scholarly journals Extensive epitranscriptomic methylation of A and C residues on murine leukemia virus transcripts enhances viral gene expression

2019 ◽  
Author(s):  
David G. Courtney ◽  
Andrea Chalem ◽  
Hal P. Bogerd ◽  
Brittany A. Law ◽  
Edward M. Kennedy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Positive Role ◽  
Viral Rnas ◽  
Order Of Magnitude ◽  
Murine Leukemia

AbstractWhile it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels ofN6-methyladenosine (m6A), 5-methylcytosine (m5C) and 2’O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m6A and m5C residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m6A and m5C in viral replication, we also demonstrate that overexpression of the key m6A reader protein YTHDF2 enhances MLV replication, while downregulation of the m5C writer NSUN2 inhibits MLV replication.ImportanceThe data presented in this manuscript demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications m6A, m5C and Nm, thus suggesting that these each facilitate some aspect of the viral replication cycle. Consistent with this hypothesis, we demonstrate that mutational removal of a subset of these m6A or m5C modifications from MLV transcripts inhibits MLV replication incisand a similar result was also observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors intrans. Together, these results argue that the addition of several different epitranscriptomic modifications to viral transcripts stimulates viral gene expression and suggest that MLV has therefore evolved to maximize the level of these modifications that are added to viral RNAs.

scholarly journals Extensive Epitranscriptomic Methylation of A and C Residues on Murine Leukemia Virus Transcripts Enhances Viral Gene Expression

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
David G. Courtney ◽  
Andrea Chalem ◽  
Hal P. Bogerd ◽  
Brittany A. Law ◽  
Edward M. Kennedy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Positive Role ◽  
Viral Rnas ◽  
Order Of Magnitude ◽  
Murine Leukemia

ABSTRACTWhile it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels ofN6-methyladenosine (m6A), 5-methylcytosine (m5C), and 2′O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m6A and m5C residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m6A and m5C in viral replication, we also demonstrate that overexpression of the key m6A reader protein YTHDF2 enhances MLV replication, while downregulation of the m5C writer NSUN2 inhibits MLV replication.IMPORTANCEThe data presented in the present study demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications m6A, m5C, and Nm, suggesting that these each facilitate some aspect of the viral replication cycle. Consistent with this hypothesis, we demonstrate that mutational removal of a subset of these m6A or m5C modifications from MLV transcripts inhibits MLV replication incis, and a similar result was also observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors intrans. Together, these results argue that the addition of several different epitranscriptomic modifications to viral transcripts stimulates viral gene expression and suggest that MLV has therefore evolved to maximize the level of these modifications that are added to viral RNAs.

scholarly journals Bacteriophage self-counting in the presence of viral replication

2021 ◽  
Vol 118 (51) ◽  
pp. e2104163118
Author(s):  
Tianyou Yao ◽  
Seth Coleman ◽  
Thu Vu Phuc Nguyen ◽  
Ido Golding ◽  
Oleg A. Igoshin
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Viral Gene Expression ◽  
Host Cells ◽  
Optimal Decision ◽  
Viral Gene ◽  
Viral Genomes ◽  
Viral Copy Number ◽  
Viral Copy ◽  
Type Phage

When host cells are in low abundance, temperate bacteriophages opt for dormant (lysogenic) infection. Phage lambda implements this strategy by increasing the frequency of lysogeny at higher multiplicity of infection (MOI). However, it remains unclear how the phage reliably counts infecting viral genomes even as their intracellular number increases because of replication. By combining theoretical modeling with single-cell measurements of viral copy number and gene expression, we find that instead of hindering lambda’s decision, replication facilitates it. In a nonreplicating mutant, viral gene expression simply scales with MOI rather than diverging into lytic (virulent) and lysogenic trajectories. A similar pattern is followed during early infection by wild-type phage. However, later in the infection, the modulation of viral replication by the decision genes amplifies the initially modest gene expression differences into divergent trajectories. Replication thus ensures the optimal decision—lysis upon single-phage infection and lysogeny at higher MOI.

B-Catenin Signaling Regulates the In Vivo Distribution of Hepatitis B Virus Biosynthesis across the Liver Lobule

2021 ◽  
Author(s):  
Grant Tarnow ◽  
Alan McLachlan
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Viral Replication ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Central Vein ◽  
Liver Lobule ◽  
Liver Gene ◽  
Mice Liver

β-catenin (Ctnnb1) supports high levels of liver gene expression in hepatocytes in proximity to the central vein functionally defining zone 3 of the liver lobule. This region of the liver lobule supports the highest levels of viral biosynthesis in wildtype HBV transgenic mice. Liver-specific β-catenin-null HBV transgenic mice exhibit a stark loss of high levels of pericentral viral biosynthesis. Additionally, viral replication that does not depend directly on β-catenin activity appears to expand to include hepatocytes of zone 1 of the liver lobule in proximity to the portal vein, a region of the liver that typically lacks significant HBV biosynthesis in wildtype HBV transgenic mice. While the average amount of viral RNA transcripts does not change, viral DNA replication is reduced approximately three-fold. Together, these observations demonstrate that β-catenin signaling represents a major determinant of HBV biosynthesis governing the magnitude and distribution of viral replication across the liver lobule in vivo. Additionally, these findings reveal a novel mechanism for the regulation of HBV biosynthesis that is potentially relevant to the expression of additional liver-specific genes. IMPORTANCE Viral biosynthesis is highest around the central vein in the HBV transgenic mouse model of chronic infection. The associated HBV biosynthetic gradient across the liver lobule is primarily dependent upon β-catenin. In the absence of β-catenin, the gradient of viral gene expression spanning the liver lobule is absent and HBV replication is reduced. Therefore, therapeutically manipulating β-catenin activity in the liver of chronic HBV carriers may reduce circulating infectious virions without greatly modulating viral protein production. Together, these change in viral biosynthesis might limit infection of additional hepatocytes while permitting immunological clearance of previously infected cells, potentially limiting disease persistence.

scholarly journals Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

2000 ◽  
Vol 74 (22) ◽  
pp. 10349-10358 ◽  
Author(s):  
Elias K. Halvas ◽  
Evguenia S. Svarovskaia ◽  
Vinay K. Pathak
Keyword(s):  
Amino Acids ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Deoxyribonucleoside Triphosphate ◽  
Murine Leukemia

ABSTRACT Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription.

scholarly journals An Interferon Regulatory Factor Binding Site in the U5 Region of the Bovine Leukemia Virus Long Terminal Repeat Stimulates Tax-Independent Gene Expression

1998 ◽  
Vol 72 (7) ◽  
pp. 5526-5534 ◽  
Author(s):  
Véronique Kiermer ◽  
Carine Van Lint ◽  
Delphine Briclet ◽  
Caroline Vanhulle ◽  
Richard Kettmann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Long Terminal Repeat ◽  
Binding Site ◽  
Bovine Leukemia Virus ◽  
Transcription Initiation ◽  
Leukemia Virus ◽  
Viral Gene Expression ◽  
Terminal Repeat ◽  
Viral Gene ◽  
Enhancer Activity

ABSTRACT Bovine leukemia virus (BLV) replication is controlled by bothcis- and trans-acting elements. The virus-encoded transactivator, Tax, is necessary for efficient transcription from the BLV promoter, although it is not present during the early stages of infection. Therefore, sequences that control Tax-independent transcription must play an important role in the initiation of viral gene expression. This study demonstrates that the R-U5 sequence of BLV stimulates Tax-independent reporter gene expression directed by the BLV promoter. R-U5 was also stimulatory when inserted immediately downstream from the transcription initiation site of a heterologous promoter. Progressive deletion analysis of this region revealed that a 46-bp element corresponding to the 5′ half of U5 is principally responsible for the stimulation. This element exhibited enhancer activity when inserted upstream or downstream from the herpes simplex virus thymidine kinase promoter. This enhancer contains a binding site for the interferon regulatory factors IRF-1 and IRF-2. A 3-bp mutation that destroys the IRF recognition site caused a twofold decrease in Tax-independent BLV long terminal repeat-driven gene expression. These observations suggest that the IRF binding site in the U5 region of BLV plays a role in the initiation of virus replication.

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