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

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.

CpG-creating mutations are costly in many human viruses

“…(1) Our results for A → G and T → C mutations are very similar, if this was due to a mutation rate effect, it would have to have the same effect on both of these mutation types. (2) Our results are consistent with results from epidemiological studies on polio (Stern et al 2017) and in vitro studies on HIV (Takata et al 2017;Ficarelli et al 2019). Future studies will hopefully measure viral mutation rates with more precision.…”

“…It is not entirely clear why CpG sites are costly, but it is likely, at least in part, because the mammalian immune system uses CpG sites to recognize foreign genetic material (Murphy and Weaver 2016). Recently it was shown that ZAP proteins, which inhibit the proliferation of most RNA viruses, are more effective when the CpG sites were common (Takata et al 2017;Ficarelli et al 2019).…”

Evidence for strong mutation bias towards, and selection against, T/U content in SARS-CoV2: implications for attenuated vaccine design

“…The codon pair bias attenuation effect has more recently been shown to be largely owing to increased CpG content (Tulloch, et al 2014;Gaunt, et al 2016). This is very likely to relate to the activity of the human Zinc Antiviral Protein (ZAP) as this targets transcripts with high CpG content (Takata, et al 2017;Ficarelli, et al 2020), although it is by no means the only antiviral peptide ( Supplementary Table 1). As might be expected, ZAP is under positive selection owing to host-parasite coevolution (Kerns, et al 2008).…”

Cellular Factors Targeting HIV-1 Transcription and Viral RNA Transcripts

“…ZAP-L has been reported to represent the most antivirally active isoform but is not significantly upregulated by IFN, while the shorter ZAP-S also exerts antiviral activity and is IFN-inducible but may actually act as a negative feedback regulator of the interferon response [95]. Depletion of ZAP reduced the inhibitory effect of type I IFN on CpG-enriched HIV-1, suggesting that this factor contributes to the antiviral IFN response [96]. Importantly, ZAP needs cofactors to exert antiviral activity.…”

“…Notably, the inhibitory effect of ZAP on HIV-1 and other primate lentiviruses is not only determined by CpG frequency. Using different approaches, i.e., artificial CpG-enrichment in specific regions of the HIV-1 genome [96] and analyses of a large panel of primary infectious molecular clones of HIV-1 [100], two recent studies showed that the CpG frequency in first part of the viral env gene, rather than the overall content, determines ZAP sensitivity. The latter study also showed that the genomes of different primate lentiviruses differ substantially in CpG frequencies, and that the magnitude of suppression does not correlate with ZAP sensitivity, suggesting possible viral evasion or counteraction mechanisms.…”