Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles.IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.

• Klíčová slova
• HIV-1, IP6, assembly, capsid, immature, mature, polyanion,
• MeSH
• genové produkty gag - virus lidské imunodeficience chemie   genetika   metabolismus   MeSH
• HIV-1 chemie   genetika   MeSH
• missense mutace MeSH
• polyelektrolyty MeSH
• polymery chemie   MeSH
• sestavení viru * MeSH
• substituce aminokyselin MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty gag - virus lidské imunodeficience MeSH
• polyanions MeSH Prohlížeč
• polyelektrolyty MeSH
• polymery MeSH

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.

• Klíčová slova
• CAH *, IP6 *, M-PMV *, MLV *, RSV *, SP domain *, assembly *, hexamer *, immature *, polyanion *,
• MeSH
• Alpharetrovirus fyziologie   MeSH
• Betaretrovirus fyziologie   MeSH
• buněčná membrána chemie   metabolismus   MeSH
• Gammaretrovirus fyziologie   MeSH
• genové produkty gag chemie   metabolismus   MeSH
• interakce hostitele a patogenu * MeSH
• kultivované buňky MeSH
• polyelektrolyty chemie   metabolismus   MeSH
• Retroviridae fyziologie   ultrastruktura   MeSH
• sestavení viru * MeSH
• virion MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty gag MeSH
• polyanions MeSH Prohlížeč
• polyelektrolyty MeSH

Nucleocytoplasmic large DNA viruses (NCLDVs) blur the line between viruses and cells. Melbournevirus (MelV, family Marseilleviridae) belongs to a new family of NCLDVs. Here we present an electron cryo-microscopy structure of the MelV particle, with the large triangulation number T = 309 constructed by 3080 pseudo-hexagonal capsomers. The most distinct feature of the particle is a large and dense body (LDB) consistently found inside all particles. Electron cryo-tomography of 147 particles shows that the LDB is preferentially located in proximity to the probable lipid bilayer. The LDB is 30 nm in size and its density matches that of a genome/protein complex. The observed LDB reinforces the structural complexity of MelV, setting it apart from other NCLDVs.

• Klíčová slova
• Amoeba, Capsid, Cryo-electron microscopy, Marseilleviridae, Melbournevirus, NCLDV, Protein complex, Structure, Tomography, Virus,
• MeSH
• DNA viry genetika   fyziologie   ultrastruktura   MeSH
• elektronová kryomikroskopie MeSH
• genom virový MeSH
• kapsida metabolismus   ultrastruktura   MeSH
• sestavení viru MeSH
• virion genetika   fyziologie   ultrastruktura   MeSH
• virové proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• virové proteiny MeSH

Colloidal gold nanoparticles represent technological building blocks which are easy to fabricate while keeping full control of their shape and dimensions. Here, we report on a simple two-step maskless process to assemble gold nanoparticles from a water colloidal solution at specific sites of a silicon surface. First, the silicon substrate covered by native oxide is exposed to a charged particle beam (ions or electrons) and then immersed in a HF-modified solution of colloidal nanoparticles. The irradiation of the native oxide layer by a low-fluence charged particle beam causes changes in the type of surface-terminating groups, while the large fluences induce even more profound modification of surface composition. Hence, by a proper selection of the initial substrate termination, solution pH, and beam fluence, either positive or negative deposition of the colloidal nanoparticles can be achieved.

• MeSH
• částice - urychlovače MeSH
• koloidy chemie   účinky záření   MeSH
• kovové nanočástice chemie   účinky záření   MeSH
• křemík chemie   účinky záření   MeSH
• krystalizace metody   MeSH
• povrchové vlastnosti účinky záření   MeSH
• testování materiálů MeSH
• těžké ionty * MeSH
• velikost částic MeSH
• zlato chemie   účinky záření   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• koloidy MeSH
• křemík MeSH
• zlato MeSH

Retrovirus assembly is driven mostly by Gag polyprotein oligomerization, which is mediated by inter and intra protein-protein interactions among its capsid (CA) domains. Mason-Pfizer monkey virus (M-PMV) CA contains three cysteines (C82, C193 and C213), where the latter two are highly conserved among most retroviruses. To determine the importance of these cysteines, we introduced mutations of these residues in both bacterial and proviral vectors and studied their impact on the M-PMV life cycle. These studies revealed that the presence of both conserved cysteines of M-PMV CA is necessary for both proper assembly and virus infectivity. Our findings suggest a crucial role of these cysteines in the formation of infectious mature particles.

• Klíčová slova
• Cysteine mutagenesis, M-PMV capsid, M-PMV infectivity, Retrovirus assembly, Virus core stability,
• MeSH
• buněčné linie MeSH
• cystein genetika   MeSH
• genetické vektory MeSH
• HEK293 buňky MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus genetika   fyziologie   MeSH
• mutace MeSH
• proviry genetika   MeSH
• sestavení viru * MeSH
• virion fyziologie   MeSH
• virové plášťové proteiny chemie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• cystein MeSH
• virové plášťové proteiny MeSH

Immature retroviral particles are assembled by self-association of the structural polyprotein precursor Gag. During maturation the Gag polyprotein is proteolytically cleaved, yielding mature structural proteins, matrix (MA), capsid (CA), and nucleocapsid (NC), that reassemble into a mature viral particle. Proteolytic cleavage causes the N terminus of CA to fold back to form a β-hairpin, anchored by an internal salt bridge between the N-terminal proline and the inner aspartate. Using an in vitro assembly system of capsid-nucleocapsid protein (CANC), we studied the formation of virus-like particles (VLP) of a gammaretrovirus, the xenotropic murine leukemia virus (MLV)-related virus (XMRV). We show here that, unlike other retroviruses, XMRV CA and CANC do not assemble tubular particles characteristic of mature assembly. The prevention of β-hairpin formation by the deletion of either the N-terminal proline or 10 initial amino acids enabled the assembly of ΔProCANC or Δ10CANC into immature-like spherical particles. Detailed three-dimensional (3D) structural analysis of these particles revealed that below a disordered N-terminal CA layer, the C terminus of CA assembles a typical immature lattice, which is linked by rod-like densities with the RNP.

• MeSH
• DNA primery MeSH
• elektronová kryomikroskopie MeSH
• Escherichia coli ultrastruktura   virologie   MeSH
• Fourierova analýza MeSH
• molekulární sekvence - údaje MeSH
• polymerázová řetězová reakce MeSH
• proteolýza MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• sestavení viru * MeSH
• transmisní elektronová mikroskopie MeSH
• virion fyziologie   MeSH
• virové proteiny chemie   metabolismus   MeSH
• virus myší leukemie fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA primery MeSH
• virové proteiny 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
• bodová mutace * MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   MeSH
• sestavení viru genetika   fyziologie   MeSH
• virion genetika   metabolismus   MeSH
• virové plášťové proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• virové plášťové proteiny MeSH

Due to the high number of drug-resistant HIV-1 mutants generated by highly active antiretroviral therapy (HAART), there is continuing demand for new types of inhibitors. Both the assembly of the Gag polyprotein into immature and mature HIV-1 particles are attractive candidates for the blocking of the retroviral life cycle. Currently, no therapeutically-used assembly inhibitor is available. One possible explanation is the lack of a reliable and simple assembly inhibitor screening method. To identify compounds potentially inhibiting the formation of both types of HIV-1 particles, we developed a new fluorescent high-throughput screening assay. This assay is based on the quantification of the assembly efficiency in vitro in a 96-well plate format. The key components of the assay are HIV-1 Gag-derived proteins and a dual-labelled oligonucleotide, which emits fluorescence only when the assembly of retroviral particles is inhibited. The method was validated using three (CAI, BM2, PF74) reported assembly inhibitors.

• Klíčová slova
• Assay, Assembly, CA, Capsid, HIV, High-throughput screening, Inhibitor, Retrovirus,
• MeSH
• genové produkty gag metabolismus   MeSH
• HIV infekce farmakoterapie   virologie   MeSH
• HIV-1 účinky léků   fyziologie   MeSH
• látky proti HIV farmakologie   MeSH
• lidé MeSH
• preklinické hodnocení léčiv metody   MeSH
• rychlé screeningové testy metody   MeSH
• sestavení viru účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty gag MeSH
• látky proti HIV 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
• cirkulární dichroismus MeSH
• elektronová kryomikroskopie MeSH
• konformace proteinů MeSH
• Masonův-Pfizerův opičí virus fyziologie   MeSH
• molekulární modely MeSH
• nukleokapsida - proteiny chemie   genetika   metabolismus   ultrastruktura   MeSH
• sestavení viru * MeSH
• uvolnění viru z buňky MeSH
• virové plášťové proteiny chemie   genetika   metabolismus   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nukleokapsida - proteiny MeSH
• virové plášťové proteiny MeSH

This review article is addressed to a broad community of polymer scientists. We outline and analyse the fundamentals of the dissipative particle dynamics (DPD) simulation method from the point of view of polymer physics and review the articles on polymer systems published in approximately the last two decades, focusing on their impact on macromolecular science. Special attention is devoted to polymer and polyelectrolyte self- and co-assembly and self-organisation and to the problems connected with the implementation of explicit electrostatics in DPD numerical machinery. Critical analysis of the results of a number of successful DPD studies of complex polymer systems published recently documents the importance and suitability of this coarse-grained method for studying polymer systems.

• Klíčová slova
• assembly, dissipative particle dynamics, polyelectrolyte, polymer,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH