[PMC free article] [PubMed] [CrossRef] [Google Scholar] 36. regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein complexes has implications for the study of the pathogenic effects of lingering CHIKV contamination and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne computer virus (arbovirus). IMPORTANCE Chikungunya computer virus (CHIKV) is usually a reemerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No D-(-)-Quinic acid approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Improvements in microscopy and development of noncytotoxic replicons (A. Utt, P. K. Das, M. Varjak, V. Lulla, A. Lulla, A. Merits, J Virol 89:3145C3162, 2015, https://doi.org/10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged nonstructural protein 3 (nsP3). The ability to track nsP3 within the host cell and during prolonged replication can benefit fundamental research efforts to better understand long-term effects of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV contamination and treat chronic disease symptoms. genus, causes a transient illness with debilitating symptoms (fever, headache, rash, myalgia, and arthralgia). Chronic disease is usually common, and joint pain can persist for months to years (1,C3). Half of the patients from your recent Latin American outbreak may develop chronic inflammatory rheumatism, raising the health burden of musculoskeletal disease in areas of endemicity (4, 5). During acute contamination, this cytotoxic computer virus induces apoptosis, leading to direct tissue injury and local inflammation (6,C8). Biopsies have also revealed the persistence of CHIKV antigens and RNA in synovial macrophages and muscle tissue (1, 9). CHIKV also persists in mice and nonhuman primate models (10,C13). Chronic disease may be a consequence of prolonged, replicating, and transcriptionally active CHIKV RNA (13), but an understanding of CHIKV’s long-term effect is still emerging. The 12-kb positive-sense RNA genome of CHIKV encodes four nonstructural proteins, nsP1 to nsP4, which make up the viral replication and transcription complex (Fig. 1A) (reviewed in reference 14). A subgenomic RNA expresses six structural proteins. Cellular responses to infection include D-(-)-Quinic acid apoptosis, interferon signaling, stress granule (SG) formation, unfolded protein response, host cell shutoff, and autophagy (examined in reference 15). Previous research on alphaviruses established the vital role that nsP3 plays in counteracting cellular responses (16,C20) and recognized essential protein-protein interactions between nsP3 and host proteins (16, 21,C23). However, few studies have systematically investigated the long-term effect of persistently replicating CHIKV RNA and continued expression of proteins such as nsP3 on human cells. D-(-)-Quinic acid Although recent studies characterize the CASP3 formation of organelles that contain nsP3 during acute contamination and transient replication (16, 24,C27), a corresponding characterization during prolonged CHIKV replication is usually missing. To address these gaps, we sought to further develop CHIKV replicons capable of prolonged replication in human cells and to harness this system for analysis by subdiffraction multicolor microscopy. Open in a separate windows FIG 1 nsP3 has a granular distribution in stable CHIKV cells and infected HuH-7 cells. (A) Schematic representation of tagged reporter viruses and noncytotoxic replicon encoding D-(-)-Quinic acid SNAP-nsP3. SGP, subgenomic promoter; PAC, puromycin-luciferase (Rluc) flanked by SpeI restriction sites was inserted into nsP3. The SNAP-tagged replicon, which has a SNAP sequence (also flanked by SpeI.