There are essentially two approaches for identifying novel host processes involved in antiviral functions and that can be exploited therapeutically. One is to learn as much as possible about the host mechanisms required by the virus and then test the effects of inhibiting them. The other is to take an unbiased approach and screen for chemical inhibitors of virus functions, or host genes required by the virus. Taking the chemical genetics version of the second approach, we conducted a screen for compounds that antagonized the inhibition of gene expression by NS1 and identified napthalimides that inhibited replication of influenza viruses and vesicular stomatitis virus. These compounds functioned by increasing expression of REDD1, a major negative regulator of the mTORC1 pathway, and in cells lacking REDD1, the compound lost its antiviral activity.
Many viruses activate AKT through stimulating PI3K 28,29
. The NS1 protein of influenza virus directly binds PI3K, resulting in activation of AKT 30–33
. This has been interpreted either as functioning to inhibit apoptosis, preventing the cell from dying prematurely during infection, or as necessary in some way to promote virus replication. A recent genome-wide siRNA screen implicated mTORC1 in influenza virus replication34
, suggesting that activating that pathway might be one of the functions of elevated AKT1 signaling. Our results imply that an important consequence of AKT signaling for influenza virus replication is activation of the mTORC1 effector S6K through phosphorylation, as the anti-viral napthalimides we identified inhibited phosphorylation of p70 S6K by mTORC1. We showed that the protein up-regulated by our napthalimides, the mTORC1 inhibitor REDD1, is a novel host defense factor. REDD1 was induced by influenza virus or VSV, but was then successfully suppressed by the virus. REDD1 suppression by viruses promoted virus replication as REDD1 knock-out cells were highly permissive to virus replication.
REDD1 is induced by various environmental conditions, including cell confluency, glucocorticoid treatment, hypoxia, and other stress-response pathways, such as ER (endoplasmic reticulum) stress 35
. Both ER stress and the hypoxia inducible factor (HIF) play a role in immunity and infection36,37
. ER stress was shown to promote plasma cell development, and absence of key components of this pathway results in sensitization to viral infection36
. Mouse embryonic fibroblasts deficient in the ER protein kinase PERK/PEK, which is activated by accumulated unfolded proteins in the ER, are more permissive to VSV replication than wild-type cells 38
. Up-regulation of REDD1 in response to ER stress39,40
occurs via the transcription factor ATF440
. HIF activation by the hypoxia mimetic cobalt chloride promotes cellular resistance to VSV infection, whereas inhibition of HIF activity by RNAi or by a small molecule antagonist showed increased sensitivity to viral infection as measured by enhanced VSV cytotoxicity and replication37
; however, the mechanism is not known. During hypoxia, REDD1 was shown to be a direct target of the HIF-1 alpha transcription factor20
, which induces REDD1 expression. Thus, activating a stress response pathway or promoting the expression of a stress response protein to a certain extent may induce resistance to pathogens and decrease host cytotoxicity. However, coordination of a stress response to promote cellular resistance without significantly damaging the host upon pathogen invasion remains to be further investigated. We showed that induction of REDD1 by small molecules is an efficient strategy for interfering with the functions of the mTORC1 pathway that are required by viruses.
The effect of the napthalimide on influenza virus was a sharp attenuation of the production of virus proteins early in infection. We found no effect of the napthalimide on global protein synthesis and no induction of an interferon response. In addition, in cells lacking REDD1, in which expression of influenza virus proteins is enhanced, rapamycin inhibited expression of influenza virus proteins at a concentration in which bulk protein synthesis is known to be unaltered. Thus, this indicates selective translational regulation, which has been documented in a number of conditions, including the general amino acid control response41
and other types of processes, such as survival and proliferation42
. In addition, during nuclear mRNA processing and export, specific sequences either within the UTRs or in the coding region can dictate differential binding by RNA-binding proteins (hnRNPs), which will regulate processing and export of specific subsets of mRNAs, resulting in differential expression43,44
. This raises the possibility that the inhibition of the mTORC1 pathway may alter translation in some way unfavorable to initiation of specific viral mRNAs relative to host messages. In cells infected at a low multiplicity of infection, the first viral messages must compete with the far larger volume of host messages for access to ribosomes. In this respect, the early viral messages would have the same problem as a host cell message of low abundance, such as mRNAs encoding certain transcription factors. However, at the earliest phases of infection, viruses are largely dependent upon normal host processes and it is these processes that are likely to be the most useful therapeutic targets.
Although many viruses can be controlled by vaccination, there is still an important need for antiviral drugs. For viruses, such as influenza virus, that can infect other animals, vaccination will never eradicate the virus. Other viruses, such as small pox or measles can potentially be eradicated by global immunization. However, once the incidence of disease is very low, global vaccination is inevitably discontinued, leaving the human population vulnerable to reemergence of the virus. The long lead times required to produce sufficient vaccine to protect the human population means that appearance of a new or re-occurring highly infectious virus can lead to a pandemic of disease before the vaccine is available. However, antiviral drugs that target viral proteins have the disadvantage that resistance to the drug will arise due to the high rates of mutation inherent in viruses and the large numbers of progeny that they produce. A strategy targeting host processes that are essential for virus replication, such as the one discussed here, would avoid this problem, although it would be limited by the possibility of toxic side effects. Thus, combinations of non-cytotoxic small molecules that target both viral and host proteins are desirable. Recently, influenza A nucleoprotein was identified as an antiviral target45
. A small molecule that triggered nucleoprotein aggregation and prevented its import into the nucleus protected against influenza virus replication45
. In addition, a chemical compound that inhibited host pyrimidine biosynthesis has been recently shown to reduce influenza virus replication46
In sum, the strategy of chemically inducing host antiviral activities targeting host pathways without causing significant short-term toxic effects will likely have an important impact in antiviral therapy. One such strategy was identified here with the induction of the mTORC1 inhibitor REDD1 by naphthalimides. Furthermore, small molecules that inhibit the mTORC1 pathway in different ways have the potential for anti-cancer therapy as the mTORC1 pathway is a major regulator of cell proliferation and cancer 47