Graf1 Controls the Growth of Human Parainfluenza Virus Type 2 through Inactivation of RhoA Signaling

Abstract

Rho GTPases are involved in a variety of cellular activities and are regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We found that the activation of Rho GTPases by lysophosphatidic acid promotes the growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, hPIV-2 infection causes activation of RhoA, a Rho GTPase. We hypothesized that Graf1 (also known as ARHGAP26), a GAP, regulates hPIV-2 growth by controlling RhoA signaling. Immunofluorescence analysis showed that hPIV-2 infection altered Graf1 localization from a homogenous distribution within the cytoplasm to granules. Graf1 colocalized with hPIV-2 P, NP, and L proteins. Graf1 interacts with P and V proteins via their N-terminal common region, and the C-terminal Src homology 3 domain-containing region of Graf1 is important for these interactions. In HEK293 cells constitutively expressing Graf1, hPIV-2 growth was inhibited, and RhoA activation was not observed during hPIV-2 infection. In contrast, Graf1 knockdown restored hPIV-2 growth and RhoA activation. Overexpression of hPIV-2 P and V proteins enhanced hPIV-2-induced RhoA activation. These results collectively suggested that hPIV-2 P and V proteins enhanced hPIV-2 growth by binding to Graf1 and that Graf1 inhibits hPIV-2 growth through RhoA inactivation. IMPORTANCE Robust growth of hPIV-2 requires Rho activation. hPIV-2 infection causesRhoA activation, which is suppressed by Graf1. Graf1 colocalizes with viral RNP (vRNP) in hPIV-2-infected cells. We found that Graf1 interacts with hPIV-2 P and V proteins. We also identified regions in these proteins which are important for this interaction. hPIV-2 P and V proteins enhanced the hPIV-2 growth via binding to Graf1, while Graf1 inhibited hPIV-2 growth through RhoA inactivation. Rho GTPases are members of the Ras superfamily of 20-to 30-kDa small GTPases. They are highly conserved in eukaryotes and act as molecular switches to regulate essential cellular functions. To date, at least 22 members of the Rho GTPases have been identified in mammalian cells (1, 2). The most well characterized members, namely, RhoA, Cdc42, and Rac1, affect a variety of cellular activities, including actin reorganization, apoptosis, intracellular trafficking, and cell polarity (1-5). Rho GTPases regulate cellular activities by coordinating with other host proteins such as focal adhesion kinase (FAK) and Akt. It is important for viruses to establish an environment that facilitates their growth by controlling these cellular activities. Rho GTPases and their related proteins affect the life cycles of some viruses, including respiratory syncytial virus (RSV) (6, 7), Ebola virus (8), vesicular stomatitis virus (8), Epstein-Barr virus (9), influenza A virus (IAV) (10, 11), and rotavirus (12). The relationship between herpesvirus and Rho GTPases has been well investigated (13). We previously reported that Rho activation promotes syncytium formation induced by human parainfluenza virus type 2 (hPIV-2) (14). However, it remains unknown whe...

The role of GTPase-activating protein ARHGAP26 in human cancers

“…The ARHGAP26 molecule includes the GAP domain in the central region, the src homology 3 (SH3) domain in the C segment, and the Bin/amphiphysin/Rvs (BAR) and pleckstrin homology (PH) domains at the N-terminal. These domains are highly conserved among GAPs and GEFs [ 47 , 48 ]. The GAP domain mainly inactivates Rho GTPases by enhancing the hydrolysis of GTPases [ 1 , 2 ].…”

Inhibition of Cavin3 Degradation by the Human Parainfluenza Virus Type 2 V Protein Is Important for Efficient Viral Growth

“…Although V protein is not essential for hPIV-2 replication, the growth of V-deficient hPIV-2 is remarkably decreased relative to wt hPIV-2 (Nishio et al, 2005;Ohta et al, 2016b) (Table 1). V protein has been found to interact with several host proteins, such as STATs (Nishio et al, 2005), AIP1/Alix (Nishio et al, 2007), TRAF6 (Kitagawa et al, 2013), tetherin (Ohta et al, 2016b), Graf1 (Ohta et al, 2016a), caspase1 (Ohta et al, 2018a), inactive RhoA (Ohta et al, 2018c), and profilin2 (Ohta et al, 2019). Most of these V partners interact with the C-terminal region of V protein containing three Trp and seven Cys residues that form a bowl-shaped depression (Figure 1) (Li et al, 2006).…”

“…Most of these V partners interact with the C-terminal region of V protein containing three Trp and seven Cys residues that form a bowl-shaped depression (Figure 1) (Li et al, 2006). The one exception is Graf1 whose interaction site is the N-terminal V/P common region (Ohta et al, 2016a). Nishio et al (2005) and Ohta et al (2016b) Nishio et al (2005) Nishio et al (2005 Ohta et al (2018a) Ohta et al (2018c) FIGURE 1 | Amino acid sequences of the V-specific region of hPIV-2 V proteins.…”

“…The one exception is Graf1 whose interaction site is the N-terminal V/P common region (Ohta et al, 2016a). Nishio et al (2005) and Ohta et al (2016b) Nishio et al (2005) Nishio et al (2005 Ohta et al (2018a) Ohta et al (2018c) FIGURE 1 | Amino acid sequences of the V-specific region of hPIV-2 V proteins. Trp and Cys residues are marked with filled circles and filled squares, respectively.…”

SMURF1-mediated ubiquitination of ARHGAP26 promotes ovarian cancer cell invasion and migration

“…Rho GTPase-activating protein 26 (ARHGAP26) belongs to the small G protein family that can hydrolyze active Rho GTPase into inactive Rho GDP and negatively regulates RhoA 6 . RhoA belongs to the Rho GTPase superfamily and is abnormally expressed in many malignant tumors and involved in the tumor invasion and metastasis 7,8 , contributing to the occurrence and development of cancer.…”