scholarly journals KHNYN is essential for ZAP-mediated restriction of HIV-1 containing clustered CpG dinucleotides

2019 ◽  
Author(s):  
Mattia Ficarelli ◽  
Harry Wilson ◽  
Rui Pedro Galão ◽  
Stuart J D Neil ◽  
Chad M Swanson
Keyword(s):  
Infectious Virus ◽  
Rna Virus ◽  
Virus Production ◽  
Innate Immune ◽  
Viral Rna ◽  
Antiviral Protein ◽  
Cpg Dinucleotides ◽  
Endonuclease Domain ◽  
Virus Genomes ◽  
Hiv 1

AbstractCpG dinucleotides are suppressed in most vertebrate RNA viruses, including HIV-1, and introducing CpGs into RNA virus genomes inhibits their replication. The zinc-finger antiviral protein (ZAP) binds regions of viral RNA containing CpGs and targets them for degradation. ZAP does not have enzymatic activity and recruits other cellular proteins to inhibit viral replication. Here we show that KHNYN, a protein with no previously known function, interacts with ZAP. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides and this requires ZAP and its cofactor TRIM25. KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity. Crucially, depletion of KHNYN eliminated the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production indicating that KHNYN is required for this antiviral pathway. Overall, we have identified KHNYN as a novel ZAP cofactor that is essential for innate immune destruction of CpG containing viral RNA.

scholarly journals KHNYN is essential for the zinc finger antiviral protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mattia Ficarelli ◽  
Harry Wilson ◽  
Rui Pedro Galão ◽  
Michela Mazzon ◽  
Irati Antzin-Anduetza ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Infectious Virus ◽  
Rna Virus ◽  
Leukemia Virus ◽  
Virus Production ◽  
Antiviral Protein ◽  
Cpg Dinucleotides ◽  
Endonuclease Domain ◽  
Virus Genomes ◽  
Hiv 1

CpG dinucleotides are suppressed in most vertebrate RNA viruses, including HIV-1, and introducing CpGs into RNA virus genomes inhibits their replication. The zinc finger antiviral protein (ZAP) binds regions of viral RNA containing CpGs and targets them for degradation. ZAP does not have enzymatic activity and recruits other cellular proteins to inhibit viral replication. We found that KHNYN, a protein with no previously known function, interacts with ZAP. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides and this requires ZAP and its cofactor TRIM25. KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity. Crucially, depletion of KHNYN eliminated the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production and also enhanced the production of murine leukemia virus. Overall, we have identified KHNYN as a novel cofactor for ZAP to target CpG-containing retroviral RNA for degradation.

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

2019 ◽  
Vol 94 (6) ◽  
Author(s):  
Mattia Ficarelli ◽  
Irati Antzin-Anduetza ◽  
Rupert Hugh-White ◽  
Andrew E. Firth ◽  
Helin Sertkaya ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Zinc Finger ◽  
Splice Site ◽  
Viral Genome ◽  
Rna Virus ◽  
Antiviral Protein ◽  
Cpg Dinucleotides ◽  
Viral Genomes ◽  
Hiv 1 ◽  
Cpg Suppression

ABSTRACT 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.

scholarly journals Characterization of HIV-1 Infection and Innate Sensing in Different Types of Primary Human Monocyte-Derived Macrophages

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Elisabeth A. Diget ◽  
Kaja Zuwala ◽  
Randi K. Berg ◽  
Rune R. Laursen ◽  
Stine Søby ◽  
...  
Keyword(s):  
Virus Transmission ◽  
Virus Production ◽  
Human Monocyte ◽  
Innate Immune ◽  
Viral Reservoir ◽  
Low Grade ◽  
Interferon Stimulated Genes ◽  
Differentiation Protocol ◽  
Macrophage Colony ◽  
Hiv 1

Macrophages play an important role in human immunodeficiency virus (HIV) pathogenesis and contribute to establishment of a viral reservoir responsible for continuous virus production and virus transmission to T cells. In this study, we investigated the differences between various monocyte-derived macrophages (MDMs) generated through different differentiation protocols and evaluated different cellular, immunological, and virological properties. We found that elevated and persistent HIV-1 pWT/BaL replication could be obtained only in MDMs grown in RPMI containing macrophage colony-stimulating factor (M-CSF). Interestingly, this MDM type was also most responsive to toll-like receptor stimulation. By contrast, all MDM types were activated to a comparable extent by intracellular DNA, and the macrophage serum-free medium-(Mac-SFM-)differentiated MDMs responded strongly to membrane fusion through expression of CXCL10. Finally, we found that HIV infection of RPMI/M-CSF-differentiated MDMs induced low-grade expression of two interferon-stimulated genes in some donors. In conclusion, our study demonstrates that the differentiation protocol used greatly influences the ability of MDMs to activate innate immune reactions and support HIV-1 replication. Paradoxically, the data show that the MDMs with the strongest innate immune response were also the most permissive for HIV-1 replication.

scholarly journals Excessive RNA Splicing and Inhibition of HIV-1 Replication Induced by Modified U1 Small Nuclear RNAs

2010 ◽  
Vol 84 (24) ◽  
pp. 12790-12800 ◽  
Author(s):  
Dibyakanti Mandal ◽  
Zehua Feng ◽  
C. Martin Stoltzfus
Keyword(s):  
Rna Splicing ◽  
Virus Production ◽  
Viral Rna ◽  
Splice Sites ◽  
Rna Transcripts ◽  
Cellular Factors ◽  
Novel Strategy ◽  
Definition Of ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT HIV-1 RNA undergoes a complex splicing process whereby over 40 different mRNA species are produced by alternative splicing. In addition, approximately half of the RNA transcripts remain unspliced and either are used to encode Gag and Gag-Pol proteins or are packaged into virions as genomic RNA. It has previously been shown that HIV-1 splicing is regulated by cis elements that bind to cellular factors. These factors either enhance or repress definition of exons that are flanked by the HIV-1 3′ splice sites. Here we report that expression of modified U1 snRNPs with increased affinity to HIV-1 downstream 5′ splice sites and to sequences within the first tat coding exon act to selectively increase splicing at the upstream 3′ splice sites in cotransfected 293T cells. This results in a decrease of unspliced viral RNA levels and an approximately 10-fold decrease in virus production. In addition, excessive splicing of viral RNA is concomitant with a striking reduction in the relative amounts of Gag processing intermediates and products. We also show that T cell lines expressing modified U1 snRNAs exhibit reduced HIV-1 replication. Our results suggest that induction of excessive HIV-1 RNA splicing may be a novel strategy to inhibit virus replication in human patients.

scholarly journals Inhibition of Cellular Autophagy Deranges Dengue Virion Maturation

2012 ◽  
Vol 87 (3) ◽  
pp. 1312-1321 ◽  
Author(s):  
Roberto Mateo ◽  
Claude M. Nagamine ◽  
Jeannie Spagnolo ◽  
Ernesto Méndez ◽  
Michael Rahe ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Virus Production ◽  
Innate Immune ◽  
Viral Rna ◽  
Virus Particles ◽  
Cellular Autophagy ◽  
Autophagy Pathway ◽  
Viral Maturation ◽  
Rna Accumulation ◽  
Selection Of

ABSTRACTAutophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 (specific andpotentautophagyinhibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatment-resistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.

scholarly journals Pokeweed antiviral protein alters splicing of HIV-1 RNAs, resulting in reduced virus production

RNA ◽  
2014 ◽  
Vol 20 (8) ◽  
pp. 1238-1247 ◽  
Author(s):  
Alice Zhabokritsky ◽  
Sheila Mansouri ◽  
Katalin A. Hudak
Keyword(s):  
Virus Production ◽  
Pokeweed Antiviral Protein ◽  
Antiviral Protein ◽  
Hiv 1

scholarly journals Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

2018 ◽  
Author(s):  
Adrian Viehweger ◽  
Sebastian Krautwurst ◽  
Kevin Lamkiewicz ◽  
Ramakanth Madhugiri ◽  
John Ziebuhr ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Virus ◽  
Consensus Sequence ◽  
Full Length ◽  
Viral Rna ◽  
Genome Replication ◽  
Nanopore Sequencing ◽  
Human Coronavirus ◽  
Viral Rnas ◽  
Virus Genomes

Sequence analyses of RNA virus genomes remain challenging due to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called 'quasispecies'. Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (i) RNA virus genomes and (ii) subgenome-length (sg) RNAs comprised of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. Using DRS, we were able to map the longest (~26 kb) contiguous read to the viral reference genome. By combining Illumina and nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV) 229E genome (27.3 kb). Furthermore, using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by demonstrating the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that direct RNA sequencing may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

scholarly journals Cellular Factors Targeting HIV-1 Transcription and Viral RNA Transcripts

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 495
Author(s):  
Rayhane Nchioua ◽  
Matteo Bosso ◽  
Dorota Kmiec ◽  
Frank Kirchhoff
Keyword(s):  
Defense Mechanisms ◽  
Viral Gene Expression ◽  
Immune Defense ◽  
Viral Rna ◽  
Viral Gene ◽  
Restriction Factors ◽  
Antiviral Protein ◽  
Rna Transcripts ◽  
Cellular Factors ◽  
Hiv 1

Restriction factors are structurally and functionally diverse cellular proteins that constitute a first line of defense against viral pathogens. Exceptions exist, but typically these proteins are upregulated by interferons (IFNs), target viral components, and are rapidly evolving due to the continuous virus–host arms race. Restriction factors may target HIV replication at essentially each step of the retroviral replication cycle, and the suppression of viral transcription and the degradation of viral RNA transcripts are emerging as major innate immune defense mechanisms. Recent data show that some antiviral factors, such as the tripartite motif-containing protein 22 (TRIM22) and the γ-IFN-inducible protein 16 (IFI16), do not target HIV-1 itself but limit the availability of the cellular transcription factor specificity protein 1 (Sp1), which is critical for effective viral gene expression. In addition, several RNA-interacting cellular factors including RNAse L, the NEDD4-binding protein 1 (N4BP1), and the zinc finger antiviral protein (ZAP) have been identified as important immune effectors against HIV-1 that may be involved in the maintenance of the latent viral reservoirs, representing the major obstacle against viral elimination and cure. Here, we review recent findings on specific cellular antiviral factors targeting HIV-1 transcription or viral RNA transcripts and discuss their potential role in viral latency.

Expression of an RNA glycosidase inhibits HIV-1 transactivation of transcription

2017 ◽  
Vol 474 (20) ◽  
pp. 3471-3483 ◽  
Author(s):  
Meherzad Kutky ◽  
Katalin A. Hudak
Keyword(s):  
Mechanistic Explanation ◽  
Viral Rna ◽  
Mrna Levels ◽  
Activator Protein 1 ◽  
Tat Protein ◽  
Antiviral Protein ◽  
Jnk Pathway ◽  
Culture Cells ◽  
Virus Transcription ◽  
Hiv 1

HIV-1 (human immunodeficiency virus) transcription is primarily controlled by the virally encoded Tat (transactivator of transcription) protein and its interaction with the viral TAR (transcription response element) RNA element. Specifically, binding of a Tat-containing complex to TAR recruits cellular factors that promote elongation of the host RNA polymerase engaging the viral DNA template. Disruption of this interaction halts viral RNA transcription. In the present study, we investigated the effect of pokeweed antiviral protein (PAP), an RNA glycosidase (EC#: 3.2.2.22) synthesized by the pokeweed plant (Phytolacca americana), on transcription of HIV-1 mRNA. We show that co-expression of PAP with a proviral clone in culture cells resulted in a Tat-dependent decrease in viral mRNA levels. PAP reduced HIV-1 transcriptional activity by inhibiting Tat protein synthesis. The effects of PAP expression on host factors AP-1 (activator protein 1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) and specificity protein 1, which modulate HIV-1 transcription by binding to the viral LTR (5′-long terminal repeat), were also investigated. Only AP-1 showed a modest JNK pathway-dependent increase in activity in the presence of PAP; however, this activation was not sufficient to significantly enhance transcription from a partial viral LTR containing AP-1 binding sites. Therefore, the primary effect of PAP on HIV-1 transcription is to reduce viral RNA synthesis by decreasing the abundance of Tat. These findings provide a mechanistic explanation for the observed decrease in viral RNAs in cells expressing PAP and contribute to our understanding of the antiviral effects of this plant protein.

Sign in / Sign up

Export Citation Format

Share Document