APOBEC3G-Regulated Host Factors Interfere with Measles Virus Replication: Role of REDD1 and Mammalian TORC1 Inhibition

Abstract

We found earlier that ectopic expression of the cytidine deaminase APOBEC3G (A3G) in Vero cells inhibits measles virus (MV), respiratory syncytial virus, and mumps virus, while the mechanism of inhibition remained unclear. A microarray analysis revealed that in A3G-transduced Vero cells, several cellular transcripts were differentially expressed, suggesting that A3G regulates the expression of host factors. One of the most upregulated host cell factors, REDD1 (regulated in development and DNA damage response-1, also called DDIT4), reduced MV replication ∼10-fold upon overexpression in Vero cells. REDD1 is an endogenous inhibitor of mTORC1 (mammalian target of rapamycin complex-1), the central regulator of cellular metabolism. Interestingly, rapamycin reduced the MV replication similarly to REDD1 overexpression, while the combination of both did not lead to further inhibition, suggesting that the same pathway is affected. REDD1 silencing in A3G-expressing Vero cells abolished the inhibitory effect of A3G. In addition, silencing of A3G led to reduced REDD1 expression, confirming that its expression is regulated by A3G. In primary human peripheral blood lymphocytes (PBL), expression of A3G and REDD1 was found to be stimulated by phytohemagglutinin (PHA) and interleukin-2. Small interfering RNA (siRNA)-mediated depletion of A3G in PHA-stimulated PBL reduced REDD1 expression and increased viral titers, which corroborates our findings in Vero cells. Silencing of REDD1 also increased viral titers, confirming the antiviral role of REDD1. Finally, pharmacological inhibition of mTORC1 by rapamycin in PHA-stimulated PBL reduced viral replication to the level found in unstimulated lymphocytes, indicating that mTORC1 activity supports MV replication as a proviral host factor. Knowledge about host factors supporting or restricting virus replication is required for a deeper understanding of virus-cell interactions and may eventually provide the basis for therapeutic intervention. This work was undertaken predominantly to explain the mechanism of A3G-mediated inhibition of MV, a negative-strand RNA virus that is not affected by the deaminase activity of A3G acting on single-stranded DNA. We found that A3G regulates the expression of several cellular proteins, which influences the capacity of the host cell to replicate MV. One of these, REDD1, which modulates the cellular metabolism in a central position by regulating the kinase complex mTORC1, was identified as the major cellular factor impairing MV replication. These findings show interesting aspects of the function of A3G and the dependence of the MV replication on the metabolic state of the cell. Interestingly, pharmacological inhibition of mTORC1 can be utilized to inhibit MV replication in Vero cells and primary human peripheral blood lymphocytes.

Global analysis of polysome-associated mRNA in vesicular stomatitis virus infected cells

“…Among the 138 cellular mRNAs that exhibit reduced polysome association following infection was DNA-damage inducible transcript 4 (DDIT4) which encodes regulated in development and DNA-damage response 1 (Redd1) [61][62][63]. Existing studies demonstrate that DDIT4/Redd1 restricts the replication of negative-strand RNA viruses, including VSV.…”

“…Several host mRNAs increased in polysome association include genes with known "proviral" functions for entry and replication including heparan sulfate, clathrin, and HSP90 [56,[72][73][74]. Similarly, host mRNAs with decreased polysome association included genes with published roles in restricting VSV replication such as interferon regulatory factor 1 (IRF1), DDIT4, and TXNIP [62,63,75]. It is difficult to definitively determine whether this reflects evolution of the virus to contend with the environment in which it finds itself or a bona-fide pro and antiviral effect of a given host protein.…”

KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport

“…Real time qPCR showed a tendency of increased KDELR2 mRNA expression in Vero-A3G cells when compared to Vero-023 cells (empty vector control) (Figure 1A). Accordingly, the protein expression of KDELR2 was increased in Vero-A3G cells 1.3-fold [2] (Figure 1B, lane 2). To investigate the potential role of KDELR2 in the inhibition of MV replication, Vero cells were transduced with a KDELR2 expressing lentiviral vector.…”

“…Measles virus (MV) replication is reduced by more than 90% in ectopically A3G expressing Vero cells [1]. We recently described that one of the A3G regulated host cell factors, REDD1 (regulated in development and DNA damage response-1), affects MV replication via regulation of mTORC1 [2]. Another host cell factor, KDELR2, was significantly increased upon the ectopic expression of A3G [2], requiring closer investigation.…”

“…We recently described that one of the A3G regulated host cell factors, REDD1 (regulated in development and DNA damage response-1), affects MV replication via regulation of mTORC1 [2]. Another host cell factor, KDELR2, was significantly increased upon the ectopic expression of A3G [2], requiring closer investigation. The KDELR family of receptors plays an important role in the retrieval of ER resident chaperones containing the C-terminal tetra peptide sequence KDEL (Lys-Asp-Glu-Leu) [3].…”