scholarly journals Comparative Analysis of HIV-1 and Murine Leukemia Virus Three-Dimensional Nuclear Distributions

2016 ◽  
Vol 90 (10) ◽  
pp. 5205-5209 ◽  
Author(s):  
Valentina Quercioli ◽  
Cristina Di Primio ◽  
Antonio Casini ◽  
Lubbertus C. F. Mulder ◽  
Lenard S. Vranckx ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Murine Leukemia Virus ◽  
Fluorescent Protein ◽  
Leukemia Virus ◽  
Three Dimensional ◽  
Moloney Murine Leukemia Virus ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Hiv 1 ◽  
Murine Leukemia

Recent advances in fluorescence microscopy allow three-dimensional analysis of HIV-1 preintegration complexes in the nuclei of infected cells. To extend this investigation to gammaretroviruses, we engineered a fluorescent Moloney murine leukemia virus (MLV) system consisting of MLV-integrase fused to enhanced green fluorescent protein (MLV-IN-EGFP). A comparative analysis of lentiviral (HIV-1) and gammaretroviral (MLV) fluorescent complexes in the nuclei of infected cells revealed their different spatial distributions. This research tool has the potential to achieve new insight into the nuclear biology of these retroviruses.

scholarly journals Small Dumbbell Oligonucleotides Inhibitors of RNase H Activity of MOMULV Reverse Transcriptase

2011 ◽  
Vol 8 (2) ◽  
pp. 629-634
Author(s):  
Ajay Kumar
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Dissociation Constants ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Mobility Shift ◽  
Hiv 1 ◽  
Murine Leukemia

The small dumbbell oligonucleotides containing loops of phosphodiester (OL-1), two trimethylene, C3moieties in each loop (OL-2) and phosphorothioate (OL-3) linkages were synthesized. Incubation of OL-1 and OL-2 with S-1 nuclease generated break down products whereas incubation of OL-3 did not result in significant cleavage. Their binding to moloney murine leukemia virus reverse transcriptase was evaluated by PAGE band mobility shift assays. The OL-3 bound more strongly to the reverse transcriptase than OL-1 and OL-2. The dissociation constants evaluated using PAGE band mobility shift assays were of the order of 10-7. Investigation of inhibition of RNase H activity of reverse transcriptase showed that the OL-3 is a better inhibitor of the retroviral RNase H activity than both OL-1 and OL-2. Thus OL-3 may be used as RNase H inhibitor. Our studies demonstrated that this particularly designed oligonucleotide (OL-3) displays an IC50of 25 nM in its inhibition on the reverse transcriptase RNase H activity, a magnitude lower than that of first nucleotide reverse transcriptase of HIV-1, tenofovir, introduced by Gilead Science in the market.

scholarly journals Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells

2001 ◽  
Vol 75 (23) ◽  
pp. 11365-11372 ◽  
Author(s):  
Lilin Lai ◽  
Hongmei Liu ◽  
Xiaoyun Wu ◽  
John C. Kappes
Keyword(s):  
Dna Synthesis ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Specific Interaction ◽  
Proteolytic Processing ◽  
Moloney Murine Leukemia Virus ◽  
Wild Type ◽  
Viral Dna ◽  
Infected Cells ◽  
Murine Leukemia

ABSTRACT Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (ΔIN) or 34 C-terminal amino acid residues (Δ34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Δ34 and ΔIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Δ34 and ΔIN mutants intrans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of ΔIN mutant virions could not be complemented with the Δ34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.

scholarly journals Identification of Directly Infected Cells in the Bone Marrow of Neonatal Moloney Murine Leukemia Virus-Infected Mice by Use of a Moloney Murine Leukemia Virus-Based Vector

1999 ◽  
Vol 73 (2) ◽  
pp. 1617-1623 ◽  
Author(s):  
Michael A. Okimoto ◽  
Hung Fan
Keyword(s):  
Bone Marrow ◽  
Murine Leukemia Virus ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Small Cells ◽  
Nonspecific Esterase ◽  
Hematopoietic Progenitors ◽  
Infected Cells ◽  
Murine Leukemia

ABSTRACT Early bone marrow infection of Moloney murine leukemia virus (M-MuLV)-infected mice was studied. Previous experiments indicated that early bone marrow infection is essential for the efficient development of T lymphoma. In order to identify the cellular pathway of infection in the bone marrow, infection of mice with a helper-free replication-defective M-MuLV-based retroviral vector was carried out. Such a vector will undergo only one round of infection, without spreading to other cells; thus, cells infected by the initially injected virus (directly infected cells) can be identified. For these experiments, the BAG vector that expresses bacterial β-galactosidase was employed. Neonatal NIH/Swiss mice were inoculated intraperitoneally with ca. 106 infectious units of a BAG vector pseudotyped with M-MuLV proteins, and bone marrow cells were recovered 2 to 12 days postinfection. Single-cell suspensions were tested for infection by staining with X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) or by immunofluorescence with an anti-β-galactosidase antibody. Two sizes of infected cells were evident: large multinucleated cells and small nondescript (presumptively hematopoietic) cells. Secondary stains for lineage-specific markers indicated that the large cells were osteoclasts. Some of the small cells expressed nonspecific esterase, which placed them in the myeloid lineage, but they lacked markers for hematopoietic progenitors (mac-1, gr-1, sca-1, and CD34). These results provide evidence for primary M-MuLV infection of osteoclasts or osteoclast progenitors in the bone marrow, and they suggest that known hematopoietic progenitors are not primary targets for infection. However, the subsequent spread of infection to hematopoietic progenitors was indicated, since bone marrow from mice infected in parallel with replication-competent wild-type M-MuLV showed detectable infection in small cells positive for mac-1 or CD34, as well as in osteoclasts.

scholarly journals Crystal Structure of the Moloney Murine Leukemia Virus RNase H Domain

2006 ◽  
Vol 80 (17) ◽  
pp. 8379-8389 ◽  
Author(s):  
David Lim ◽  
G. Glenn Gregorio ◽  
Craig Bingman ◽  
Erik Martinez-Hackert ◽  
Wayne A. Hendrickson ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Bacillus Halodurans ◽  
Catalytic Core ◽  
E Coli ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT A crystallographic study of the Moloney murine leukemia virus (Mo-MLV) RNase H domain was performed to provide information about its structure and mechanism of action. These efforts resulted in the crystallization of a mutant Mo-MLV RNase H lacking the putative helix C (ΔC). The 1.6-Å resolution structure resembles the known structures of the human immunodeficiency virus type 1 (HIV-1) and Escherichia coli RNase H. The structure revealed the coordination of a magnesium ion within the catalytic core comprised of the highly conserved acidic residues D524, E562, and D583. Surface charge mapping of the Mo-MLV structure revealed a high density of basic charges on one side of the enzyme. Using a model of the Mo-MLV structure superimposed upon a structure of HIV-1 reverse transcriptase bound to an RNA/DNA hybrid substrate, Mo-MLV RNase H secondary structures and individual amino acids were examined for their potential roles in binding substrate. Identified regions included Mo-MLV RNase H β1-β2, αA, and αB and residues from αB to αD and its following loop. Most of the identified substrate-binding residues corresponded with residues directly binding nucleotides in an RNase H from Bacillus halodurans as observed in a cocrystal structure with RNA/DNA. Finally, superimposition of RNases H of Mo-MLV, E. coli, and HIV-1 revealed that a loop of the HIV-1 connection domain resides within the same region of the Mo-MLV and E. coli C-helix. The HIV-1 connection domain may serve to recognize and bind the RNA/DNA substrate major groove.

scholarly journals The Retroviruses Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Adopt Radically Different Strategies To Regulate Promoter-Proximal Polyadenylation

2001 ◽  
Vol 75 (23) ◽  
pp. 11735-11746 ◽  
Author(s):  
Andre Furger ◽  
Joan Monks ◽  
Nick J. Proudfoot
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Immunodeficiency Virus ◽  
A Site ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT Maximal gene expression in retroviruses requires that polyadenylation in the 5′ long terminal repeat (LTR) is suppressed. In human immunodeficiency virus type 1 (HIV-1) the promoter-proximal poly(A) site is blocked by interaction of U1 snRNP with the closely positioned major splice donor site (MSD) 200 nucleotides downstream. Here we investigated whether the same mechanism applies to down-regulate 5′ LTR polyadenylation in Moloney murine leukemia virus (MoMLV). Although the same molecular architecture is present in both viruses, the MoMLV poly(A) signal in the 5′ LTR is active whether or not the MSD is mutated. This surprising difference between the two retroviruses is not due to their actual poly(A) signals or MSD sequences, since exchange of either element between the two viral sequences does not alter their ability to regulate 5′ LTR poly(A) site use. Instead we demonstrate that sequence between the cap and AAUAAA is required for MSD-dependent poly(A) regulation in HIV-1, indicating a key role for this part of the LTR in poly(A) site suppression. We also show that the MoMLV poly(A) signal is an intrinsically weak RNA-processing signal. This suggests that in the absence of a poly(A) site suppression mechanism, MoMLV is forced to use a weak poly(A) signal.

scholarly journals Astrocytes Survive Chronic Infection and Cytopathic Effects of the ts1 Mutant of the Retrovirus Moloney Murine Leukemia Virus by Upregulation of Antioxidant Defenses

2006 ◽  
Vol 80 (7) ◽  
pp. 3273-3284 ◽  
Author(s):  
Wenan Qiang ◽  
Xianghong Kuang ◽  
Jinrong Liu ◽  
Na Liu ◽  
Virginia L. Scofield ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Murine Leukemia Virus ◽  
Target Genes ◽  
Leukemia Virus ◽  
Antioxidant Defenses ◽  
Moloney Murine Leukemia Virus ◽  
Related Factor ◽  
Infected Cells ◽  
Thiol Redox ◽  
Murine Leukemia

ABSTRACT The ts1 mutant of Moloney murine leukemia virus (MoMuLV) induces a neurodegenerative disease in mice, in which glial cells are infected by the retrovirus but neurons are not. ts1 infection of primary astrocytes, or of the immortalized astrocytic cell line C1, results in accumulation of the ts1 gPr80 env envelope protein in the endoplasmic reticulum (ER), with ER and oxidative stress. Notably, only about half of the infected astrocytes die in these cultures, while the other half survive, continue to proliferate, and continue to produce virus. To determine how these astrocytes survive ts1 infection in culture, we established a chronically infected subline of the living cells remaining after the death of all acutely infected cells in an infected C1 cell culture (C1-ts1-S). We report here that C1-ts1-S cells proliferate more slowly, produce less virus, show reduced H2O2 levels, increase their uptake of cystine, and maintain higher levels of intracellular GSH and cysteine compared to acutely infected or uninfected C1 cells. C1-ts1-S cells also upregulate their thiol antioxidant defenses by activation of the transcription factor NF-E2-related factor 2 (Nrf2) and its target genes. Interestingly, despite maintenance of higher levels of intracellular reduced thiols, C1-ts1-S cells are more sensitive to cystine deprivation than uninfected C1 cells. We conclude that some ts1-infected astrocytes survive and adapt to virus-induced oxidative stress by successfully mobilizing their thiol redox defenses.

scholarly journals Neutralizing activity and cellular immune responses induced in mice after immunization with apoptotic HIV-1/murine leukemia virus infected cells

Vaccine ◽  
2009 ◽  
Vol 27 (46) ◽  
pp. 6424-6431 ◽  
Author(s):  
Jorma Hinkula ◽  
Lilian Walther-Jallow ◽  
Anna Laurén ◽  
Barbro Mäkitalo ◽  
Monica Öberg ◽  
...  
Keyword(s):  
Immune Responses ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Neutralizing Activity ◽  
Cellular Immune Responses ◽  
Infected Cells ◽  
Cellular Immune ◽  
Hiv 1 ◽  
Murine Leukemia
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