Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression

Courtney, David; Tsai, Kevin; Bogerd, Hal P.; Kennedy, Edward M.; Law, Brittany A.; Emery, Ann; Swanstrom, Ronald; Holley, Christopher L.; Cullen, Bryan R.

doi:10.1016/j.chom.2019.07.005

Cited by 146 publications

(165 citation statements)

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“…, A3G) more strongly associated with genomes than others. While we have yet to define the RNA signature(s) that specify genome detection by A3G, our observations may be informative in the context of recent CLIP-seq studies demonstrating A3G’s RNA-binding preference to be relatively sequence non-specific 19,20 coupled to compelling recent work showing that HIV-1 genomes are selectively enriched in selective post-transcriptional regulatory marks including N 6 methyladenosine (m6a) 48–50 , 5-methylcytosine (m5c), and 2’O-methylation 51 . Artificially tethering A3G to membranes restricts HIV-1 genome trafficking and virus particle assembly (Fig.…”

Section: Discussionmentioning

confidence: 62%

HIV-1 RNA genomes initiate host protein packaging in the cytosol independently of Gag capsid proteins

Becker

Evans

Benner

et al. 2019

Preprint

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17HIV-1 RNA genomes interact with diverse RNA binding proteins in the cytoplasm 18 including antiviral factor APOBEC3G (A3G) that, in the absence of viral Vif proteins, is 19 packaged into virions. When and where genome-A3G interactions are initiated in the host 20 cell is unknown. Here we use quantitative long-term (>24 h) live cell fluorescence video 21 microscopy and a new in-cell RNA-protein interaction assay (the "IC-IP") to describe 22 subcellular viral and A3G trafficking behaviors over the entire HIV-1 productive phase. 23Among other findings, we demonstrate that genome-A3G interactions are initiated in the 24 cytosol soon if not immediately after genome nuclear export; that A3G-genome 25 interactions are sufficiently strong so that tethering either factor to membranes inhibits 26 trafficking of the reciprocal binding partner; and that selective recognition of genomes 27 promotes consistent delivery of A3G to sites of virion assembly. Further elucidation of 28 RNA signature(s) detected by A3G may inform development of RNA-targeted antivirals. 29 RNA-and NC-dependent15-20. However, the relative contributions of genomes vs. host 50RNAs to this process remains controversial. On the one hand, A3G-RNA binding is 51 relatively promiscuous, with A3G incorporated into virus particles even when packageable 52 genomes are not expressed17,19. On the other hand, A3G incorporation levels are 53 moderately enhanced when genomes are present16,21; and A3G has been shown to 54 exhibit selective RNA-binding characteristics19,22 including a reported preference for G-55 rich segments of the HIV-1 genome20. 56Where and when A3G interfaces with Gag and/or genomes in the cell has also been 57 under investigation for some time with conflicting results. At steady-state, A3G is 58 distributed throughout the cytosol (the aqueous phase of the cytoplasm) and accumulates 59 to high levels at non-membranous cytoplasmic sites of mRNA decay known as processing 60 bodies (PBs)23,24. Cytosolic A3G is also rapidly re-localized to sites of translational 61 repression known as stress granules (SGs) in response to heat shock or oxidative 62 stress23,24. Initial studies proposed a functional link between PBs and A3G's antiviral 63 activity23,25,26. By contrast, more recent work has shown visible PBs to have little to no 64 discernible relevance to the HIV-1 life cycle27,28. 65The dynamic and complex nature of cytoplasmic RNA trafficking during HIV-1 virion 66 genesis may have obscured consistent prior observations made in fixed cells, 67 emphasizing a need for real-time characterization the host RBP response to HIV-1 68 infection at subcellular resolution. Accordingly, herein we set out to comprehensively 69 define the behaviors of HIV-1 RNA genomes, Gag, A3G, and additional PB and SG 70 markers over the entire viral productive phase using long-term (>24h) live cell video 71 microscopy. Our results expose single-cell A3G degradation and trafficking behaviors in 72 the presence or absence of Vif; and show that HIV-1 infection has little to no ne...

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Section: Discussionmentioning

confidence: 62%

HIV-1 RNA genomes initiate host protein packaging in the cytosol independently of Gag capsid proteins

Becker

Evans

Benner

et al. 2019

Preprint

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“…The initial goal of this project was to quantify the epitranscriptomic RNA modifications present on MLV genomic RNA (gRNA) using ultra-high-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) (17) (8). This required the purification of the gRNA away from cellular tRNAs and other ncRNAs that are heavily modified.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, we reported that m 6 A residues enhance viral gene expression and replication for HIV-1, influenza A virus and the polyoma virus SV40 (9–11) and others have also reported that m 6 A residues promote HIV-1 and enterovirus 71 replication (12, 13) and play a role in the activation of lytic replication in Kaposi’s sarcoma herpesvirus (KSHV)-infected cells (14, 15). More recently, we reported that addition of m 5 C also enhances HIV-1 gene expression (8), and others have reported that Nm modifications on HIV-1 transcripts promote HIV-1 replication by inhibiting the detection of viral transcripts by the innate antiviral RNA sensor MDA5 (16). Consistent with a positive role for these epitranscriptomic modifications in the regulation of viral replication, we recently reported that HIV-1 transcripts bear a far higher level of m 6 A, m 5 C and Nm residues than does the average cellular mRNA (8).…”

Section: Introductionmentioning

confidence: 99%

“…More recently, we reported that addition of m 5 C also enhances HIV-1 gene expression (8), and others have reported that Nm modifications on HIV-1 transcripts promote HIV-1 replication by inhibiting the detection of viral transcripts by the innate antiviral RNA sensor MDA5 (16). Consistent with a positive role for these epitranscriptomic modifications in the regulation of viral replication, we recently reported that HIV-1 transcripts bear a far higher level of m 6 A, m 5 C and Nm residues than does the average cellular mRNA (8). Here, we extend these earlier findings by demonstrating that the addition of both m 6 A and m 5 C independently upregulates murine leukemia virus (MLV) gene expression and replication.…”

Section: Introductionmentioning

confidence: 99%

“…Once added, m 6 A can be recognized by several “readers”, including the nuclear YTHDC1 and cytoplasmic YTHDF2 proteins, which then regulate the splicing, translation and/or stability of that mRNA. Less is known about the m 5 C modification, although NSUN2 has been shown to add m 5 C to a handful of cellular mRNAs (4–7) and we have recently identified NSUN2 as the primary writer of m 5 C residues on the HIV-1 genome (8).…”

Section: Introductionmentioning

confidence: 99%

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

Courtney

Chalem

Bogerd

et al. 2019

Preprint

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12While it has been known for several years that viral RNAs are subject to the addition of 13 several distinct covalent modifications to individual nucleotides, collectively referred to as 14 epitranscriptomic modifications, the effect of these editing events on viral gene expression has 15 been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic 16RNA to homogeneity and show that this viral RNA contains levels of N 6 -methyladenosine (m 6 A), 17 5-methylcytosine (m 5 C) and 2'O-methylated (Nm) ribonucleotides that are an order of 18 magnitude higher than detected on bulk cellular mRNAs. Mapping of m 6 A and m 5 C residues on 19 MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV 20 variants bearing silent mutations that removed a subset of these sites. Analysis of the 21 replication potential of these mutants revealed a modest but significant attenuation in viral 22 replication in 3T3 cells in culture. Consistent with a positive role for m 6 A and m 5 C in viral 23 replication, we also demonstrate that overexpression of the key m 6 A reader protein YTHDF2 24 enhances MLV replication, while downregulation of the m 5 C writer NSUN2 inhibits MLV 25 replication. 26 Importance 27The data presented in this manuscript demonstrate that MLV RNAs bear an 28 exceptionally high level of the epitranscriptomic modifications m 6 A, m 5 C and Nm, thus 29 suggesting that these each facilitate some aspect of the viral replication cycle. Consistent with 30 this hypothesis, we demonstrate that mutational removal of a subset of these m 6 A or m 5 C 31 modifications from MLV transcripts inhibits MLV replication in cis and a similar result was also 32 observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors 33 in trans. Together, these results argue that the addition of several different epitranscriptomic 34 modifications to viral transcripts stimulates viral gene expression and suggest that MLV has 35 therefore evolved to maximize the level of these modifications that are added to viral RNAs. 36

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Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression

Cited by 146 publications

References 53 publications

HIV-1 RNA genomes initiate host protein packaging in the cytosol independently of Gag capsid proteins

HIV-1 RNA genomes initiate host protein packaging in the cytosol independently of Gag capsid proteins

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

Viral Epitranscriptomics

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