Study reveals CMPK-2 protein-coding gene as a potent inhibitor of flaviviruses

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In a recent study published in the journal PLoS Pathogens, researchers in the United States characterized cytidine/uridine monophosphate kinase-2 (CMPK-2) as a host restriction factor, specifically for flaviviruses, that exerts antiviral activity by inhibiting viral ribonucleic acid (RNA) translation.

Study: CMPK2 restricts Zika virus replication by inhibiting viral translation. Image Credit: felipe caparros / ShutterstockStudy: CMPK2 restricts Zika virus replication by inhibiting viral translation. Image Credit: felipe caparros / Shutterstock

Background

Flaviviruses are arthropod-borne viral organisms that continue to pose health threats globally, with no United States (US) Food and Drug Administration (FDA)-authorized therapeutic antiviral therapeutics for flaviviral infections. Therefore, an urgent need exists to detect viral-host interaction factors that can be targeted to develop novel antivirals and vaccines against flaviviruses.

As part of the initial immune defense against infectious pathogens, type I interferons (IFN-I) are produced in the human body in response to microbes. CMPK-2 is a type I IFN-stimulated gene (ISG) with antiviral properties. However, the biological mechanisms underlying the inhibition of viral activity by CMPK-2 are not entirely understood and warrant further research.

About the study

In the present study, researchers investigated CMPK-2 as a potential flavivirus inhibitor that can be targeted to develop novel antivirals against flaviviruses.

The team investigated the antiviral action of CMPK-2 against the Zika virus (ZIKV) and other medically important flaviviruses. The breadth of CMPK2’s antiviral activity was later examined against viruses belonging to the Coronaviridae, Flaviviridae, Herpesviridae, Paramyxoviridae, and Orthomyxoviridae viral families.

For the analysis, CMPK-2 was overexpressed and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9)-mediated CMPK-2 knockout studies were performed using human foreskin fibroblast cells (HFF). CMPK-2 was expressed in doxycycline-inducible Vero E6 cells, and the stringency of the doxycycline-inducible system was validated based on CMPK-2 RNA levels, measured using quantitative reverse-transcription polymerase chain reaction (qRT-PCR).

The IFN-stimulated radical S-adenosyl methionine domain containing 2 (RSAD-2) or viperin gene was used as a control for the experiments. CMPK-2 expression and its sub-cellular localization were verified by immunoblot analysis and confocal microscopy, respectively. In addition, immunofluorescence analysis and flow cytometry were performed.

Viral titers were determined using plaque assays. The team investigated whether CMPK-2 localization in the mitochondria was critical for antiviral activity, for which they generated a CMPK-2 mitochondrial localization sequence deletion mutant (ΔMLS) and transfected 293T cells with vectors expressing the mutant. In addition, the domain of MLS required for antiviral activity was investigated.

Results

A robust increase in CMPK-2 RNA levels was observed in the IFN-I- and doxycycline-treated and CMPK-2-expressing Vero cells, and a considerable elevation in viperin RNA was observed after adding IFN-I. CMPK-2 showed significantly lower staining for the Zika virus envelope protein than non-CMPK-2-expressing Vero cells. The findings indicated that CMPK-2 inhibited Zika virus replication by particularly viral RNA translation.

The loss of interferon-I-induced CMPK-2 gene expression loss among CMPK-2-KO HFF cells and protein expression was verified by Western blotting, indicating that interferon-I-induced CMPK-2 has a significant contribution to the antiviral responses against Zika virus. Additionally, CMPK-2 induction significantly reduced the multiplication of other flaviviruses such as dengue virus serotype-2 (DENV-2), yellow fever virus strain 17D (YFV17D), and Kunjin virus (a subtype of West Nile virus).

However, none of the coronaviruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV-2 expressing mNeonGreen (SARS-CoV-2-mNG), or avian infectious bronchitis virus (IBV) were inhibited by CMPK-2. In addition, CMPK-2 did not protect against influenza A virus (IAV), vesicular stomatitis virus (VSV), herpes simplex virus type 1 (HSV-1), and Newcastle disease virus (NDV).

CMPK-2 required MLS but not the C-terminal kinase domain for antiviral activity. Therefore, CMPK-2’s N-terminal domain (NTD) was adequate for restricting viral RNA translation. The transport of NTD of CMPK-2 by MLS into cellular mitochondria would facilitate the antiviral activity of CMPK-2. Seven preserved residues of cysteine were detected in the NTD, which were critical for the antiviral activity of CMPK-2.

The need for CMPK-2 to enable the IFN-I-regulated limitation of ZIKV proliferation could be partially due to the functional association between CMPK-2 and RSAD-2 since CMPK-2’s kinase domain provides the cytidine triphosphate (CTP) substrate for RSAD-2.

Thus, in the absence of CMPK-2, viperin might not be able to affect Zika virus multiplication due to suboptimal CTP substrate levels to produce didehydro-CTP (ddh-CTP) in sufficient amounts. Therefore, the loss of CMPK-2 activity might attenuate the antiviral functions of two constituents that can influence the replication of flaviviruses.

Overall, the study findings showed that the CMPK-2 gene, which encodes the IFN-stimulated protein cytidylate monophosphate kinase 2, might be a pan-flavivirus inhibitor, given the broad width of antiviral responses against flaviviruses. Developing new antiviral therapies based on ISG functions rather than targeting viral proteins could minimize the emergence of antiviral drug resistance, and the broad spectrum of antiviral activity could improve global preparedness for future viral pandemics.

Journal reference:
Pooja Toshniwal Paharia

Written by

Pooja Toshniwal Paharia

Pooja Toshniwal Paharia is an oral and maxillofacial physician and radiologist based in Pune, India. Her academic background is in Oral Medicine and Radiology. She has extensive experience in research and evidence-based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.

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