Retrieval of human cytomegalovirus glycoprotein B from cell surface is not required for virus envelopment in astrocytoma cells

Human cytomegalovirus (HCMV) is a prototypic member of the betaherpesvirus family. The HCMV virion is composed of a large DNA genome encapsidated within a nucleocapsid, which is wrapped within an inner proteinaceous tegument and an outer lipid envelope containing viral glycoproteins. Although genome encapsidation clearly occurs in the nucleus, the subsequent steps in the virion assembly process are unclear. HCMV glycoprotein B (gB) is a major component of the virion envelope that plays a critical role in virus entry and is essential for the production of infectious virus progeny. The aim of our present study was to identify the secretory compartment to which HCMV gB was localized and to investigate the role of endocytosis in mediating gB localization and HCMV biogenesis. We show that HCMV gB is localized to the trans-Golgi network (TGN) in HCMV-infected cells and that gB contains all of the trafficking information necessary for TGN localization. Endocytosis of gB was shown to play a role in mediating TGN localization of gB and in targeting of the protein to the site of virus envelopment. However, inhibition of endocytosis with a dominant-negative dynamin I molecule did not affect the production of infectious virus. These observations indicate that, although endocytosis is involved in the trafficking of gB to the site of glycoprotein accumulation in the TGN, endocytosis of gB is not required for the production of infectious HCMV.     

Epidermal growth factor receptor is not required for human cytomegalovirus entry or signaling

Human cytomegalovirus (HCMV) can bind, fuse, and initiate gene expression in a diverse range of vertebrate cell types. This broad cellular tropism suggests that multiple receptors and/or universally distributed receptors mediate HCMV entry. Our laboratory has recently discovered that certain beta1 and beta3 integrin heterodimers are critical mediators of HCMV entry into permissive fibroblasts (A. L. Feire, H. Koss, and T. Compton, Proc. Natl. Acad. Sci. USA 101:15470-15475, 2004). It has also been reported that epidermal growth factor receptor (EGFR) is necessary for HCMV-mediated signaling and entry (X. Wang, S. M. Huong, M. L. Chiu, N. Raab-Traub, and E. E. Huang, Nature 424:456-461, 2003). Integrins are known to signal synergistically with growth factor receptors, and this coordination was recently reported for EGFR and beta3 integrins in the context of HCMV entry (X. Wang, D. Y. Huang, S. M. Huong, and E. S. Huang, Nat. Med. 11:515-521, 2005). However, EGFR-negative cell lines, such as hematopoietic cells, are known to be infected by HCMV. Therefore, we wished to confirm a role for EGFR in HCMV entry and then examine any interaction between beta1 integrins and EGFR during the entry process. Surprisingly, we were unable to detect any role for EGFR in the process of HCMV entry into fibroblast, epithelial, or endothelial cell lines. Additionally, HCMV did not activate the EGFR kinase in fibroblast cell lines. We first examined HCMV entry into two EGFR-positive or -negative cell lines but observed no increase in entry when EGFR was expressed to high levels. Physically blocking EGFR with a neutralizing antibody in fibroblast, epithelial, or endothelial cell lines or blocking EGFR kinase signaling with a chemical inhibitor in fibroblast cells did not inhibit virus entry. Lastly, we were unable to detect phosphorylation of EGFR in fibroblasts cells in response to HCMV stimulation. Our findings demonstrate that EGFR does not play a significant role in HCMV entry or signaling. These results suggest that specific integrin heterodimers either act alone as the primary entry receptors or interact in conjunction with an additional receptor(s), other than EGFR, to facilitate virus entry.     

Multimerization of tegument protein pp28 within the assembly compartment is required for cytoplasmic envelopment of human cytomegalovirus

Human cytomegalovirus (HCMV) UL99-encoded pp28 is an essential tegument protein required for envelopment and production of infectious virus. Nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with the UL99 gene deleted. Previous results have suggested that a key function of pp28 in the envelopment of infectious HCMV is expressed after the protein localizes in the assembly compartment (AC). In this study, we investigated the potential role of pp28 multimerization in the envelopment of the infectious virion. Our results indicated that pp28 multimerized during viral infection and that interacting domains responsible for self-interaction were localized in the amino terminus of the protein (amino acids [aa] 1 to 43). The results from transient-expression and/or infection assays indicated that the self-interaction took place in the AC. A mutant pp28 molecule containing only the first 35 aa failed to accumulate in the AC, did not interact with pp28 in the AC, and could not support virus replication. In contrast, the first 50 aa of pp28 was sufficient for the self-interaction within the AC and the assembly of infectious virus. Recombinant viruses encoding an in-frame deletion of aa 26 to 33 of pp28 were replication competent, whereas infectious virus was not recovered from HCMV BACs lacking aa 26 to 43. These findings suggested that the accumulation of pp28 was a prerequisite for multimerization of pp28 within the AC and that pp28 multimerization in the AC represented an essential step in the envelopment and production of infectious virions.     

The ESCRT system is required for hepatitis C virus production

Background

Recently, lipid droplets have been found to be involved in an important cytoplasmic organelle for hepatitis C virus (HCV) production. However, the mechanisms of HCV assembly, budding, and release remain poorly understood. Retroviruses and some other enveloped viruses require an endosomal sorting complex required for transport (ESCRT) components and their associated proteins for their budding process.

Methodology/Principal Findings

To determine whether or not the ESCRT system is needed for HCV production, we examined the infectivity of HCV or the Core levels in culture supernatants as well as HCV RNA levels in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, expressing short hairpin RNA or siRNA targeted to tumor susceptibility gene 101 (TSG101), apoptosis-linked gene 2 interacting protein X (Alix), Vps4B, charged multivesicular body protein 4b (CHMP4b), or Brox, all of which are components of the ESCRT system. We found that the infectivity of HCV in the supernatants was significantly suppressed in these knockdown cells. Consequently, the release of the HCV Core into the culture supernatants was significantly suppressed in these knockdown cells after HCV-JFH1 infection, while the intracellular infectivity and the RNA replication of HCV-JFH1 were not significantly affected. Furthermore, the HCV Core mostly colocalized with CHMP4b, a component of ESCRT-III. In this context, HCV Core could bind to CHMP4b. Nevertheless, we failed to find the conserved viral late domain motif, which is required for interaction with the ESCRT component, in the HCV-JFH1 Core, suggesting that HCV Core has a novel motif required for HCV production.

Conclusions/Significance

These results suggest that the ESCRT system is required for infectious HCV production.     

Rab27a is required for human cytomegalovirus assembly

Human cytomegalovirus (HCMV) completes its final envelopment on intracellular membranes before it is released from the cell. The mechanisms underlying these processes are not understood. Here we studied the role of Rab27a, a regulator of lysosome-related organelle transport, in HCMV production. HCMV infection increased Rab27a expression, and recruitment of Rab27a to membranous strutures at the assembly site. Immuno-gold labelling demonstrated association of Rab27a with viral envelopes. CMV production was reduced after knock-down of Rab27a, and in Rab27a-deficient ashen melanocytes. This study shows a requirement for Rab27a in the CMV life cycle and suggests that CMV and LRO biogenesis share common molecular mechanisms.     

Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids

The human cytomegalovirus UL99-encoded pp28 is a myristylated phosphoprotein that is a constituent of the virion. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. In the infected cell, pp28 is found in a cytoplasmic compartment derived from the Golgi apparatus, where the virus buds into vesicles to acquire its final membrane. We have constructed two mutants of human cytomegalovirus that fail to produce the pp28 protein, a substitution mutant (BADsubUL99) and a point mutant (BADpmUL99), and we have propagated them by complementation in pp28-expressing fibroblasts. Both mutant viruses are profoundly defective for growth in normal fibroblasts; no infectious virus could be detected after infection. Whereas normal levels of viral DNA and late proteins were observed in mutant virus-infected cells, large numbers of tegument-associated capsids accumulated in the cytoplasm that failed to acquire an envelope. We conclude that pp28 is required for the final envelopment of the human cytomegalovirus virion in the cytoplasm.     

Cellular autophagy machinery is not required for vaccinia virus replication and maturation

The origin of the primary membrane of the vaccinia virus, a double-membrane structure that surrounds the immature virions (IV), is not fully understood. Here we investigated whether the primary membrane originates from the autophagic membrane. Morphologic studies by electron microscopy (EM) showed no apparent difference in viral maturation in the autophagy-deficient cell lines, the atg5(-/-) mouse embryonic fibroblasts (MEFs) and the beclin1(-/-) embryonic stem (ES) cells, compared to their isogenic wild-type counterparts. Moreover, viral growth curves demonstrated that vaccinia viruses replicate and mature in the autophagy-deficient cell lines as efficiently as they do in their isogenic wild type counterpart cells. This study indicates that the cellular autophagy machinery is not required for the life-cycle of vaccinia virus, suggesting that the primary vaccinia viral membrane does not originate from the autophagic membrane.     

Functional ESCRT machinery is required for constitutive recycling of claudin-1 and maintenance of polarity in vertebrate epithelial cells

Genetic screens in Drosophila have identified regulators of endocytic trafficking as neoplastic tumor suppressor genes. For example, Drosophila endosomal sorting complex required for transport (ESCRT) mutants lose epithelial polarity and show increased cell proliferation, suggesting that ESCRT proteins could function as tumor suppressors. In this study, we show for the for the first time to our knowledge that ESCRT proteins are required to maintain polarity in mammalian epithelial cells. Inhibition of ESCRT function caused the tight junction protein claudin-1 to accumulate in intracellular vesicles. In contrast E-cadherin and occludin localization was unaffected. We investigated the cause of this accumulation and show that claudin-1 is constitutively recycled in kidney, colon, and lung epithelial cells, identifying claudin-1 recycling as a newly described feature of diverse epithelial cell types. This recycling requires ESCRT function, explaining the accumulation of intracellular claudin-1 when ESCRT function is inhibited. We further demonstrate that small interfering RNA knockdown of the ESCRT protein Tsg101 causes epithelial monolayers to lose their polarized organization and interferes with the establishment of a normal epithelial permeability barrier. ESCRT knockdown also reduces the formation of correctly polarized three-dimensional cysts. Thus, in mammalian epithelial cells, ESCRT function is required for claudin-1 trafficking and for epithelial cell polarity, supporting the hypothesis that ESCRT proteins function as tumor suppressors.     

The emerging shape of the ESCRT machinery

The past two years have seen an explosion in the structural understanding of the endosomal sorting complex required for transport (ESCRT) machinery that facilitates the trafficking of ubiquitylated proteins from endosomes to lysosomes via multivesicular bodies (MVBs). A common organization of all ESCRTs is a rigid core attached to flexibly connected modules that recognize other components of the MVB pathway. Several previously unsuspected key links between multiple ESCRT subunits, phospholipids and ubiquitin have now been elucidated, which, together with the detailed morphological analyses of ESCRT-depletion phenotypes, provide new insights into the mechanism of MVB biogenesis.     

The carboxy-terminal domain of glycoprotein N of human cytomegalovirus is required for virion morphogenesis

Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.     

The human cytomegalovirus (HCMV) tegument protein UL94 is essential for secondary envelopment of HCMV virions

Human cytomegalovirus (HCMV) virions are structurally complex, and the mechanisms by which they are assembled are poorly understood. However, several tegument proteins are known to be essential for proper particle assembly and maturation. Despite intense investigation, the function of many tegument proteins remains unknown. The HCMV UL94 gene is conserved among all herpesviruses and encodes a virion protein of unknown function. We demonstrate here that UL94 is a tegument protein that is expressed with true-late kinetics and localizes to the viral assembly complex during infection. To elucidate the function of UL94, we constructed a UL94-null mutant, designated UL94stop. This mutant is completely defective for replication, demonstrating that UL94 is essential. Phenotypic analysis of the UL94stop mutant shows that in the absence of UL94, viral gene expression and genome synthesis occur at wild-type levels. However, analysis of the localization of viral proteins to the cytoplasmic assembly complex shows that the essential tegument protein UL99 (pp28) exhibits aberrant localization in cells infected with the UL94stop mutant. Finally, we show that there is a complete block in secondary envelopment in the absence of UL94. Taken together, our data suggest that UL94 functions late in infection to direct UL99 to the assembly complex, thereby facilitating secondary envelopment of virions.     

Antigenic domain 1 is required for oligomerization of human cytomegalovirus glycoprotein B

Human cytomegalovirus (HCMV) glycoprotein B (gB) is an abundant virion envelope protein that has been shown to be essential for the infectivity of HCMV. HCMV gB is also one of the most immunogenic virus-encoded proteins, and a significant fraction of virus neutralizing antibodies are directed at gB. A linear domain of gB designated AD-1 (antigenic domain 1) represents a dominant antibody binding site on this protein. AD-1 from clinical isolates of HCMV exhibits little sequence variation, suggesting that AD-1 plays an essential role in gB structure or function. We investigated this possibility by examining the role of AD-1 in early steps of gB synthesis. Our results from studies using eukaryotic cells indicated that amino acid (aa) 635 of the gB sequence represented the carboxyl-terminal limit of this domain and that deletion of aa 560 to 640 of the gB sequence resulted in loss of AD-1 expression. AD-1 was shown to be required for oligomerization of gB. Mutation of cysteine at either position 573 or 610 in AD-1 resulted in loss of its reactivity with AD-1-specific monoclonal antibodies and gB oligomerization. Infectious virus could not be recovered from HCMV bacterial artificial chromosomes following introduction of these mutations into the HCMV genome, suggesting that AD-1 was an essential structural domain required for gB function in the replicative cycle of HCMV. Sequence alignment of AD-1 with homologous regions of gBs from other herpesviruses demonstrated significant relatedness, raising the possibility that this domain may contribute to multimerization of gBs in other herpesviruses.     

The ESCRT machinery: a cellular apparatus for sorting and scission

The ESCRT (endosomal sorting complex required for transport) machinery is a group of multisubunit protein complexes conserved across phyla that are involved in a range of diverse cellular processes. ESCRT proteins regulate the biogenesis of MVBs (multivesicular bodies) and the sorting of ubiquitinated cargos on to ILVs (intraluminal vesicles) within these MVBs. These proteins are also recruited to sites of retroviral particle assembly, where they provide an activity that allows release of these retroviruses. More recently, these proteins have been shown to be recruited to the intracellular bridge linking daughter cells at the end of mitosis, where they act to ensure the separation of these cells through the process of cytokinesis. Although these cellular processes are diverse, they share a requirement for a topologically unique membrane-fission step for their completion. Current models suggest that the ESCRT machinery catalyses this membrane fission.     

The essential human cytomegalovirus gene UL52 is required for cleavage-packaging of the viral genome

Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.     

M94 is essential for the secondary envelopment of murine cytomegalovirus

The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle, a library of random M94 mutants was generated by modified transposon-based linker scanning mutagenesis. A comprehensive set of M94 mutants was reinserted into the MCMV genome and tested for their capacity to complement the M94 null mutant. Thereby, 34 loss-of-function mutants of M94 were identified, which were tested in a second screen for their capacity to inhibit virus replication. This analysis identified two N-terminal insertion mutants of M94 with a dominant negative effect. We compared phenotypes induced by the conditional expression of these dominant negative M94 alleles with the null phenotype of the M94 deletion. The viral gene expression cascade and the nuclear morphogenesis steps were not affected in either setting. In both cases, however, secondary envelopment did not proceed in the absence of functional M94, and capsids subsequently accumulated in the center of the cytoplasmic assembly complex. In addition, deletion of M94 resulted in a block of cell-to-cell spread. Moreover, the dominant negative mutant of M94 demonstrated a defect in interacting with M99, the UL11 homologue of MCMV.