Mol Cell │A new mechanism of antiviral infection independent of the innate immune pathway

Written by | Sister Xian

Innate immunity is the first line of defense against all infectious microorganisms, when the body encounters a viral infection, the abnormal nucleic acid structure encoded by the virus can be "perceived" by the germline-encoded pattern recognition receptor (PRR) family, such as retinoic acid induction gene I (RIG-I)-like receptors (RLR) and Toll-like receptors (TLR), which leads to the activation of certain signaling articulation proteins, stimulating downstream signaling pathways and deficient transcription of various innate immune effectors, such as the typical family of antiviral cytokines.
Interferon type I (IFN), which can function in both autocrine and paracrine ways, upmodulates hundreds of interferon-stimulating genes (ISGs) that inhibit viral infections
[1].
Of course, viruses are also extremely clever, and many have evolved ways to counter and evade IFN signals
.
For example, chikungunya virus (CHIKV), a mosquito-borne virus, has a positive single-stranded RNA genome of about 11.
8 kb that can replicate
in the cytoplasm of the infected host cell.
CHIVV exhibits a broad tendency to infect epithelial, endothelial, and myeloid subsets, leading to diseases characterized by weakness and chronic arthralgia, which can cause acute disease in millions of people, and utilizes effective mechanisms to combat and bypass innate immune pathways, including the IFN pathway [2, 3
].
It can be seen that these known classical signaling pathways have become the central battlefield of a never-ending evolutionary arms competition, where viruses continue to evolve mechanisms to circumvent antiviral immunity, while hosts try to overcome virus-mediated innate immune antagonism
.
However, evil prevails, and no matter how tragic, most viruses will eventually be eliminated, which has to be believed that the body must have some undiscovered additional immunomodulation and function to combat these virus escape strategies
.

Recently, Sara Cherry's team from the University of Pennsylvania published an article titled The lncRNA ALPHA specifically targets chikungunya virus to control infection on Molecular Cell, using high-throughput depletion screening to find cytoplasmic antiviral lncRNA ALPHA induced by A-virus infection.
It specifically inhibits the replication of chikungunya virus (CHIKV) and its close relative Aryalian virus (ONNV) without affecting the replication of other alpha viruses, and confirms that ALPHA can function independently of IFN to prevent viral RNA replication by directly binding to viral genomic RNA, suggesting that ALPHA and other potential lncRNAs can mediate atypical antiviral immune responses
against specific viruses.

The main goal of this study was to find additional mechanisms for host cells to fight against CHIKV strategies to evade innate immunity, so the researchers performed RNA sequencing of human brain microvascular endothelial cells (HBMEC) infected with CHIKV, starting with exploring the transcriptional response of CHIKV infection, with another class of arboviruses Zika virus (ZIKV) as a control to distinguish between virus-specific pathways and universally induced pathways of viral infection (e.
g.
, IFN).

。 The experimental results showed that the total number of transcripts significantly upregulated in CHINKV-infected cells and the relative levels of many induced typical innate immune genes were reduced compared to ZIKV, suggesting that CHIKV circumvents classical signals
to a greater extent than ZIKV.
Notably, the researchers found that lncRNAs make up a large portion of all transcripts induced by infection, and unlike classical immunocoding genes, these lncRNAs are either CHIKV-specific or zikv-specific
.
This suggests that lncRNA may be a key medium
for virus-specific antiviral responses.

To identify lncRNAs with anti-CHIKV activity, the researchers performed high-throughput RNAi screening of 2200 lncRNAs in HBMEC and then infected with CHIKV
that expressed mKate fluorescence reporter during viral replication.
Binding RNA sequencing data, the researchers found 9 potential antiviral lncRNAs that have not yet been characterized, and their deletions can lead to an increase
in the percentage of cells infected with CHIVV 。 After secondary screening and nucleoplasmic isolation and identification, the researchers found one of the lncRNAs located in the cytoplasm and named it ALPHA (Antiviral lncRNA Prohibiting Human Alphaviruses), and confirmed that after knocking down ALPHA in HBMEC, the viral RNA, titer, and percentage of infected cells increased significantly, and overexpression of ALPHA significantly weakened CHIVV in cells.
This confirms that ALPHA is a cytoplasmic lncRNA with strong antiviral activity against CHIKV
.

Further exploring the transcriptional regulation of ALPHA during CHIVV infection, the researchers found that CHIVV infection can effectively induce ALPHA RNA in a dose-dependent manner, and is induced in a specific and viral replication-dependent manner of virus A infection, and is independent of classical cytokines, typical innate immune signaling pathways, and other specific CHIVV-induced pathways
。 In addition, infection with CHIVV in other types of cells (such as primary human monocytes, pulmonary epithelial cancer cells, and osteosarcoma cells) does not induce ALPHA expression, suggesting that ALPHA expression is regulated in a viral and cell type-specific manner
.

So, how exactly does ALPHA attenuate CHIVV infection? First, the researchers found that ALPHA's anti-CHIVV activity is independent of the transcription or translation of IFN and ISG, as well as the IFN signaling pathway
.
It is worth mentioning that the antiviral activity of ALPHA is also highly limited to the closely related A viruses CHIVV and ONNV, while the slightly more distant A viruses or other types of unrelated viruses are not affected
by it.
Next, the researchers explored how ALPHA interferes with the replication cycle of CHIVV
.
The experimental results found that whether it is inhibition of viral translation with cycloheximide (CHX) (the first step in intracellular viral replication) or the administration of ammonium chloride (_NH4Cl) after the virus enters the cell to inhibit the endocytic acidification required for secondary infection, it does not affect the regulation of the virus by ALPHA, indicating that ALPHA activity neither regulates the early entry of the virus nor the spread of the virus, but rather regulates an intracellular step
in the life cycle of CHIKV 。 Further experimental data showed that ALPHA can attenuate the replication of early viral RNA, which is achieved by inhibiting the anti-genome generation of viruses
.
In-depth research confirms that ALPHA can interact with CHIKV genomic RNA, and that this potential interaction occurs in the cytoplasm without the involvement of other cytokines, and that the 3'-terminal of ALPHA's exon 1 is the minimum requirement for binding to the CHIKV genome, mainly within nsp1 at the 5'-end of the CHIKV genome
.


In summary, this study is the first to identify ALPHA as a novel antiviral lncRNA required to identify and control CHIVV infection in the cytoplasm, providing evidence that cytoplasmic lncRNA has direct antiviral activity against closely related viruses, and at the same time proving that host lncRNA can physically interact with the viral genome and disrupt viral replication.
This suggests that lncRNA can play a greater role as an antiviral effector and can directly inhibit viral infection
independently of the typical innate immune response.

Original link:

https://doi.
org/10.
1016/j.
molcel.
2022.
08.
030

Plate Maker: Eleven

References

1.
Chow, K.
T.
, Gale, M.
, Jr.
, and Loo, Y.
M.
(2018).
RIG-I and other RNA sensors in antiviral immunity.
Annu.
Rev.
Immunol.
36, 667–694.

2.
Fox, J.
M.
, and Diamond, M.
S.
(2016).
Immune-mediated protection and pathogenesis of Chikungunya virus.
J.
Immunol.
197, 4210–4218.

3.
Akhrymuk, I.
, Kulemzin, S.
V.
, and Frolova, E.
I.
(2012).
Evasion of the innate immune response: the Old World Alphavirus nsP2 protein induces rapid degradation of Rpb1, a catalytic subunit of RNA polymerase II.
J.
Virol.
86, 7180–7191.

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