Sindbis virus induced phosphorylation of IRF3 in human embryonic kidney cells is not dependent on mTOR.

The mammalian target of rapamycin (mTOR) plays critical roles in immunity. We previously showed that infection of human embryonic kidney (HEK) cells with Sindbis virus (SIN), an enveloped RNA alphavirus, profoundly suppresses Akt/mTOR signaling, and host translation late during infection. To understand how SIN mediated suppression of mTOR affects innate response, we analyzed phosphorylation of interferon regulatory factor 3 (IRF3) and expression of two antiviral genes. Here we show strong phosphorylation of IRF3, and an increase in mRNA levels for antiviral genes interferon stimulated gene (ISG)56 and interferon gamma inducible protein (IP)-10 when intracellular viral RNA levels are high during late infection. The mTOR inhibitors rapamycin and torin1 do not block, but mildly upregulate these responses. Even after prolonged treatment with Ly294002, the PI3K inhibitor only partially blocks SIN induced phosphorylation of IRF3. While Ly294002 treatment downregulated the SIN induced expression of ISG56 mRNA levels, it had no effect on SIN induced upregulation of IP-10 expression. These results point to SIN replication-mediated activation of IRF3, independent of mTOR function, when host protein synthesis is severely suppressed by virus infection.

Lipopolysaccharide inhibits Sindbis virus-induced IP-10 release in human peripheral blood mononuclear cells.

Chemokines play a pivotal role in the innate response to both bacterial and viral infections, and in mixed infections. To determine chemokine responses to Sindbis virus (SIN) in a co-infection model, peripheral blood mononuclear cells (PBMCs) derived from healthy volunteers were exposed to SIN in the presence and absence of lipopolysaccharide (LPS). Culture supernatants recovered at 2, 24, and 72 h post-exposure were evaluated for virus replication and analyzed for chemokines by ELISA. None of the PBMC cultures showed new virus release, GFP reporter expression, or viral RNA synthesis. While SIN had little effect on the induction of IL-8 and RANTES, the chemokines MCP-1, MIP1-α (p < 0.001), and MIP1-ß (p < 0.0004) were drastically upregulated by SIN as well as LPS. Both live and UV-inactivated SIN induced secretion of IP-10 and I-TAC. Although LPS did not induce release of IP-10, it sharply inhibited (p = 0.004) SIN-mediated IP-10 secretion. On the contrary, the release of SLC was blocked by SIN. The adjuvant activity of IP-10, its antiangiogenic function, and antagonism between SIN and LPS for the release of select chemokines may be useful in understanding the pathogenesis of mixed infections, cross-talk between cellular pathways, and may have applications in cancer and sepsis.

Sindbis virus replication, is insensitive to rapamycin and torin1, and suppresses Akt/mTOR pathway late during infection in HEK cells.

Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002--a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors; (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of eIF2α and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions.

Efficient replication, and evolution of Sindbis virus genomes with non-canonical 3'A/U-rich elements (NC3ARE) in neonatal mice.

Sindbis virus (SIN) is a mosquito-transmitted animal RNA virus. We previously reported that SIN genomes lacking a canonical 19 nt 3'CSE undergo novel repair processes in BHK cells to generate a library of stable atypical SIN genomes with non-canonical 3'A/U-rich elements (NC3AREs) adjacent to the 3' poly(A) tail [1]. To determine the stability and evolutionary pressures on the SIN genomes with NC3AREs to regain a 3'CSE, five representative SIN isolates and a wild type SIN were tested in newborn mice. The key findings of this study are: (a) all six SIN isolates, including those that have extensive NC3AREs in the 3'NTRs, replicate well and produce high titer viremia in newborn mice; (b) 7-9 successive passages of these isolates in newborn mice produced comparable levels of viremia; (c) while all isolates produced only small-sized plaques during primary infection in animals, both small- and large-sized plaques were generated in all other passages; (d) polymerase stuttering occurs on select 3' oligo(U) motifs to add more U residues within the NC3AREs; (e) the S3-8 isolate with an internal UAUUU motif in the 3'poly(A) tail maintains this element even after 9 passages in animals; (f) despite differences in 3'NTRs and variable tissue distribution, all SIN isolates appear to produce similar tissue pathology in infected animals. Competition experiments with wt SIN and atypical SIN isolates in BHK cells show dominance of wt SIN. As shown for BHK cells in culture, the 3'CSE of the SIN genome is not required for virus replication and genome stability in live animals. Since the NC3AREs of atypical SIN genomes are not specific to SIN replicases, alternate RNA motifs of alphavirus genome must confer specificity in template selection. These studies fulfill the need to confirm the long-term viability of atypical SIN genomes in newborn mice and offer a basis for exploring the use of atypical SIN genomes in biotechnology.