viral genome. In some orthoretroviruses, including Rous Sarcoma Virus (RSV), CA carries a short and hydrophobic spacer peptide (SP) at its C-terminus early in the maturation process, which is progressively removed as maturation proceeds. In this work, we show that RSV CA assembles in vitro at near-physiological temperatures, forming hexamer tubes that effectively model the mature capsid surface. Tube assembly is strongly influenced by electrostatic effects, and is a nucleated process that remains thermodynamically favored at lower temperatures, but is effectively arrested by the large Gibbs energy barrier associated with nucleation. RSV CA tubes are multi-layered, being formed by nested and concentric tubes of capsid hexamers. However the spacer peptide acts as a layering determinant during tube assembly. If only a minor fraction of CA-SP is present, multi-layered tube formation is blocked, and single-layered tubes predominate. This likely prevents formation of biologically aberrant multi-layered capsids in the virion. The generation of single-layered hexamer tubes facilitated 3D helical image reconstruction from cryo-electron microscopy data, revealing the basic tube architecture.

Peptide-Based Cancer-Targeted DDS and Molecular Imaging.

Targeting cancer cell-surface receptors is an attractive approach for cancer treatment and diagnosis. Peptides having high binding affinities to receptors overexpressed in cancer cells are useful because of their simple structure, low immunogenicity, and easy, cost-effective chemical synthesis. A number of peptide ligands have been developed for cancer cell-surface receptors and applied to nanoparticles with anticancer drugs, genes, small interfering RNAs (siRNAs), and molecular imaging agents. In particular, recent findings have revealed that peptide-modified PEGylated liposome-encapsulated drugs are effective in cancer-targeted therapy and cancer cell-specific imaging. This review discusses peptide-modified nanoparticles for drug delivery systems (DDS) and molecular imaging, focusing on peptide ligands for somatostatin receptors, integrin, transferrin receptor, human epidermal growth factor 2 (HER2), etc. In addition, methods to improve binding affinity or endosomal escape with spacer peptides and stimuli (internal and external) are discussed.

Mechanistic Studies and Modeling Reveal the Origin of Differential Inhibition of Gag Polymorphic Viruses by HIV-1 Maturation Inhibitors.

HIV-1 maturation inhibitors (MIs) disrupt the final step in the HIV-1 protease-mediated cleavage of the Gag polyprotein between capsid p24 capsid (CA) and spacer peptide 1 (SP1), leading to the production of infectious virus. BMS-955176 is a second generation MI with improved antiviral activity toward polymorphic Gag variants compared to a first generation MI bevirimat (BVM). The underlying mechanistic reasons for the differences in polymorphic coverage were studied using antiviral assays, an LC/MS assay that quantitatively characterizes CA/SP1 cleavage kinetics of virus like particles (VLPs) and a radiolabel binding assay to determine VLP/MI affinities and dissociation kinetics. Antiviral assay data indicates that BVM does not achieve 100% inhibition of certain polymorphs, even at saturating concentrations. This results in the breakthrough of infectious virus (partial antagonism) regardless of BVM concentration. Reduced maximal percent inhibition (MPI) values for BVM correlated with elevated EC50 values, while rates of HIV-1 protease cleavage at CA/SP1 correlated inversely with the ability of BVM to inhibit HIV-1 Gag polymorphic viruses: genotypes with more rapid CA/SP1 cleavage kinetics were less sensitive to BVM. In vitro inhibition of wild type VLP CA/SP1 cleavage by BVM was not maintained at longer cleavage times. BMS-955176 exhibited greatly improved MPI against polymorphic Gag viruses, binds to Gag polymorphs with higher affinity/longer dissociation half-lives and exhibits greater time-independent inhibition of CA/SP1 cleavage compared to BVM. Virological (MPI) and biochemical (CA/SP1 cleavage rates, MI-specific Gag affinities) data were used to create an integrated semi-quantitative model that quantifies CA/SP1 cleavage rates as a function of both MI and Gag polymorph. The model outputs are in accord with in vitro antiviral observations and correlate with observed in vivo MI efficacies. Overall, these findings may be useful to further understand antiviral profiles and clinical responses of MIs at a basic level, potentially facilitating further improvements to MI potency and coverage.