Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2021.09.29.462334

Enhancement of alphavirus replication in mammalian cells at sub-physiological temperatures

Guo, J.·Harris, M.
View original source ↗Export metadata ↓

Abstract

Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes mosquitoes, causing fever, rash and arthralgia in mammals. The function of the CHIKV non-structural protein 3 (nsP3) remains enigmatic. Building on previous studies (Gao et al, 2019) [1], we generated a panel of mutants in a conserved and surface-exposed cluster in the nsP3 alphavirus unique domain (AUD) and tested their replication using a sub-genomic replicon (SGR). Three of these SGR mutants replicated well in mosquito cells but poorly in mammalian cells. We observed that this difference was due to culture temperature (mosquito cells: 28{degrees}C; mammalian cells: 37{degrees}C), as the mutants exhibited no replication defect in mammalian cells grown at a sub-physiological temperature (28{degrees}C). Similar phenotypes were observed for infectious CHIKV and the closely related ONyong Nyong virus. Intriguingly, the wildtype SGR replicated more efficiently in mammalian cells at 28{degrees}C compared to 37{degrees}C. To explore the mechanism behind this difference, we focused on two known antiviral pathways: interferon-stimulated genes (ISGs) and stress granules (SGs). SGR replication was concomitant with increased expression of ISGs at 37{degrees}C, but this response was impaired at 28{degrees}C. We also observed enhanced recruitment of the SG component G3BP1 into cytoplasmic sites of viral genome replication at 28{degrees}C. These findings may have real-world implications as when a mosquito bites a mammal, the virus first infects cells in peripheral tissues which are often at sub-physiological temperatures. We propose that alphaviruses such as CHIKV have evolved mechanisms to both promote viral genome replication and concomitantly limit antiviral responses in these cells. Significance statementChikungunya virus (CHIKV) is a re-emerging arbovirus that is transmitted by Aedes mosquitos and poses epidemic threats. Arboviruses must be able to grow efficiently in both the mosquito vector and the mammalian host, which have different body temperatures. Following a mosquito bite, the first cells infected are typically in the skin and are at sub-physiological temperatures, ie lower than the internal body temperature (37{degrees}C). Here we show that CHIKV grows more efficiently in mammalian cells cultured at 28{degrees}C, compared to 37{degrees}C. We investigated the mechanism behind this increase in growth efficiency and demonstrated that at the lower temperature the production of antiviral genes was impaired. Additionally, proteins that respond to cellular stress and are needed for virus replication were more abundantly recruited to sites in the cell where the virus was replicating. We propose that CHIKV has evolved mechanisms to promote its replication in mammalian cells at sub-physiological temperatures to facilitate infection of mammals via a mosquito bite.

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Guo, J., Harris, M.. 2021-09-29. Enhancement of alphavirus replication in mammalian cells at sub-physiological temperatures. https://doi.org/10.1101/2021.09.29.462334

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A conserved cysteine-histidine-glutamate metal site identifies DUF501 (Rv1025), an essential uncharacterised protein family of Mycobacterium tuberculosis, as a candidate metalloenzyme and drug target

A substantial fraction of the Mycobacterium tuberculosis proteome remains functionally uncharacterised. Rv1025, a 155-residue protein carrying the domain of unknown function DUF501 (Pfam PF04417), is essential by transposon mutagenesis and vulnerable by CRISPR interference, an attractive but neglected drug target, yet has never been functionally described. The family (4,370 proteins, no Gene Ontology term, no solved structure) is uncharacterised across all organisms and essential in three Actinobacterial genera. A Foldseek search of the AlphaFold model against complete structural databases finds no significant homolog, indicating a novel fold. The operon eno-divIC-Rv1025-ppx2 is conserved across the Actinobacteria phylum, yet AlphaFold-Multimer finds no direct complex between Rv1025 and its neighbour DivIC. Instead, conservation across 8,700 homologous sequences reveals a near-invariant Cys113-His115-Glu59 cluster forming a pocket. Holo AlphaFold3 predictions with Zn, Fe and Mn confidently place a divalent metal on this triad at 2.25-2.47 A; mutating the triad relocates the metal, and an independent backbone-geometry predictor recovers the same site, confirming specificity. The triad is universal across the family: present in all 1,472 near-complete bacterial sequences of the Pfam alignment, with no non-conservative substitution among the 2,228 sequences examined, a defining feature of bacterial DUF501 rather than a mycobacterial peculiarity. We propose that DUF501 is a metal-binding protein and candidate metalloenzyme, the first functional hypothesis for this family, whose conserved, essential metal pocket is a promising drug target. As the predictions build on a conservation-defined site within a fully computational study, they are supportive rather than proof of metal occupancy and warrant experimental validation.

microbiology↗
bioRxiv2026

Mycoplasmal endosymbionts of Trichomonas vaginalis are associated with reduced risk for Chlamydia trachomatis endometrial infection in asymptomatic, coinfected, women.

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, the most common curable non-viral sexually transmitted infection, and Chlamydia trachomatis is a bacterial pathogen that can ascend to the upper genital tract and cause pelvic inflammatory disease, infertility, and ectopic pregnancy. T. vaginalis harbors bacterial endosymbionts, including Candidatus Malacoplasma girerdii, an obligate symbiont, and Metamycoplasma hominis, which can live freely or symbiotically. In a 16S rRNA sequencing study of the cervicovaginal microbiome of women at high risk for chlamydial infection, Ca. M. girerdii abundance was one of 13 features predicting lack of chlamydial spread to the endometrium, despite no direct association between T. vaginalis infection and reduced chlamydial ascension. Investigating the relationship between these microorganisms further, we found that T. vaginalis vaginal abundance correlated positively with chlamydial burden in women whose infection was confined to the cervix, while a nonsignificant inverse relationship was seen in women with endometrial spread. Among participants with high chlamydial burden, Ca. M. girerdii was detected exclusively in women without endometrial infection. Both endosymbionts trended toward more frequent detection, and higher abundance, in coinfected women without endometrial spread, while M. hominis abundance correlated strongly with T. vaginalis burden in this group. These findings suggest that mycoplasmal endosymbionts of T. vaginalis, rather than T. vaginalis itself, are microbial factors limiting chlamydial ascension, and point to a three-way interaction between parasite, endosymbiont, and bacterial pathogen that shapes upper genital tract C. trachomatis infection risk.

microbiology↗
bioRxiv2026

Understanding the physiological alterations of Vibrio cholerae upon exposure to L-ascorbic acid

The scourge of cholera remains a major global public health threat. It affects up to 4 million people worldwide and causes tens of thousands of deaths each year. The disease is experiencing a concerning resurgence in many parts of Africa, the Middle East, and Asia. To effectively tackle cholera and circumvent rising antimicrobial resistance, targeted biological and preventive approaches, complementing traditional rehydration, are urgently needed. In this regard, our group has demonstrated the efficacy of L-ascorbic acid in controlling the growth and pathogenesis of Vibrio cholerae in vitro. The present work further provides a mechanistic elucidation of the L-ascorbic acid-mediated physiological changes in V. cholerae and also bolsters such a non-antibiotic approach to control cholera.

microbiology↗