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

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

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