Endocytic processes are critical for cellular entry of several viruses; however, the role of endocytosis in cellular trafficking of viruses beyond virus entry is only partially understood. Here, we utilized two laboratory strains (AD169 and Towne) of human cytomegalovirus (HCMV), which are known to use cell membrane fusion rather than endocytosis to enter fibroblasts, in order to study a post-entry role of endocytosis in HCMV life cycle. Upon pharmacological inhibition of dynamin-2 or clathrin terminal domain (TD) ligand association, these strains entered the cells successfully based on the expression of immediate early viral protein. However, both the inhibitors significantly reduced the growth rates and final virus yields of viruses without inhibiting the expression of early to late viral proteins. Clathrin accumulated in the cytoplasmic virus assembly compartment (vAC) of infected cells co-localizing with virus tegument protein pp150 and the formation of vAC was compromised upon endocytic inhibition. Transmission electron micrographs (TEM) of infected cells treated with endocytosis inhibitors showed intact nuclear stages of nucleocapsid assembly but the cytoplasmic virus maturation was greatly compromised. Thus, the data presented here implicate endocytic pathways in HCMV maturation and egress.

Cytomegalovirus secondary envelopment occurs in a virus-induced cytoplasmic assembly compartment (vAC) generated via a drastic reorganization of the membranes of the secretory and endocytic systems. Dynamin is a eukaryotic GTPase that is implicated in membrane remodeling and endocytic membrane fission events; however, the role of dynamin in cellular trafficking of viruses beyond virus entry is only partially understood. Mouse embryonic fibroblasts (MEF) engineered to excise all three isoforms of dynamin were infected with mouse cytomegalovirus (MCMV-K181). Immediate-early (IE1; m123) viral protein was detected in these triple dynamin knockout (TKO) cells, as well as in mock-induced parental MEF, at early times postinfection, although levels were reduced in TKO cells, indicating that virus entry was affected but not eliminated. Levels of IE1 protein and another viral early protein (m04) were normalized by 48 h postinfection; however, late protein (m55; gB) expression was reduced in infected TKO cells compared to parental MEF. Ultrastructural analysis revealed intact stages of nuclear virus maturation in both cases with equivalent numbers of nucleocapsids containing packaged viral DNA (C-capsids), indicating successful viral DNA replication, capsid assembly, and genome packaging. Most importantly, severe defects in virus envelopment were visualized in TKO cells but not in parental cells. Dynamin inhibitor (dynasore)-treated MEF showed a phenotype similar to TKO cells upon mouse cytomegalovirus infection, confirming the role of dynamin in late maturation processes. In summary, dynamin-mediated endocytic pathways are critical for the completion of cytoplasmic stages of cytomegalovirus maturation.IMPORTANCE Viruses are known to exploit specific cellular functions at different stages of their life cycle in order to replicate, avoid immune recognition by the host and to establish a successful infection. Cytomegalovirus (CMV)-infected cells are characterized by a prominent cytoplasmic inclusion (virus assembly compartment [vAC]) that is the site of virus maturation and envelopment. While endocytic membranes are known to be the functional components of vAC, knowledge of specific endocytic pathways implicated in CMV maturation and envelopment is lacking. We show here that dynamin, which is an integral part of host endocytic machinery, is largely dispensable for early stages of CMV infection but is required at a late stage of CMV maturation. Studies on dynamin function in CMV infection will help us understand the host-virus interaction pathways amenable to targeting by conventional small molecules, as well as by newer generation nucleotide-based therapeutics (e.g., small interfering RNA, CRISPR/CAS gRNA, etc.).

17 18 Cytomegalovirus secondary envelopment occurs in a virus-induced cytoplasmic 19assembly compartment (vAC) generated via a drastic reorganization of the membranes 20 of the secretory and endocytic systems. Dynamin is a eukaryotic GTPase that is 21 implicated in membrane remodeling and endocytic membrane fission events; however, 22 the role of dynamin in cellular trafficking of viruses beyond virus entry is only partially 23 understood. Mouse embryonic fibroblasts (MEF) engineered to excise all three isoforms 24 of dynamin were infected with mouse cytomegalovirus (MCMV-K181). Immediate early 25 (IE1; m123) viral protein was detected in these triple dynamin knockout (TKO) cells as 26 well as in mock-induced parental MEF at early times post infection although levels were 27 reduced in TKO cells, indicating that virus entry was affected but not eliminated. Levels 28 of IE1 protein and another viral early protein (m04) were normalized by 48 hours post 29 infection; however, late protein (m55; gB) expression was significantly reduced in 30 infected TKO cells compared to parental MEF. Ultrastructural analysis revealed intact 31 stages of nuclear virus maturation in both cases with equivalent numbers of 32 nucleocapsids containing packaged viral DNA (C-capsids) indicating successful viral 33 DNA replication, capsid assembly and genome packaging. Most importantly, severe 34 defects in virus envelopment were visualized in TKO cells but not in parental cells. 35Dynamin inhibitor (dynasore) treated MEF showed a phenotype similar to TKO cells 36 upon MCMV infection confirming the role of dynamin in late maturation processes. In 37 summary, dynamin-mediated endocytic pathways are critical for the completion of 38 cytoplasmic stages of cytomegalovirus maturation. Importance: 41 42Viruses are known to exploit specific cellular functions at different stages of their life 43 cycle in order to replicate, avoid immune recognition by the host and to establish a 44 successful infection. Cytomegalovirus (CMV) infected cells are characterized by a 45 prominent cytoplasmic inclusion (virus assembly compartment; vAC) that is the site of 46 virus maturation and envelopment. While endocytic membranes are known to be the 47 functional components of vAC, knowledge of specific endocytic pathways implicated in 48 CMV maturation and envelopment is lacking. Here we show that dynamin, which is an 49 integral part of host endocytic machinery, is largely dispensable for early stages of CMV 50 infection but is required at a late stage of CMV maturation. Studies on dynamin function 51 in CMV infection will help us understand the host-virus interaction pathways amenable 52 to targeting by conventional small molecules as well as by newer generation nucleotide-53 based therapeutics (e.g. siRNA, CRISPR/CAS gRNA, etc.). Introduction: 55 56Endocytic pathways are important for cellular entry of several viruses (1-5); however, 57 their role in post-entry stages of virus replication is far from resolved. Maturing 58 herpesvirus nucleocapsids undergo primar...