A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein-protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

• Keywords
• HIV-1 CA inhibitor, PF74 derivatives, disassembly, uncoating,
• MeSH
• HIV-1 drug effects   MeSH
• Indoles chemical synthesis   chemistry   pharmacology   MeSH
• Anti-HIV Agents chemical synthesis   chemistry   pharmacology   MeSH
• Humans MeSH
• Magnetic Resonance Spectroscopy MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Drug Design MeSH
• Recombinant Proteins MeSH
• Virus Assembly drug effects   MeSH
• Chemistry Techniques, Synthetic MeSH
• Virion drug effects   ultrastructure   MeSH
• Capsid Proteins antagonists & inhibitors   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Indoles MeSH
• Anti-HIV Agents MeSH
• Recombinant Proteins MeSH
• Capsid Proteins MeSH

Despite successful vaccination programs and effective treatments for some viral infections, humans are still losing the battle with viruses. Persisting human pandemics, emerging and re-emerging viruses, and evolution of drug-resistant strains impose continuous search for new antiviral drugs. A combination of detailed information about the molecular organization of viruses and progress in molecular biology and computer technologies has enabled rational antivirals design. Initial step in establishing efficacy of new antivirals is based on simple methods assessing inhibition of the intended target. We provide here an overview of biochemical and cell-based assays evaluating the activity of inhibitors of clinically important viruses.

• Keywords
• Assay, Assembly, Cell-based, Entry, High-throughput screening, In vitro, Inhibitor, Method, Replication, Virus,
• MeSH
• Antiviral Agents pharmacology   MeSH
• Virus Physiological Phenomena drug effects   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Host-Pathogen Interactions drug effects   MeSH
• Virus Internalization drug effects   MeSH
• Capsid drug effects   metabolism   MeSH
• Humans MeSH
• Drug Evaluation, Preclinical methods   MeSH
• Virus Replication drug effects   MeSH
• High-Throughput Screening Assays methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Antiviral Agents MeSH
• Enzyme Inhibitors MeSH

The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.

• Keywords
• Mason-Pfizer monkey virus, endosomes, envelope, intracellular trafficking, transport, virus-like particles,
• MeSH
• Simian Acquired Immunodeficiency Syndrome virology   MeSH
• Cell Membrane metabolism   virology   MeSH
• Endosomes metabolism   virology   MeSH
• Gene Products, env genetics   metabolism   MeSH
• Mason-Pfizer monkey virus genetics   physiology   MeSH
• Virus Assembly MeSH
• Protein Transport MeSH
• Transport Vesicles metabolism   virology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gene Products, env MeSH

The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins.

• Keywords
• capsid protein (CA), charged assembly helix (CAH), circular dichroism (CD), electron microscopy (EM), murine leukemia virus (MLV), nuclear magnetic resonance (NMR), retrovirus, single alpha-helix (SAH), spacer peptide (SP), virus assembly,
• MeSH
• Mutagenesis MeSH
• Mice MeSH
• Protein Domains MeSH
• Protein Structure, Secondary MeSH
• Capsid Proteins chemistry   genetics   MeSH
• Leukemia Virus, Murine chemistry   genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Capsid Proteins MeSH

UNLABELLED: The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-Å cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly. IMPORTANCE: Retroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses, and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroviruses, such as HIV, Rous sarcoma virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.

• MeSH
• Circular Dichroism MeSH
• Cryoelectron Microscopy MeSH
• Protein Conformation MeSH
• Mason-Pfizer monkey virus physiology   MeSH
• Models, Molecular MeSH
• Nucleocapsid Proteins chemistry   genetics   metabolism   ultrastructure   MeSH
• Virus Assembly * MeSH
• Virus Release MeSH
• Capsid Proteins chemistry   genetics   metabolism   ultrastructure   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nucleocapsid Proteins MeSH
• Capsid Proteins MeSH

BACKGROUND: Formation of a mature core is a crucial event for infectivity of retroviruses such as Mason-Pfizer monkey virus (M-PMV). The process is triggered by proteolytic cleavage of the polyprotein precursor Gag, which releases matrix, capsid (CA), and nucleocapsid proteins. Once released, CA assembles to form a mature core - a hexameric lattice protein shell that protects retroviral genomic RNA. Subtle conformational changes within CA induce the transition from the immature lattice to the mature lattice. Upon release from the precursor, the initially unstructured N-terminus of CA is refolded to form a β-hairpin stabilized by a salt bridge between the N-terminal proline and conserved aspartate. Although the crucial role of the β-hairpin in the mature core assembly has been confirmed, its precise structural function remains poorly understood. RESULTS: Based on a previous NMR analysis of the N-terminal part of M-PMV CA, which suggested the role of additional interactions besides the proline-aspartate salt bridge in stabilization of the β-hairpin, we introduced a series of mutations into the CA sequence. The effect of the mutations on virus assembly and infectivity was analyzed. In addition, the structural consequences of selected mutations were determined by NMR spectroscopy. We identified a network of interactions critical for proper formation of the M-PMV core. This network involves residue R14, located in the N-terminal β-hairpin; residue W52 in the loop connecting helices 2 and 3; and residues Q113, Q115, and Y116 in helix 5. CONCLUSION: Combining functional and structural analyses, we identified a network of supportive interactions that stabilize the β-hairpin in mature M-PMV CA.

• MeSH
• Simian Acquired Immunodeficiency Syndrome genetics   metabolism   MeSH
• Cell Line MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mason-Pfizer monkey virus genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Mutation genetics   MeSH
• Protein Structure, Secondary genetics   MeSH
• Amino Acid Sequence MeSH
• Virus Assembly genetics   MeSH
• Virion genetics   metabolism   MeSH
• Capsid Proteins metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Capsid Proteins MeSH

BACKGROUND: Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. RESULTS: Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. CONCLUSION: In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Apoptosis genetics   MeSH
• Cell Line MeSH
• CD4-Positive T-Lymphocytes metabolism   MeSH
• DNA Fragmentation MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• HIV Infections genetics   metabolism   MeSH
• HIV-1 genetics   metabolism   MeSH
• HIV Protease genetics   metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Promoter Regions, Genetic genetics   MeSH
• bcl-2-Associated X Protein genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• AKIP1 protein, human MeSH Browser
• BAX protein, human MeSH Browser
• HIV Protease MeSH
• Nuclear Proteins MeSH
• Mitochondrial Proteins MeSH
• Tumor Suppressor Protein p53 MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Browser
• bcl-2-Associated X Protein MeSH
• TP53 protein, human MeSH Browser

Heterologous proteins are frequently purified by immobilized metal ion affinity chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e., CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state.

• MeSH
• Chromatography, Affinity methods   MeSH
• Escherichia coli genetics   MeSH
• Gene Expression MeSH
• HIV-1 chemistry   genetics   MeSH
• Metals chemistry   MeSH
• Mason-Pfizer monkey virus chemistry   genetics   MeSH
• Viral Proteins chemistry   genetics   isolation & purification   MeSH
• Zinc analysis   MeSH
• Zinc Fingers * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Metals MeSH
• Viral Proteins MeSH
• Zinc MeSH

Assembly of immature retroviral particles is a complex process involving interactions of several specific domains of the Gag polyprotein localized mainly within capsid protein (CA), spacer peptide (SP), and nucleocapsid protein (NC). In the present work we focus on the contribution of NC to the oligomerization of CA leading to assembly of Mason-Pfizer monkey virus (M-PMV) and HIV-1. Analyzing in vitro assembly of substitution and deletion mutants of DeltaProCANC, we identified a "spacer-like" sequence (NC(15)) at the M-PMV NC N terminus. This NC(15) domain is indispensable for the assembly and cannot be replaced with oligomerization domains of GCN4 or CREB proteins. Although the M-PMV NC(15) occupies a position analogous to that of the HIV-1 spacer peptide, it could not be replaced by the latter one. To induce the assembly, both M-PMV NC(15) and HIV-1 SP1 must be followed by a short peptide that is rich in basic residues. This region either can be specific, i.e., derived from the downstream NC sequence, or can be a nonspecific positively charged peptide. However, it cannot be replaced by heterologous interaction domains either from GCN4 or from CREB. In summary, we report here a novel M-PMV spacer-like domain that is functionally similar to other retroviral spacer peptides and contributes to the assembly of immature-virus-like particles.

• MeSH
• Cell Line MeSH
• DNA Primers genetics   MeSH
• DNA, Viral genetics   MeSH
• Escherichia coli genetics   ultrastructure   virology   MeSH
• HIV-1 genetics   physiology   MeSH
• Humans MeSH
• Mason-Pfizer monkey virus genetics   physiology   ultrastructure   MeSH
• Molecular Sequence Data MeSH
• Protein Multimerization MeSH
• Mutagenesis MeSH
• Nucleocapsid Proteins chemistry   genetics   physiology   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Virus Assembly genetics   physiology   MeSH
• Protein Structure, Tertiary MeSH
• Microscopy, Electron, Transmission MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA Primers MeSH
• DNA, Viral MeSH
• Nucleocapsid Proteins MeSH
• Recombinant Proteins MeSH

Retroviral capsid protein (CA) mediates protein interactions driving the assembly of both immature viral particles and the core of the mature virions. Structurally conserved N-terminal domains of several retroviruses refold after proteolytic cleavage into a beta-hairpin, stabilized by a salt bridge between conserved N-terminal Pro and Asp residues. Based on comparison with other retroviral CA, we identified Asp50 and Asp57 as putative interacting partners for Pro1 in Mason-Pfizer monkey virus (M-PMV) CA. To investigate the importance of CA Pro1 and its interacting Asp in M-PMV core assembly and infectivity, P1A, P1Y, D50A, T54A and D57A mutations were introduced into M-PMV. The P1A and D57A mutations partially blocked Gag processing and the released viral particles exhibited aberrant cores and were non-infectious. These data indicate that the region spanning residues Asp50-Asp57 plays an important role in stabilization of the beta-hairpin and that Asp57 likely forms a salt-bridge with P1 in M-PMV CA.

• MeSH
• Point Mutation * MeSH
• Mason-Pfizer monkey virus genetics   metabolism   MeSH
• Virus Assembly genetics   physiology   MeSH
• Virion genetics   metabolism   MeSH
• Capsid Proteins chemistry   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Capsid Proteins MeSH