Efficient functional screening of a cellular cDNA library to identify severe fever with thrombocytopenia syndrome virus entry factors

Abstract

The identification of host cell factors for virus entry is useful for the molecular explanation of viral tropisms and often leads to a more profound understanding of virus-induced diseases. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus. No countermeasures against the disease exist. In this report, we show an efficient method using virus-like particles for the functional screening of a cellular cDNA library to identify SFTS virus entry factors. Two variants encoding dendritic cell-specific ICAM-3 grabbing non-integrin related (DC-SIGNR), a calciumdependent lectin known to enhance SFTS virus infection, were successfully identified from a human liver cDNA library. We will discuss applications for yet unidentified factor(s) for SFTS virus entry and for entry factor(s) for other viruses related to SFTS virus.Among different organisms, many similar but non-identical cellular molecules exist that show the same functions. Even in a single species, the expressed molecules are dependent on cell types. This is the case with virus entry factors, which are cellular components involved in viral attachment to cells and invasion into cells for viral replication 1,2 . Thus, viral infections show specificity with regards to host ranges, tissues, and cell types, indicating that the identification of virus entry factors is useful for the molecular explanation of viral tropisms 3 . Because target tissues or cell types of viruses are strongly associated with virus-induced diseases, the identification of virus entry factors often leads to a more profound understanding of the associated diseases. Furthermore, the identification of viral entry factors might aid the efficient development of countermeasures against the induced diseases 4,5 .Severe fever with thrombocytopenia syndrome (SFTS) is a recently recognised tick-borne infectious disease with a high case fatality rate of up to 30%. SFTS virus, whose species name is Huaiyangshan banyangvirus (see below for details), is the causative agent 6,7 . There are no specific countermeasures against the disease. In SFTS patients, thrombocytopenia and leukopenia are frequently observed and viral antigens are often detected in the lymphoid organs in fatal cases [8][9][10][11] . In vitro, most lymphoid cell lines (e.g., Jurkat and Raji cells, which originate from T and B cells, respectively) show little susceptibility to SFTS virus infection. In contrast, most non-lymphoid cell lines (e.g., Vero cells, which originate from the kidney epithelium) show high susceptibility to the viral infection 8,12-14 . Calcium-dependent (C-type) lectins have been shown to enhance SFTS virus infection when expressed in cells that otherwise show little susceptibility 13,14 ; however, this cannot fully explain the viral tropisms because most non-lymphoid cell lines do not generally express them. Nonmuscle myosin heavy chain IIA (NMMHC-IIA) has been reported as a key entry factor of SFTS virus 15 ; however, the molecule is expressed even in Jurkat cells and ...

An integrated pipeline for mammalian genetic screening

“…Gene perturbation in mammalian cells can be performed either at the endogenous locus of a specific target via effector proteins that activate transcription, such as CRISPR-Cas ( Chavez et al., 2016 ) or exogenously through cDNA expression via inducible or constitutive promoters ( Arnoldo et al., 2014 ). Both CRISPR and cDNA technologies have been leveraged in recent mammalian screens to effectively target genes of interest in an individual or combinatorial manner ( Chavez et al., 2015 , 2016 ; Konermann et al., 2014 ; Qi et al., 2013 ; Horlbeck et al., 2016 ; Bikard et al., 2013 ; Gilbert et al., 2013 ; Zalatan et al., 2015 ; Tak et al., 2017 ; Zhang et al., 2017 ; Liu et al., 2019 ; Yeo et al., 2018 ; Shimojima et al., 2020 ; Parekh et al., 2018 ).…”

Entry of Phenuiviruses into Mammalian Host Cells

“…The same is probably true for the interactions between DC-SIGN and phenuiviruses. In addition, the human C-type lectins L-SIGN and LSECtin, both closely related to DC-SIGN but expressed on the surface of the liver endothelium [55], are also used as receptors by several phenuiviruses, including RVFV, TOSV, UUKV, and DABV (Table 2) [42,43,46,47]. It is possible that L-SIGN and LSECtin, by acting as receptors on the surface of the liver endothelium, contribute to the hepatic tropism of some phenuiviruses.…”

N-glycosylation of viral glycoprotein is a novel determinant for the tropism and virulence of highly pathogenic tick-borne bunyaviruses

“…No deletions were observed in the Hp50-4 strain genome. Based on the determined sequences, plasmids for the reverse genetics of the SFTS virus [ 27 , 41 ] were prepared and used for the preparation of recombinant viruses (recOri for the original and recHp50-4 for Hp50-4) and chimeric viruses (L Hp M Ori whose L and M segments originated from Hp50-4 and the original strains, respectively, and L Ori M Hp whose L and M segments originated from the original and Hp50-4 strains, respectively) ( Fig 2A ). Growth of recOri and recHp50-4 in HeLa cells ( Fig 2B ) and viral antigen distribution in inoculated cells ( Fig 2C ) resembled those of the original and Hp50-4 strains, respectively ( Fig 1A and 1B ).…”

“…In vitro , but not in vivo , SFTS virus proliferates well in various adherent cell lines including epithelial kidney Vero and hepatocellular HuH-7 cell lines but not in lymphoid cell lines including Raji (B cell origin) and Jurkat (T cell origin) [ 23 – 25 ]. It has been reported that the expressions of calcium-dependent lectins (C-type lectins, e.g., DC-SIGN, DC-SIGNR, LSECtin) in lymphoid cell lines increased SFTS virus susceptibility, suggesting that SFTS virus infects macrophages via lectins [ 25 – 27 ]. A recent report on the molecular mechanism(s) of B cell infection suggested increased expression levels of C-C motif chemokine receptor 2, CCR2, were involved [ 28 ].…”