We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

• Keywords
• DEAH-box RNA helicase, DHX15, G-patch, gRNA packaging, retrovirus,
• MeSH
• Cell Nucleus metabolism   virology   MeSH
• DEAD-box RNA Helicases metabolism   genetics   MeSH
• Genome, Viral MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mason-Pfizer monkey virus * genetics   metabolism   physiology   MeSH
• Virus Replication genetics   physiology   MeSH
• RNA, Viral * metabolism   genetics   MeSH
• RNA Helicases metabolism   genetics   MeSH
• Virus Assembly * genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DEAD-box RNA Helicases MeSH
• DHX15 protein, human MeSH Browser
• RNA, Viral * MeSH
• RNA Helicases MeSH

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

• Keywords
• HIV-1, RNA packaging, fullerene, inhibition, nucleocapsid,
• MeSH
• Fullerenes metabolism   pharmacology   MeSH
• Genome, Viral drug effects   MeSH
• gag Gene Products, Human Immunodeficiency Virus metabolism   MeSH
• HEK293 Cells MeSH
• HIV-1 drug effects   genetics   metabolism   physiology   MeSH
• Anti-HIV Agents metabolism   pharmacology   MeSH
• Humans MeSH
• Nucleocapsid Proteins metabolism   MeSH
• Reverse Transcription MeSH
• RNA, Viral metabolism   MeSH
• Virus Uncoating drug effects   MeSH
• Protein Binding MeSH
• Virion metabolism   MeSH
• Viral Genome Packaging drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fullerenes MeSH
• gag Gene Products, Human Immunodeficiency Virus MeSH
• Anti-HIV Agents MeSH
• Nucleocapsid Proteins MeSH
• RNA, Viral MeSH

Betulinic acid (BA) is a potent triterpene, which has shown promising potential in cancer and HIV-1 treatment. Here, we report a synthesis and biological evaluation of 17 new compounds, including BODIPY labelled analogues derived from BA. The analogues terminated by amino moiety showed increased cytotoxicity (e.g., BA had on CCRF-CEM IC50 > 50 μM, amine 3 IC50 0.21 and amine 14 IC50 0.29). The cell-cycle arrest was evaluated and did not show general features for all the tested compounds. A fluorescence microscopy study of six derivatives revealed that only 4 and 6 were detected in living cells. These compounds were colocalized with the endoplasmic reticulum and mitochondria, indicating possible targets in these organelles. The study of anti-HIV-1 activity showed that 8, 10, 16, 17 and 18 have had IC50i > 10 μM. Only completely processed p24 CA was identified in the viruses formed in the presence of compounds 4 and 12. In the cases of 2, 8, 9, 10, 16, 17 and 18, we identified not fully processed p24 CA and p25 CA-SP1 protein. This observation suggests a similar mechanism of inhibition as described for bevirimat.

• Keywords
• BODIPY, betulinic acid, bevirimat, cancer, cell-cycle, cytotoxicity, fluorescent microscopy, maturation inhibitor,
• Publication type
• Journal Article MeSH

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.

• Keywords
• HIV-1, IP6, assembly, capsid, immature, mature, polyanion,
• MeSH
• gag Gene Products, Human Immunodeficiency Virus chemistry   genetics   metabolism   MeSH
• HIV-1 chemistry   genetics   MeSH
• Mutation, Missense MeSH
• Polyelectrolytes MeSH
• Polymers chemistry   MeSH
• Virus Assembly * MeSH
• Amino Acid Substitution MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• gag Gene Products, Human Immunodeficiency Virus MeSH
• polyanions MeSH Browser
• Polyelectrolytes MeSH
• Polymers MeSH

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

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.

• Keywords
• Cysteine mutagenesis, M-PMV capsid, M-PMV infectivity, Retrovirus assembly, Virus core stability,
• MeSH
• Cell Line MeSH
• Cysteine genetics   MeSH
• Genetic Vectors MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mason-Pfizer monkey virus genetics   physiology   MeSH
• Mutation MeSH
• Proviruses genetics   MeSH
• Virus Assembly * MeSH
• Virion physiology   MeSH
• Capsid Proteins chemistry   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cysteine MeSH
• Capsid Proteins MeSH

In addition to specific RNA-binding zinc finger domains, the retroviral Gag polyprotein contains clusters of basic amino acid residues that are thought to support Gag-viral genomic RNA (gRNA) interactions. One of these clusters is the basic K16NK18EK20 region, located upstream of the first zinc finger of the Mason-Pfizer monkey virus (M-PMV) nucleocapsid (NC) protein. To investigate the role of this basic region in the M-PMV life cycle, we used a combination of in vivo and in vitro methods to study a series of mutants in which the overall charge of this region was more positive (RNRER), more negative (AEAEA), or neutral (AAAAA). The mutations markedly affected gRNA incorporation and the onset of reverse transcription. The introduction of a more negative charge (AEAEA) significantly reduced the incorporation of M-PMV gRNA into nascent particles. Moreover, the assembly of immature particles of the AEAEA Gag mutant was relocated from the perinuclear region to the plasma membrane. In contrast, an enhancement of the basicity of this region of M-PMV NC (RNRER) caused a substantially more efficient incorporation of gRNA, subsequently resulting in an increase in M-PMV RNRER infectivity. Nevertheless, despite the larger amount of gRNA packaged by the RNRER mutant, the onset of reverse transcription was delayed in comparison to that of the wild type. Our data clearly show the requirement for certain positively charged amino acid residues upstream of the first zinc finger for proper gRNA incorporation, assembly of immature particles, and proceeding of reverse transcription.IMPORTANCE We identified a short sequence within the Gag polyprotein that, together with the zinc finger domains and the previously identified RKK motif, contributes to the packaging of genomic RNA (gRNA) of Mason-Pfizer monkey virus (M-PMV). Importantly, in addition to gRNA incorporation, this basic region (KNKEK) at the N terminus of the nucleocapsid protein is crucial for the onset of reverse transcription. Mutations that change the positive charge of the region to a negative one significantly reduced specific gRNA packaging. The assembly of immature particles of this mutant was reoriented from the perinuclear region to the plasma membrane. On the contrary, an enhancement of the basic character of this region increased both the efficiency of gRNA packaging and the infectivity of the virus. However, the onset of reverse transcription was delayed even in this mutant. In summary, the basic region in M-PMV Gag plays a key role in the packaging of genomic RNA and, consequently, in assembly and reverse transcription.

• Keywords
• M-PMV, RNA packaging, assembly, basic residues, human immunodeficiency virus, infectivity, nucleocapsid, retroviruses, reverse transcription,
• MeSH
• Cell Line MeSH
• Gene Products, gag genetics   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mason-Pfizer monkey virus genetics   physiology   MeSH
• Mutation genetics   MeSH
• Nucleocapsid Proteins genetics   MeSH
• Reverse Transcription genetics   MeSH
• RNA, Viral genetics   MeSH
• Amino Acid Sequence genetics   MeSH
• Virus Assembly genetics   MeSH
• Zinc Fingers genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gene Products, gag MeSH
• Nucleocapsid Proteins MeSH
• RNA, Viral MeSH

The cellular role of breast carcinoma-associated protein (BCA3), also known as A-kinase-interacting protein 1 (AKIP-1), is not fully understood. Recently, we reported that full-length, but not C-terminally truncated, BCA3 is incorporated into virions of Mason-Pfizer monkey virus, and that BCA3 enhances HIV-1 protease-induced apoptosis. In the present study, we report that BCA3 is associated with purified and subtilisin-treated HIV particles. Using a combination of immune-based methods and confocal microscopy, we show that the C-terminus of BCA3 is required for packaging into HIV-1 particles. However, we were unable to identify an HIV-1 binding domain for BCA3, and we did not observe any effect of incorporated BCA3 on HIV-1 infectivity. Interestingly, the BCA3 C-terminus was previously identified as a binding site for the catalytic subunit of protein kinase A (PKAc), a cellular protein that is specifically packaged into HIV-1 particles. Based on our analysis of PKAc⁻BCA3 interactions, we suggest that BCA3 incorporation into HIV-1 particles is mediated by its ability to interact with PKAc.

• Keywords
• AKIP-1, BCA3, HIV-1, M-PMV, PKAc, virus incorporation,
• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• HIV-1 metabolism   physiology   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• Virus Replication genetics   MeSH
• Virus Assembly MeSH
• Protein Binding MeSH
• Virion 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
• Nuclear Proteins MeSH
• Cyclic AMP-Dependent Protein Kinases MeSH

UNLABELLED: The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCE: Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV.

• MeSH
• Cell Line MeSH
• Cryoelectron Microscopy MeSH
• Genome, Viral * MeSH
• Gene Products, gag MeSH
• Humans MeSH
• Mason-Pfizer monkey virus physiology   ultrastructure   MeSH
• Mutation MeSH
• Recombinant Proteins MeSH
• RNA, Viral metabolism   MeSH
• Amino Acid Sequence MeSH
• Virus Assembly * genetics   MeSH
• Amino Acid Substitution MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Capsid Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gene Products, gag MeSH
• Recombinant Proteins MeSH
• RNA, Viral MeSH
• 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