Abstract
Shortly after entering the cells, cytomegaloviruses (CMVs) initiate massive reorganization of cellular endocytic and secretory pathways, which results in the forming of the cytoplasmic virion assembly compartment (AC). We have previously shown that the formation of AC in murine CMV- (MCMV) infected cells begins in the early phase of infection (at 4–6 hpi) with the pre-AC establishment. Pre-AC comprises membranes derived from the endosomal recycling compartment, early endosomes, and the trans-Golgi network, which is surrounded by fragmented Golgi cisterns. To explore the importance of Arf GTPases in the biogenesis of the pre-AC, we infected Balb 3T3 cells with MCMV and analyzed the expression and intracellular localization of Arf proteins in the early phases (up to 16 hpi) of infection and the development of pre-AC in cells with a knockdown of Arf protein expression by small interfering RNAs (siRNAs). Herein, we show that even in the early phase, MCMVs cause massive reorganization of the Arf system of the host cells and induce the over-recruitment of Arf proteins onto the membranes of pre-AC. Knockdown of Arf1, Arf3, Arf4, or Arf6 impaired the establishment of pre-AC. However, the knockdown of Arf1 and Arf6 also abolished the establishment of infection. Our study demonstrates that Arf GTPases are required for different steps of early cytomegalovirus infection, including the establishment of the pre-AC.
Highlights
Cytomegaloviruses (CMVs) are widespread double-stranded DNA betaherpesviruses that mostly cause asymptomatic infection followed by a long-lasting latent state
Our study revealed that the contribution of Arf1 and Arf6 is most likely associated with the earliest stages of infection, either during virion entry or during the establishment of infection
Given that many processes of the AC biogenesis are conserved among beta-herpesviruses, our data may contribute to understanding the biogenesis of the HCMV assembly compartment and to identifying the host cell processes that may be targeted for the development of antivirals
Summary
Cytomegaloviruses (CMVs) are widespread double-stranded DNA betaherpesviruses that mostly cause asymptomatic infection followed by a long-lasting latent state. Murine CMV (MCMV) shares many similarities with other betaherpesviruses and is used as a model for studying betaherpesvirus biology in vitro and in vivo. MCMV, upon entry, activates a gene-expression program, which is executed through three phases: immediate–early (IE), which lasts up to 2 h post-infection (hpi), early (E; from 2 to 16 hpi), and late (L; after 16 hpi) [4]. In the early stages of infection, CMV induces the reorganization of intracellular membranes and hijacks many cellular proteins as well as endocytic and secretory pathways of the host cells to form the virion assembly compartment (AC). The onset of AC formation in murine MCMV-infected cells can be identified at 4–6 hpi as an extensive reorganization of the Golgi and the endosomal system [6,7]. A similar set of events was executed during HCMV infection with significantly delayed kinetics [8,9,10,11]
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