Moloney Murine Leukemia Virus p12 Is Required for Histone Loading onto Retroviral DNAs.

During retrovirus infection, a histone-free DNA copy of the viral RNA genome is synthesized and rapidly loaded with nucleosomes de novo upon nuclear entry. The potential role of viral accessory proteins in histone loading onto retroviral DNAs has not been extensively investigated. The p12 protein of Moloney murine leukemia virus (MMLV) is a virion protein that is critical for tethering the incoming viral DNA to host chromatin in the early stages of infection. Infection by virions containing a mutant p12 (PM14) defective in chromatin tethering results in the formation of viral DNAs that do not accumulate in the nucleus. In this report, we show that viral DNAs of these mutants are not loaded with histones. Moreover, the DNA genomes delivered by mutant p12 show prolonged association with viral structural proteins nucleocapsid (NC) and capsid (CA). The histone-poor viral DNA genomes do not become associated with the host RNA polymerase II machinery. These findings provide insights into fundamental aspects of retroviral biology, indicating that tethering to host chromatin by p12 and retention in the nucleus are required to allow loading of histones onto the viral DNA. IMPORTANCE Incoming retroviral DNAs are rapidly loaded with nucleosomal histones upon entry into the nucleus and before integration into the host genome. The entry of murine leukemia virus DNA into the nucleus occurs only upon dissolution of the nuclear membrane in mitosis, and retention in the nucleus requires the action of a viral protein, p12, which tethers the DNA to host chromatin. Data presented here show that the tethering activity of p12 is required for the loading of histones onto the viral DNA. p12 mutants lacking tethering activity fail to acquire histones, retain capsid and nucleocapsid proteins, and are poorly transcribed. The work defines a new requirement for a viral protein to allow chromatinization of viral DNA.

Repression of the Chromatin-Tethering Domain of Murine Leukemia Virus p12.

Murine leukemia virus (MLV) p12, encoded within Gag, binds the viral preintegration complex (PIC) to the mitotic chromatin. This acts to anchor the viral PIC in the nucleus as the nuclear envelope re-forms postmitosis. Mutations within the p12 C terminus (p12 PM13 to PM15) block early stages in viral replication. Within the p12 PM13 region (p12 60PSPMA65), our studies indicated that chromatin tethering was not detected when the wild-type (WT) p12 protein (M63) was expressed as a green fluorescent protein (GFP) fusion; however, constructs bearing p12-I63 were tethered. N-terminal truncations of the activated p12-I63-GFP indicated that tethering increased further upon deletion of p12 25DLLTEDPPPY34, which includes the late domain required for viral assembly. The p12 PM15 sequence (p12 70RREPP74) is critical for wild-type viral viability; however, virions bearing the PM15 mutation (p12 70AAAAA74) with a second M63I mutant were viable, with a titer 18-fold lower than that of the WT. The p12 M63I mutation amplified chromatin tethering and compensated for the loss of chromatin binding of p12 PM15. Rescue of the p12-M63-PM15 nonviable mutant with prototype foamy virus (PFV) and Kaposi's sarcoma herpesvirus (KSHV) tethering sequences confirmed the function of p1270-74 in chromatin binding. Minimally, full-strength tethering was seen with only p12 61SPIASRLRGRR71 fused to GFP. These results indicate that the p12 C terminus alone is sufficient for chromatin binding and that the presence of the p12 25DLLTEDPPPY34 motif in the N terminus suppresses the ability to tether. IMPORTANCE: This study defines a regulatory mechanism controlling the differential roles of the MLV p12 protein in early and late replication. During viral assembly and egress, the late domain within the p12 N terminus functions to bind host vesicle release factors. During viral entry, the C terminus of p12 is required for tethering to host mitotic chromosomes. Our studies indicate that the p12 domain including the PPPY late sequence temporally represses the p12 chromatin tethering motif. Maximal p12 tethering was identified with only an 11-amino-acid minimal chromatin tethering motif encoded at p1261-71 Within this region, the p12-M63I substitution switches p12 into a tethering-competent state, partially rescuing the p12-PM15 tethering mutant. A model for how this conformational change regulates early versus late functions is presented.

Phosphorylation Requirement of Murine Leukemia Virus p12.

The p12 protein of murine leukemia virus (MLV) Gag is associated with the preintegration complex (PIC), and mutants of p12 (PM14) exhibit defects in nuclear entry/retention. Mutants of the phosphorylated serine 61 also have been reported to have defects in the early life cycle. Here we show that a phosphorylated peptide motif derived from human papillomavirus 8 (HPV-8), the E2 hinge region including residues 240 to 255, can functionally replace the main phosphorylated motif of MLV p12 and can rescue the viral titer of a strain with the lethal p12-PM14 mutation. Complementation with the HPV-8 E2 hinge motif generated multiple second-site mutations in live viral passage assays. Additional p12 phosphorylation sites were detected, including the late domain of p12 (PPPY) as well as the late domain/protease cleavage site of matrix (LYPAL), by mass spectrometry and Western blotting. Chromatin binding of p12-green fluorescent protein (GFP) fusion protein and functional complementation of p12-PM14 occurred in a manner independent of the E2 hinge region phosphorylation. Replacement of serine 61 by alanine within the minimal tethering domain (61SPMASRLRGRR71) maintained tethering, but in the context of the full-length p12, mutants with substitutions in S61 remained untethered and lost infectivity, indicating phosphorylation of p12 serine 61 functions to temporally regulate early and late p12 functions. IMPORTANCE: The p12 protein, required for both early and late viral functions, is the predominant phosphorylated viral protein of Moloney MLV and is required for virus viability. Our studies indicate that the N terminus of p12 represses the early function of the chromatin binding domain and that deletion of the N terminus activates chromatin binding in the wild-type Moloney MLV p12 protein. Mass spectrometry and mutagenesis studies suggest that phosphorylation of both the repression domain and the chromatin binding domain acts to temporally regulate this process at the appropriate stages during infection.

Innate immunity protein Tag7 (PGRP-S) activates lymphocytes capable of Fasl-Fas-dependent contact killing of virus-infected cells.

The innate immunity protein Tag7 (PGRP-S, PGLYRP1) is involved in antimicrobial and antitumor defense. As shown in our previous studies, Tag7 specifically interacts with the major heat shock protein Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against tumor cells. A stable complex of Tag7 with the calcium-binding protein Mts1 (S100A4) stimulates migration of lymphocytes. Moreover, Tag7 can activate cytotoxic lymphocytes that recognize and kill HLA-negative tumor cells. Here, we have shown that Tag 7 treatment of human peripheral blood mononuclear cells (PBMCs) results in activation of different cytotoxic lymphocyte populations-natural killer (NK) cells and CD8+ NKG2D+ T lymphocytes-that kill Moloney murine leukemia virus (MMLV) infected SC-1 cells using different mechanisms of cell death induction. This mechanism in NK cells is based on the release of granzymes, which activate apoptosis in target cells, while CD8+ NKG2D+ T lymphocytes recognize the noncanonical MicA antigen on the surface of virus-containing cells and kill them via the FasL-Fas interaction, triggering the apoptotic or necroptotic cell death pathway. Preliminary incubation of PBMCs with virus-infected cells and following incubation with Tag7 results in activation of lymphocytes with a different phenotype. These lymphocytes change the spectrum of target cells and the mechanism of cell death induction, and their interaction with target cells is not species-specific. © 2017 IUBMB Life, 69(12):971-977, 2017.

Targeting of Moloney murine leukemia virus gag precursor to the site of virus budding.

Retrovirus Moloney murine leukemia virus (M-MuLV) matures by budding at the cell surface. Central to the budding process is the myristoylated viral core protein precursor Gag which, even in the absence of all other viral components, is capable of associating with the cytoplasmic leaflet of the plasma membrane and assembling into extracellular virus-like particles. In this paper we have used heterologous, Semliki Forest virus-driven, expression of M-MuLV Gag to study the mechanism by which this protein is targeted to the cell surface. In pulse-chase experiments, BFA, monensin, and 20 degrees C block did not affect incorporation of Gag into extracellular particles thereby indicating that the secretory pathway is not involved in targeting of Gag to the cell surface. Subcellular fractionation studies demonstrated that newly synthesized Gag became rapidly and efficiently associated with membranes which had a density similar to that of plasma membrane-derived vesicles. Protease-protection studies confirmed that the Gag-containing membranes were of plasma membrane origin, since in crude cell homogenates, the bulk of newly synthesized Gag was protease-resistant as expected of a protein that binds to the cytoplasmic leaflet of the plasma membrane. Taken together these data indicate that targeting of M-MuLV Gag to the cell surface proceeds via direct insertion of the protein to the cytoplasmic side of the plasma membrane. Furthermore, since the membrane insertion reaction is highly efficient and specific, this suggests that the reaction is dependent on as-yet-unidentified cellular factors.

Proviral DNA of Moloney leukemia virus: purification and visualization.

Closed-circular proviral DNA of Moloney leukemia virus has been purified from a 10(7) excess of cellular and mitochondrial DNA. The DNA can be visualized in the electron microscope and has the contour length of a molecule with a moleculecular weight of about 5.5 + 10(6). Electron microscopic observation of a hybrid between viral RNA and this circular DNA confirms the viral origin of this molecule.

Murine leukemia virus: restriction in fused permissive and nonpermissive cells.

Cultures of human cells nonpermissive for mouse leukemia virus replication could not be induced to support virus replication by homologous fusion in the presence of Moloney leukemia virus. Human cells were also fused with permissive mouse cells, and the fate of the virus in heterokaryons was determined by a simultaneous autoradiography and fluorescent antibody technique. Heterokaryons containing the full chromosome complement of both cells were likewise nonpermissive for virus synthesis, but hybrids of human and mouse cells, which lacked up to half of the human chromosome complement, were permissive for virus synthesis. The results suggest that human cell genes can direct a repressive control over mouse leukemia virus replication.

Production of pseudotyped retrovirus and the generation of proviral transgenic zebrafish.

This chapter describes a method for generation of the high-titer pseudotyped Moloney murine leukemia virus (MLV) that efficiently infects zebrafish embryos (i.e., more than 25 retroviral copies per cell). Injection techniques are also described for production of the retrovirus-infected mosaic "founder" fish. We describe a quantitative PCR (qPCR)-based assay as a quick way to assess the infectivity after each round of viral production and injection. Most of the required equipment is commercially available and commonly present in most research laboratories.

Engineering of a stable retroviral gene delivery vector by directed evolution.

The lack of safe and effective delivery vectors continues to be a critical limitation facing human gene therapy. Viruses offer excellent efficiency but can be difficult and expensive to produce and purify. For example, the production and efficiency of murine leukemia virus (MLV) are limited by its inherent instability; the half-life of infectivity is 5-8 hours at 37 degrees C. In order to generate a stable MLV, we randomly mutated the virus genome and selected for infectivity after prolonged incubation at 37 degrees C. After seven rounds of incubation and infection, we isolated a pool of MLV variants with double the half-life of wild-type MLV. Remarkably, a single mutation in the viral protease (PR), G119E, was responsible for the enhanced stability. Saturation mutagenesis at residue 119 revealed variants with half-lives of approximately 24 hours at 37 degrees C. Double mutants combining the changes at position 119 of the PR and substitutions in the PR substrate-binding pocket exhibited half-lives of up to approximately 40 hours. MLV variants provided two- to fourfold higher viral titers and exhibited increased stability with various wild-type envelope proteins. The improved stability of the variant MLVs will provide more facile virus production and increased transduction efficiency.

Directly monitoring individual retrovirus budding events using atomic force microscopy.

Retrovirus budding is a key step in the virus replication cycle. Nonetheless, very little is known about the underlying mechanism of budding, primarily due to technical limitations preventing visualization of bud formation in real time. Methods capable of monitoring budding dynamics suffer from insufficient resolution, whereas other methods, such as electron microscopy, do not have the ability to operate under physiological conditions. Here we applied atomic force microscopy to real-time visualization of individual Moloney murine leukemia virus budding events. By using a single-particle analysis approach, we were able to observe distinct patterns in budding that otherwise remain transparent. We find that bud formation follows at least two kinetically distinct pathways. The majority of virions (74%) are produced in a slow process (>45 min), and the remaining particles (26%) assemble via a fast process (<25 min). Interestingly, repetitive budding from the same site was seen to occur in only two locations. This finding challenges the hypothesis that viral budding occurs from distinct sites and suggests that budding is not restricted laterally. In this study, we established a method to monitor the fine dynamics of the virus budding process. Using this single-particle analysis to study mutated viruses will enable us to gain additional insight into the mechanisms of viral budding.

Tissue- and stage-specific activation of an endogenous provirus after transcription through its integration site in the opposite orientation.

Endogenous proviruses of the Moloney murine leukemia retrovirus (Mo-MuLV) are transcriptionally blocked in early embryos and in general remain silent even when the tissues have become permissive to the expression of newly integrated copies. Eventually, activation in presumably very few cells initiates rapid superinfection leading to viremia and leukemia, but the processes leading to provirus activation are unknown. Differences in the onset and development of viremia between several mouse strains carrying an endogenous Mo-MuLV (Mov lines) are attributed to a chromosomal position effect, but neither cell type nor stage of provirus activation is known for any strain. We have now monitored the appearance of viral transcripts and particles in the Mov13 strain, which carries the Mo-MuLV provirus in inverted orientation in the first intron of a collagen gene (Col1a1) with well-characterized transcriptional activity. We report obligatory tissue- and stage-specific virus activation in osteoblasts and odontoblasts. The significance of this activation pattern is indicated by the fact that of the great variety of cells expressing the wild-type collagen gene, only these two cell types can also transcribe the mutant allele including its viral insert. We propose that this transcription of the proviral genome, albeit in the opposite direction, leads to the activation of the viral promoter.

The role of the membrane-spanning domain sequence in glycoprotein-mediated membrane fusion.

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.

Novel retroviral vectors to facilitate expression screens in mammalian cells.

As tools for functional genomics, expression profiling and proteomics provide correlative data, while expression cloning screens can link genes directly to biological function. However, technical limitations of gene transfer, expression, and recovery of candidate genes have limited wider application of genome-wide expression screens. Here we describe the pEYK retroviral vectors, which maintain high titers and robust gene expression while addressing the major bottleneck of expression cloning--efficient candidate gene recovery. By exploiting schemes for enhanced PCR rescue or strategies for direct isolation of proviral DNA as plasmids in bacterial hosts, the pEYK vectors facilitate cDNA isolation from selected cells and enable rapid iteration of screens and genetic reversion analyses to validate gene candidates. These vectors have proven useful to identify genes linked to cell proliferation, senescence and apoptosis.

Immunosuppression by Moloney leukemia virus: lack of correlation between virus replication and the immunosuppressive effect.

Young adult mice were infected with 10(4) plaque-forming units (PFU) or Moloney murine leukemia virus M-MuLV. Two different virus preparations were used: a) M-MuLV obtained from serial passage in mice [animal passage (AP)] and b) tissue culture (TC)-grown virus harvested after three in vitro passages of the AP M-MuLV in fibroblasts. Replication of TC and AP M-MuLV in spleen cells was determined by an infectious center (IC) assay at 1 and 2 weeks after the infection. Immune responsiveness of spleen cells was evaluated in challenge with sheep red blood cells (SRBC) and subsequent enumeration of antibody plaque-forming cells (PFC). TC M-MuLV replicated faster in the spleen than did AP M-MuLV and reached about 10- to 100-fold higher titers. However, the response of anti-SRBC PFC, suppressed to the same degree in the spleens of mice infected with TC or AP virus, was from 10 to 50% of the control response. A comparison of virus replication with the anti-SRBC response in aliquots from the same spleens showed no correlation between virus IC and antibody PFC. Both TC and AP M-MuLV induced the expression of virus-specific, cell membrane antigen on spleen cells. These findings indicated a divergence between virus replication on the one hand and the immunosuppressive effect and the cell membrane alteration on the other.

Post-entry restriction of retroviral infections.

Pathogenic retroviruses have driven the evolution of several dominant-acting mechanisms able to block infection and protect the host. These are exemplified by the mouse gene Fv1, which encodes a Gag-like protein able to protect against murine leukemia virus (MLV) infection. The block is saturable, occurs after reverse transcription and is directed against the viral capsid gene. Several other mammalian species are also able to block MLV infection with the same capsid specificity. A human gene with this activity has been named Ref1. Recently, primates have been shown to restrict a variety of retroviruses only very distantly related to MLVs through a gene named Lv1. Restricted viruses include MLV as well as lentiviruses such as human immunodeficiency viruses 1 and 2, simian immunodeficiency virus and equine infectious anemia virus. In each case the block to one retrovirus can be saturated by co-infection with a second restricted virus. The possible mechanisms of action, and evolutionary consequences of restriction, are reviewed.

Purification and Injection of Retroviral Vectors.

Retroviral vectors are powerful tools for genetic manipulation. This protocol discusses the production, purification, and use of replication-deficient retroviral vectors based on Moloney murine leukemia virus and lentivirus. It also describes the injection of a retroviral vector into the dentate gyrus of young adult mice to fluorescently label live murine brain tissue.

Preparation and Use of Retroviral Vectors for Labeling, Imaging, and Genetically Manipulating Cells.

Retroviral vectors are a powerful technology for achieving long-term genetic manipulation. This introduction provides some background on replication-deficient retroviral vectors based on Moloney murine leukemia virus and lentivirus. Details, examples, and associated protocols are provided for using these vectors to fluorescently label, genetically alter, and image both live and fixed murine brain tissue.

Characterization of retrovirus-induced SC-1 cell fusion.

Virus-mediated cell-cell fusion with Moloney MLV and SC-1 cells was characterized. The level of fusion was highly dependent on the cell line used for propagation of the virus. Efficient fusion appeared to be very sensitive to negative charges on the cell surface and surroundings. Addition of polycations, removal of serum, and treatment with neuraminidase or hyaluronidase all stimulated fusion. Conversely, fusion was inhibited by fibronectin. Kinetic results and the time of action of inhibitors indicated that virus particles (or virus material) on the cell surface lead directly to fusion. The fusion then proceeded rapidly and required actin movement as shown by cytochalasin inhibition.

The effect of cerulenin on Moloney murine leukemia virus morphogenesis.

Cerulenin is an antibiotic that interferes with fatty acid synthesis in eukaryotic cells. It had been shown by Schultz and Oroszlan (1983), that murine leukemia virus (MuLV) Pr65gag, the polyprotein precursor to the virion core proteins contains the fatty acid myristate at its NH2 terminus. We showed that when 20 micrograms/ml of cerulenin is added for 3 h to mouse fibroblasts chronically infected with Moloney (M)-MuLV it causes a greater than 4-fold decrease in virus production. This is accompanied by an accumulation of uncleaved Pr65gag in the infected cells. Further, thin-section electron micrographs of cerulenin-treated cells show a 2-fold increase in the number of nascent-budding forms, as well as the appearance of aberrant viral forms at the cell membrane. This suggests that the failure to add myristic acid to Pr65gag prevents their proper assembly into viral particles.

Replication of lactate dehydrogenase-elevating virus in various species cell lines infected with dual-, ampho- and xenotropic murine leukaemia viruses in vitro.

Lactate dehydrogenase-elevating virus (LDV) replicates in mouse macrophages in vivo and in vitro. It has been shown that LDV infects and replicates in motorneurons of the spinal cord of old, immunosuppressed C58 mice, which results in an acute poliomyelitis. In spite of extensive study, cells or cell lines other than macrophages which could support LDV infection and replication in vitro have not yet been detected. We have shown that LDV can replicate in mouse or rat cell lines which were previously infected with ecotropic murine leukaemia virus (MuLV). It was examined in this study whether other types of MuLV (dualtropic, amphotropic and xenotropic viruses) can also render the mouse cells or cells of other species susceptible to LDV infection as well as the ecotropic viruses. LDV infection and replication were seen in mouse cells infected with ecotropic, dual-tropic and amphotropic viruses. These were also seen in mink, rabbit and human cell lines infected with dual-, ampho- and xenotropic viruses. These results suggested that virtually all four classes of MuLV have the ability to elicit, in mouse cells or cells from heterologous species, permissiveness to LDV infection. The percent of LDV-infected cells increased up to approximately 80% in concentrated neurovirulent LDV-C-infected ecotropic MuLV-infected-mouse cells. The susceptibility of the cells gradually declined when they were maintained for more than one month. The LDV antigen-positive cells appeared as early as 6-8 h p.i., when a large amount of LDV and MuLV were added simultaneously. The replication of LDV was inhibited in MuLV-infected cells which had been treated previously with actinomycin D and cycloheximide, but not with zidovudine (AZT). A small percent of mouse cells became susceptible to LDV, when the cells were treated with iododeoxyuridine. This suggested that the induction of endogenous MuLV or part(s) of its genome from mouse chromosomes resulted in cells that were permissive to LDV.