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Platinum_GP Retroviral Packaging Cell Line, Pantropic

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[#RV-103] Platinum_GP Retroviral Packaging Cell Line, Pantropic


RV-103 | Platinum_GP Retroviral Packaging Cell Line, Pantropic, 1 vial
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(1) Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77 Expressed in Prokaryotic and Eukaryotic Cells.[TOP]

Pubmed ID :29912170
Publication Date : //
The mouse mammary tumor virus (MMTV) Pr77 polypeptide is an essential retroviral structural protein without which infectious viral particles cannot be formed. This process requires specific recognition and packaging of dimerized genomic RNA (gRNA) by Gag during virus assembly. Most of the previous work on retroviral assembly has used either the nucleocapsid portion of Gag, or other truncated Gag derivatives—not the natural substrate for virus assembly. In order to understand the molecular mechanism of MMTV gRNA packaging process, we expressed and purified full-length recombinant Pr77-His₆-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77-His₆-tag protein retained the ability to assemble virus-like particles (VLPs) in vitro with morphologically similar immature intracellular particles. The recombinant proteins (with and without His₆-tag) could both be expressed in prokaryotic and eukaryotic cells and had the ability to form VLPs in vivo. Most importantly, the recombinant Pr77-His₆-tag fusion proteins capable of making VLPs in eukaryotic cells were competent for packaging sub-genomic MMTV RNAs. The successful expression and purification of a biologically active, full-length MMTV Pr77 should lay down the foundation towards performing RNA⁻protein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes.

Authors : Chameettachal Akhil, Pillai Vineeta Narayana, Ali Lizna Mohamed, Pitchai Fathima Nuzra Nagoor, Ardah Mustafa Taleb, Mustafa Farah, Marquet Roland, Rizvi Tahir Aziz,

(2) HIV-1 adaptation studies reveal a novel Env-mediated homeostasis mechanism for evading lethal hypermutation by APOBEC3G.[TOP]

Pubmed ID :29677220
Publication Date : //
HIV-1 replication normally requires Vif-mediated neutralization of APOBEC3 antiviral enzymes. Viruses lacking Vif succumb to deamination-dependent and -independent restriction processes. Here, HIV-1 adaptation studies were leveraged to ask whether viruses with an irreparable vif deletion could develop resistance to restrictive levels of APOBEC3G. Several resistant viruses were recovered with multiple amino acid substitutions in Env, and these changes alone are sufficient to protect Vif-null viruses from APOBEC3G-dependent restriction in T cell lines. Env adaptations cause decreased fusogenicity, which results in higher levels of Gag-Pol packaging. Increased concentrations of packaged Pol in turn enable faster virus DNA replication and protection from APOBEC3G-mediated hypermutation of viral replication intermediates. Taken together, these studies reveal that a moderate decrease in one essential viral activity, namely Env-mediated fusogenicity, enables the virus to change other activities, here, Gag-Pol packaging during particle production, and thereby escape restriction by the antiviral factor APOBEC3G. We propose a new paradigm in which alterations in viral homeostasis, through compensatory small changes, constitute a general mechanism used by HIV-1 and other viral pathogens to escape innate antiviral responses and other inhibitions including antiviral drugs.

Authors : Ikeda Terumasa, Symeonides Menelaos, Albin John S, Li Ming, Thali Markus, Harris Reuben S,

(3) Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.[TOP]

Pubmed ID :29664940
Publication Date : //
During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.

Authors : Barajas Brook C, Tanaka Motoko, Robinson Bridget A, Phuong Daryl J, Chutiraka Kasana, Reed Jonathan C, Lingappa Jaisri R,

(4) Retroviral RNA Dimerization: From Structure to Functions.[TOP]

Pubmed ID :29623074
Publication Date : //
The genome of the retroviruses is a dimer composed by two homologous copies of genomic RNA (gRNA) molecules of positive polarity. The dimerization process allows two gRNA molecules to be non-covalently linked together through intermolecular base-pairing. This step is critical for the viral life cycle and is highly conserved among retroviruses with the exception of spumaretroviruses. Furthermore, packaging of two gRNA copies into viral particles presents an important evolutionary advantage for immune system evasion and drug resistance. Recent studies reported RNA switches models regulating not only gRNA dimerization, but also translation and packaging, and a spatio-temporal characterization of viral gRNA dimerization within cells are now at hand. This review summarizes our current understanding on the structural features of the dimerization signals for a variety of retroviruses (HIVs, MLV, RSV, BLV, MMTV, MPMV…), the mechanisms of RNA dimer formation and functional implications in the retroviral cycle.

Authors : Dubois Noé, Marquet Roland, Paillart Jean-Christophe, Bernacchi Serena,

(5) Deaminase-Dead Mouse APOBEC3 Is an Retroviral Restriction Factor.[TOP]

Pubmed ID :29593034
Publication Date : //
The apolipoprotein B editing complex 3 (APOBEC3) proteins are potent retroviral restriction factors that are under strong positive selection, both in terms of gene copy number and sequence diversity. A common feature of all the members of the APOBEC3 family is the presence of one or two cytidine deamination domains, essential for cytidine deamination of retroviral reverse transcripts as well as packaging into virions. Several studies have indicated that human and mouse APOBEC3 proteins restrict retrovirus infection via cytidine deaminase (CD)-dependent and -independent means. To understand the relative contribution of CD-independent restriction , we created strains of transgenic mice on an APOBEC3 knockout background that express a deaminase-dead mouse APOBEC3 due to point mutations in both CD domains (E73Q/E253Q). Here, we show that the CD-dead APOBEC3 can restrict murine retroviruses Moreover, unlike the wild-type protein, the mutant APOBEC3 is not packaged into virions but acts only as a cell-intrinsic restriction factor that blocks reverse transcription by incoming viruses. Finally, we show that wild-type and CD-dead mouse APOBEC3 can bind to murine leukemia virus (MLV) reverse transcriptase. Our findings suggest that the mouse APOBEC3 cytidine deaminase activity is not required for retrovirus restriction. APOBEC3 proteins are important host cellular restriction factors essential for restricting retrovirus infection by causing mutations in the virus genome and by blocking reverse transcription. While both methods of restriction function , little is known about their role during infection. By developing transgenic mice with mutations in the cytidine deamination domains needed for enzymatic activity and interaction with viral RNA, we show that APOBEC3 proteins can still restrict infection by interacting with reverse transcriptase and blocking its activity. These studies demonstrate that APOBEC3 proteins have evolved multiple means for blocking retrovirus infection and that all of these means function .

Authors : Stavrou Spyridon, Zhao Wenming, Blouch Kristin, Ross Susan R,

(6) Retroviral Gag protein-RNA interactions: Implications for specific genomic RNA packaging and virion assembly.[TOP]

Pubmed ID :29580971
Publication Date : //
Retroviral Gag proteins are responsible for coordinating many aspects of virion assembly. Gag possesses two distinct nucleic acid binding domains, matrix (MA) and nucleocapsid (NC). One of the critical functions of Gag is to specifically recognize, bind, and package the retroviral genomic RNA (gRNA) into assembling virions. Gag interactions with cellular RNAs have also been shown to regulate aspects of assembly. Recent results have shed light on the role of MA and NC domain interactions with nucleic acids, and how they jointly function to ensure packaging of the retroviral gRNA. Here, we will review the literature regarding RNA interactions with NC, MA, as well as overall mechanisms employed by Gag to interact with RNA. The discussion focuses on human immunodeficiency virus type-1, but other retroviruses will also be discussed. A model is presented combining all of the available data summarizing the various factors and layers of selection Gag employs to ensure specific gRNA packaging and correct virion assembly.

Authors : Olson Erik D, Musier-Forsyth Karin,

(7) Mutations in the Basic Region of the Mason-Pfizer Monkey Virus Nucleocapsid Protein Affect Reverse Transcription, Genomic RNA Packaging, and the Virus Assembly Site.[TOP]

Pubmed ID :29491167
Publication Date : //
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 KNKEK 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 and 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. 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.

Authors : Dostálková Alžběta, Kaufman Filip, Křížová Ivana, Kultová Anna, Strohalmová Karolína, Hadravová Romana, Ruml Tomáš, Rumlová Michaela,

(8) [Preparation of recombinant retrovirus pRevTRE-E77.43 and its protective effect in a mouse model of infection].[TOP]

Pubmed ID :29469452
Publication Date : //
To explore the biological functions of E77.43, a gene segment of , in treating infection.

Authors : Fan-Sheng Zeng, Sai-Qun Luo, De-Hui Xiong, Yuan-Jing Yu, Ying-Jun Qian, Zhi-Qiang Qin,

(9) Identification of a Constitutively Active Mutant Mouse IRAK2 by Retroviral Expression Screening.[TOP]

Pubmed ID :29468521
Publication Date : //
To identify the importance of IRAK2 kinase activity in TLR-mediated signaling pathways, we constructed a retroviral vector harboring either a mouse IRAK2 gene (IRAK2-WT) or with its mutant with loss of function of its ATP-binding site (IRAK2-KD). Further, we comparatively analyzed for the gain of function and modulations in TLR-mediated signaling pathways in IRAK2 knockout (IRAK2-KO) macrophages upon introduction of the IRAK2-WT retroviral constructs. The pBS/IRAK2-KD with the ATP-binding site mutation in IRAK2 was obtained by using site-specific mutagenesis. The recombinants were identified with appropriate double digestion and sequence analysis. The recombinant vector constructs were transfected by lipofection into phoenix packaging cells. The viral vectors (10 cfu/mL) with the construct were allowed to infect IRAK2-KO macrophages. The results showed that IRAK2-WT gene overexpressed in the IRAK2-KO macrophages exhibited a modified IRAK2 expression upon LPS induction. However, the modification was absent with IRAK2-KD construct on LPS stimulation; instead, the IRAK2 protein stability was reduced considerably. The results further show that the LPS-induced effect on the stability of IRAK2 is dependent of IRAK4 stimulation.

Authors : Liu Yanmei, Yin Weilan, Xu Lingqing, Zhang Helin, Liu Qian, Yin Weiguo,

(10) Global synonymous mutagenesis identifies cis-acting RNA elements that regulate HIV-1 splicing and replication.[TOP]

Pubmed ID :29377940
Publication Date : //
The ~9.5 kilobase HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication, including transcription, splicing, nuclear export, translation, packaging and reverse transcription. Nonetheless, chemical probing and other approaches suggest that the HIV-1 genome may contain many more RNA secondary structures of unknown importance and function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we undertook a global silent mutagenesis experiment. Sixteen mutant proviruses containing clusters of ~50 to ~200 synonymous mutations covering nearly the entire HIV-1 protein coding sequence were designed and synthesized. Analyses of these mutant viruses resulted in their division into three phenotypic groups. Group 1 mutants exhibited near wild-type replication, Group 2 mutants exhibited replication defects accompanied by perturbed RNA splicing, and Group 3 mutants had replication defects in the absence of obvious splicing perturbation. The three phenotypes were caused by mutations that exhibited a clear regional bias in their distribution along the viral genome, and those that caused replication defects all caused reductions in the level of unspliced RNA. We characterized in detail the underlying defects for Group 2 mutants. Second-site revertants that enabled viral replication could be derived for Group 2 mutants, and generally contained point mutations that reduced the utilization of proximal splice sites. Mapping of the changes responsible for splicing perturbations in Group 2 viruses revealed the presence of several RNA sequences that apparently suppressed the use of cryptic or canonical splice sites. Some sequences that affected splicing were diffusely distributed, while others could be mapped to discrete elements, proximal or distal to the affected splice site(s). Overall, our data indicate complex negative regulation of HIV-1 splicing by RNA elements in various regions of the HIV-1 genome that enable balanced splicing and viral replication.

Authors : Takata Matthew A, Soll Steven J, Emery Ann, Blanco-Melo Daniel, Swanstrom Ronald, Bieniasz Paul D,