Behind the scenes of HIV-1 replication: Alternative splicing as the dependency factor on the quiet

Sertznig, Helene; Hillebrand, Frank; Erkelenz, Steffen; Schaal, Heiner; Widera, Marek

doi:10.1016/j.virol.2018.01.011

Cited by 59 publications

(95 citation statements)

References 157 publications

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“…CpG dinucleotides introduced into gag can inhibit HIV-1 gene expression by modulating pre-mRNA splicing. The HIV-1 genomic RNA undergoes extensive alternative splicing to mediate expression of all of the viral genes, and synonymous mutations in gag have previously been shown to disrupt HIV-1 splicing (36,50). Since the CpGs in the 5= region of gag reduced Env expression in all sequence contexts tested ( Fig.…”

Section: Ficarelli Et Almentioning

confidence: 91%

“…Furthermore, it is important that the synonymous mutations introducing CpGs do not disrupt RNA elements that regulate viral replication. The HIV-1 open reading frames (ORFs) contain multiple cis-acting regulatory elements, including the programmed ribosomal frameshift sequence in gag (33); the Rev response element (RRE) in env (34); polypurine tracts in pol and nef (35); and splicing signals in pol, vif, vpr, tat, rev, and env (36). In addition, there could be uncharacterized elements.…”

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confidence: 99%

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CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms

Ficarelli

Antzin-Anduetza

Hugh-White

et al. 2020

J Virol

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CpG dinucleotides are suppressed in the genomes of many vertebrate RNA viruses, including HIV-1. The cellular antiviral protein ZAP (zinc finger antiviral protein) binds CpGs and inhibits HIV-1 replication when CpGs are introduced into the viral genome. However, it is not known if ZAP-mediated restriction is the only mechanism driving CpG suppression. To determine how CpG dinucleotides affect HIV-1 replication, we increased their abundance in multiple regions of the viral genome and analyzed the effect on RNA expression, protein abundance, and infectious-virus production. We found that the antiviral effect of CpGs was not correlated with their abundance. Interestingly, CpGs inserted into some regions of the genome sensitize the virus to ZAP antiviral activity more efficiently than insertions into other regions, and this sensitivity can be modulated by interferon treatment or ZAP overexpression. Furthermore, the sensitivity of the virus to endogenous ZAP was correlated with its sensitivity to the ZAP cofactor KHNYN. Finally, we show that CpGs in some contexts can also inhibit HIV-1 replication by ZAP-independent mechanisms, and one of these is the activation of a cryptic splice site at the expense of a canonical splice site. Overall, we show that the location and sequence context of the CpG in the viral genome determines its antiviral activity. IMPORTANCE Some RNA virus genomes are suppressed in the nucleotide combination of a cytosine followed by a guanosine (CpG), indicating that they are detrimental to the virus. The antiviral protein ZAP binds viral RNA containing CpGs and prevents the virus from multiplying. However, it remains unknown how the number and position of CpGs in viral genomes affect restriction by ZAP and whether CpGs have other antiviral mechanisms. Importantly, manipulating the CpG content in viral genomes could help create new vaccines. HIV-1 shows marked CpG suppression, and by introducing CpGs into its genome, we show that ZAP efficiently targets a specific region of the viral genome, that the number of CpGs does not predict the magnitude of antiviral activity, and that CpGs can inhibit HIV-1 gene expression through a ZAP-independent mechanism. Overall, the position of CpGs in the HIV-1 genome determines the magnitude and mechanism through which they inhibit the virus.

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Section: Ficarelli Et Almentioning

confidence: 91%

mentioning

confidence: 99%

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms

Ficarelli

Antzin-Anduetza

Hugh-White

et al. 2020

J Virol

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“…CAE3 begins at the RRE and ends precisely at splice acceptor 7 (SA-7), which is used for several multiply spliced HIV-1 transcripts, including vpr, tat, rev, nef (66), and implicated in viral fitness (56). While the importance of the RRE and SA-7 were known (31,67,68), RanDeL-seq showed that the entire 300 bp region from the upstream RRE to the end of SA7 is required for sustained viral replication, and cannot be provided in trans. Finally, CAE4 spans the PPT, which is necessary for reverse transcription, and the 3' LTR (18).…”

Section: Hiv-1 Deletion Landscapes Identify Cae and Taesmentioning

confidence: 99%

RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements

Notton

Glazier

Saykally

et al. 2020

Preprint

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AbstractIt has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare, spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback, and then barcoded for tracking via sequencing (i.e., Random-Deletion Library sequencing, RanDeL-seq). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1’s cis and trans elements. The resulting landscape recapitulated HIV-1’s known cis-acting elements (i.e., LTR, Ψ, and RRE) and surprisingly indicated that HIV-1’s central DNA flap (i.e., central polypurine tract, cPPT to central termination sequence, CTS) is as critical as the LTR, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼300bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV’s cis-acting regions are larger than the UTR termini, encompassing a large fraction of the non-structural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants enabling discovery of replication mechanisms and development of novel antiviral therapeutics, particularly for emerging viral infections.ImportanceRecent studies have renewed interest in developing novel antiviral therapeutics and vaccines based on defective interfering particles (DIPs)—a subset of viral deletion mutant that conditionally replicate. Identifying and engineering DIPs requires that viral cis- and trans-acting elements be accurately mapped. Here we introduce a high-throughput method (Random Deletion Library sequencing, RanDeL-seq) to comprehensively map cis- and trans-acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central poly-purine tract (cPPT) and identified a new cis region proximal to the Rev responsive element (RRE). RanDeL-seq also identified regions of Zika virus required for replication and packaging. RanDeL-seq is a versatile and comprehensive technique to rapidly map cis and trans regions of a genome.

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“…However, literature data show that host RNA splicing is altered upon HIV-1 infection, and the level of class A/B and H of hnRNP proteins changes; initially decreased 6-12 days post infection, thereafter increased [35]. At the same time, it was shown that some proteins of this cluster; such as HNRNPH1, HNRNPU and SRSF6, are so called HIV-1 dependency factors [36] and are required by HIV-1. These data are derived from later time-points, as most of the experiments do not examine such early events at 4 h or 12 h post infection.…”

Section: Go1902580 Single-organism Cellular Localization Df Decreasementioning

confidence: 99%

Analysis of networks of host proteins in the early time points following HIV transduction

et al. 2019

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Background: Utilization of quantitative proteomics data on the network level is still a challenge in proteomics data analysis. Currently existing models use sophisticated, sometimes hard to implement analysis techniques. Our aim was to generate a relatively simple strategy for quantitative proteomics data analysis in order to utilize as much of the data generated in a proteomics experiment as possible. Results: In this study, we applied label-free proteomics, and generated a network model utilizing both qualitative, and quantitative data, in order to examine the early host response to Human Immunodeficiency Virus type 1 (HIV-1). A weighted network model was generated based on the amount of proteins measured by mass spectrometry, and analysis of weighted networks and functional sub-networks revealed upregulation of proteins involved in translation, transcription, and DNA condensation in the early phase of the viral life-cycle. Conclusion: A relatively simple strategy for network analysis was created and applied to examine the effect of HIV-1 on host cellular proteome. We believe that our model may prove beneficial in creating algorithms, allowing for both quantitative and qualitative studies of proteome change in various biological and pathological processes by quantitative mass spectrometry.

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Behind the scenes of HIV-1 replication: Alternative splicing as the dependency factor on the quiet

Cited by 59 publications

References 157 publications

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms

CpG Dinucleotides Inhibit HIV-1 Replication through Zinc Finger Antiviral Protein (ZAP)-Dependent and -Independent Mechanisms

RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements

Analysis of networks of host proteins in the early time points following HIV transduction

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