Biology subjects

Kishigami, S.

Publications and source records attributed to Kishigami, S..

6 recordsLinked to original sources

bioRxiv2021

MARCH8 targets cytoplasmic lysine residues of various viral envelope glycoproteins

The host transmembrane protein MARCH8 is a RING finger E3 ubiquitin ligase that downregulates various host transmembrane proteins, such as MHC-II. We have recently reported that MARCH8 expression in virus-producing cells impairs viral infectivity by reducing virion incorporation of not only HIV-1 envelope glycoproteins but also vesicular stomatitis virus G-glycoprotein through two different pathways. However, the MARCH8 inhibition spectrum remains largely unknown. Here, we investigate the antiviral spectrum of MARCH8 using HIV-1 pseudotyped with a variety of viral envelope glycoproteins. Pseudotyping experiments revealed that viral envelopes derived from the rhabdovirus, arenavirus, coronavirus, and togavirus (alphavirus) families were sensitive to MARCH8-mediated inhibition. Lysine mutations at the cytoplasmic tails of rabies virus-G, lymphocytic choriomeningitis virus glycoproteins, SARS-CoV and SARS-CoV-2 spike proteins, and Chikungunya virus and Ross River virus E2 proteins conferred resistance to MARCH8. Immunofluorescence showed impaired downregulation of the mutants of these viral envelopes by MARCH8, followed by lysosomal degradation, suggesting that MARCH8-mediated ubiquitination leads to intracellular degradation of these envelopes. Indeed, rabies virus-G and Chikungunya virus E2 proteins proved to be clearly ubiquitinated. We conclude that MARCH8 has inhibitory activity on a variety of viral envelope glycoproteins whose cytoplasmic lysine residues are targeted by this antiviral factor.

microbiology↗

bioRxiv2020

Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the angiotensin-converting enzyme-2 (ACE2) receptor and is cleaved by transmembrane protease serine 2 (TMPRSS2). However, whether S mutations affect SARS-CoV-2 infectivity remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural observations. Nevertheless, the D614G mutant remains susceptible to neutralization by antisera against prototypic viruses. Taken together, these data indicate that the D614G mutation enhances viral infectivity while maintaining neutralization susceptibility.

microbiology↗

bioRxiv2020

Super-rapid quantitation of the production of HIV-1 harboring a luminescent peptide tag

In virological studies using HIV-1 proviral clones, virus production is normally monitored by either an RT assay or a p24 antigen capture ELISA. However, these assays are costly and time-consuming for routine handling of a large number of HIV-1 samples. In addition, sample dilution is always required in ELISA to determine p24 protein levels because of the very narrow range of detectable concentrations in this assay. Here, we establish a novel HIV-1 production assay system to solve the aforementioned problems by using a recently developed small peptide tag called HiBiT. First, we constructed a novel full-length proviral HIV-1 DNA clone and a lentiviral packaging vector in which the HiBiT tag was added to the C terminus of the integrase. Tagging the integrase with the HiBiT sequence did not impede the production or infectivity of the resultant viruses. Electron microscopy revealed normal morphology of the virus particles. Most importantly, by comparing between ELISA and the HiBiT luciferase assay, we successfully obtained an excellent linear correlation between p24 concentrations and HiBiT-based luciferase activity. Overall, we conclude that HiBiT-tagged viruses can replace the parental HIV-1 and lentiviral vectors, which enables us to perform a super-rapid, inexpensive, convenient, simple, and highly accurate quantitative assay for HIV-1 production.

microbiology↗

bioRxiv2020

MARCH8 inhibits viral infection by two different mechanisms

Membrane-associated RING-CH 8 (MARCH8) inhibits infection with both HIV-1 and vesicular stomatitis virus G-glycoprotein (VSV-G)-pseudotyped viruses by reducing virion incorporation of envelope glycoproteins. The molecular mechanisms by which MARCH8 targets envelope glycoproteins remain unknown. Here, we show two different mechanisms by which MARCH8 inhibits viral infection. Viruses pseudotyped with the VSV-G mutant, in which cytoplasmic lysine residues were mutated, were insensitive to the inhibitory effect of MARCH8, whereas those with a similar lysine mutant of HIV-1 Env remained sensitive to it. Indeed, the wild-type VSV-G, but not its lysine mutant, was ubiquitinated by MARCH8. Furthermore, the MARCH8 mutant, which had a disrupted cytoplasmic tyrosine motif that is critical for intracellular protein sorting, did not inhibit HIV-1 Env-mediated infection, while it still impaired infection by VSV-G-pseudotyped viruses. Overall, we conclude that MARCH8 reduces viral infectivity by downregulating envelope glycoproteins through two different mechanisms mediated by a ubiquitination-dependent or tyrosine motif-dependent pathway.

microbiology↗

bioRxiv2019

Small molecules for modulating protein driven liquid-liquid phase separation in treating neurodegenerative disease

Neurodegenerave diseases such as amyotrophic lateral sclerosis (ALS) are oten associated with mutaons in proteins that are associated with stress granules. Stress granules are condensates formed by liquid-liquid phase separaon which, when aberrant, can lead to altered condensaon behaviours and disease phenotypes. Here, we identified lipoamide, a small molecule which specifically prevents cytoplasmic condensaon of stress granule proteins. Thermal proteome profiling showed that lipoamide preferentially stabilises intrinsically disordered domain-containing proteins. These include SRSF1 and SFPQ, stress granule proteins necessary for lipoamide activity. The redox state of SFPQ correlates with its condensate-dissolving behaviour, in concordance with the importance of the dithiolane ring for lipoamide activity. In animals, lipoamide ameliorates aging-associated aggregaon of a stress granule reporter, improves neuronal morphology, and recovers motor defects caused by expression of ALS-associated FUS and TDP-43 mutants. In conclusion, lipoamide is a well-tolerated small molecule modulator of stress granule condensaon and dissection of its molecular mechanism identified a cellular pathway for redox regulaon of stress granule formaon.

cell biology↗

bioRxiv2019

Hyperspectral environmental illumination maps: Characterizing directional spectral variation in natural environments

Objects placed in real-world scenes receive incident light from every direction, and the spectral content of this light may vary from one direction to another. In computer graphics, environmental illumination is approximated using maps that specify illumination at a point as a function of incident angle. However, to-date, existing public databases of environmental illumination maps specify only three colour channels (RGB). We have captured a new set of 12 environmental illumination maps (eight outdoor scenes; four indoor scenes) using a hyperspectral imaging system with 33 spectral channels. The data reveal a striking directional variation of spectral distribution of lighting in natural environments. We discuss limitations of using daylight models to describe natural environmental illumination.

neuroscience↗