Rho GTPases Modulate Entry of Ebola Virus and Vesicular Stomatitis Virus Pseudotyped Vectors

Abstract

To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.Enveloped viruses enter cells by a variety of different pathways. Productive internalization of enveloped viruses with targeted cells is mediated through interactions of the viral glycoprotein(s) (GPs) with moieties on the surface of the cell. In general, enveloped viral entry occurs through viral adherence to the cell surface, interaction with a specific plasma membrane-associated receptor that results in a series of GP conformational changes leading to fusion of viral and cellular membranes, and delivery of the viral core particle into the cytoplasm. Fusion of the two membranes can occur at the plasma membrane or by uptake of the intact virions into endosomes with subsequent membrane fusion between the viral membrane and the lipid bilayer of the endocytic vesicle. Human immunodeficiency virus (HIV) is an example of a virus that fuses directly to the plasma membrane (5), whereas influenza virus must be internalized into acidified vesicles where the appropriate GP conformational changes can occur, mediating membrane fusion (21). Most enveloped viruses that enter through vesicles utilize a low-pH environment to mediate the necessary...

Filamentous Influenza Virus Enters Cells via Macropinocytosis

“…Our results suggest that filamentous influenza virus utilizes macropinocytosis as its cellular entry pathway similarly to other filamentous viruses, such as Ebola virus (33,41,47). This is especially interesting given the observation that primary human isolates of influenza virus, as well as the recent 2009 pandemic strain, appear to be predominantly filamentous (11,24,34), suggesting that macropinocytosis may be the dominant entry pathway for most strains of influenza virus.…”

“…This is especially interesting given the observation that primary human isolates of influenza virus, as well as the recent 2009 pandemic strain, appear to be predominantly filamentous (11,24,34), suggesting that macropinocytosis may be the dominant entry pathway for most strains of influenza virus. As a result, pharmaceutical inhibitors of virally induced macropinocytosis may prove to be a valuable antiviral treatment, effective against multiple different viruses, including, among others, influenza virus, Ebola virus, vaccinia virus, and Nipah virus (30,33,38,41,47), as reviewed in reference 31. Taken together, our results show that the main entry pathway for filamentous influenza virions is macropinocytosis, whereas the spherical forms of influenza virus are capable of entering cells through either macropinocytosis or clathrin-mediated endocytosis.…”

The Tyro3 Receptor Kinase Axl Enhances Macropinocytosis of Zaire Ebolavirus

“…A recent study confirmed that clathrincoated pits are indeed utilized by infectious EBOV to enter permissive cells (6). It has also been speculated that, because of their large size, filoviruses may utilize macropinocytosis to enter permissive cells (19,56). Recently, the latter mechanism of entry was demonstrated experimentally for the first time for a filovirus (51,57).…”

Small Rho GTPases and Cholesterol Biosynthetic Pathway Intermediates in African Swine Fever Virus Infection

“…Since prenylation of Rab GTPases is dependent on GGTase II, which is not affected by the inhibitor GGTI-286, the main focus of the present study was the Rho GTPase family. These are key regulators of the cell cytoskeleton, cell cycle, gene expression, and vesicle trafficking (19,51) and are involved in the entry of diverse viruses into the host cell (14,42,48,52) but also in viral morphogenesis (27,61), infection spreading (12), or virus-induced cell motility (61,66). In this regard, a general inhibition of Rho GTPase-mediated signaling could affect simultaneously diverse and relevant cellular processes that may explain the abnormal viral factory size and extensive accumulation of envelope precursors and immature virions observed when ASFV infection takes place under these conditions.…”