Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion

Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRdelta4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRdelta4 displayed a number of defects in early infection processes. Adsorption and entry of TRdelta4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRdelta4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.     

Construction and characterization of a human cytomegalovirus mutant with the UL18 (class I homolog) gene deleted.

The UL18 open reading frame of human cytomegalovirus (HCMV) (which encodes a product homologous to major histocompatibility complex class I heavy chains) has been disrupted by insertion of the beta-galactosidase gene under control of the major HCMV early promoter. The recombinant virus delta UL18 showed no phenotypic differences from wild-type HCMV in terms of single-step growth curves or particle/infectivity ratios, indicating that the UL18 gene product is dispensable for the growth of HCMV in human fibroblasts in vitro. The synthesis of the mature cellular class I heterodimer is shut down in cells infected at a high multiplicity with wild-type HCMV, and a similar effect was seen in delta UL18-infected fibroblasts, suggesting that although the UL18 gene product can associate with beta 2 microglobulin, it is not directly involved in the disruption of class I assembly.     

Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65

UL97 is a protein kinase encoded by human cytomegalovirus (HCMV) and is an important target for antiviral drugs against this ubiquitous herpesvirus, which is a major cause of life-threatening opportunistic infections in the immunocompromised host. In an effort to better understand the function(s) of UL97 during HCMV replication, a recombinant HCMV, NTAP97, which expresses a tandem affinity purification (TAP) tag at the amino terminus of UL97, was used to obtain UL97 protein complexes from infected cells. pp65 (also known as UL83), the 65-kDa virion tegument phosphoprotein, specifically copurified with UL97 during TAP, as shown by mass spectrometry and Western blot analyses. Reciprocal coimmunoprecipitation experiments using lysates of infected cells also indicated an interaction between UL97 and pp65. Moreover, in a glutathione S-transferase (GST) pull-down experiment, purified GST-pp65 fusion protein specifically bound in vitro-translated UL97, suggesting that UL97 and pp65 do not require other viral proteins to form a complex and may directly interact. Notably, pp65 has been previously reported to form unusual aggregates during viral replication when UL97 is pharmacologically inhibited or genetically ablated, and a pp65 deletion mutant was observed to exhibit modest resistance to a UL97 inhibitor (M. N. Prichard, W. J. Britt, S. L. Daily, C. B. Hartline, and E. R. Kern, J. Virol. 79:15494-15502, 2005). A stable protein-protein interaction between pp65 and UL97 may be relevant to incorporation of these proteins into HCMV particles during virion morphogenesis, with potential implications for immunomodulation by HCMV, and may also be a mechanism by which UL97 is negatively regulated during HCMV replication.     

Cytomegalovirus pUL96 is critical for the stability of pp150-associated nucleocapsids

Maturation of human cytomegalovirus (HCMV) initiates with nucleocapsids that egress from the nucleus and associate with a juxtanuclear cytoplasmic assembly compartment, where virion envelopment and release are orchestrated. Betaherpesvirus conserved proteins pp150 (encoded by UL32) and pUL96 are critical for HCMV growth in cell culture. pp150 is a capsid-proximal tegument protein that preserves the integrity of nucleocapsids during maturation. pUL96, although expressed as an early protein, acts late during virus maturation, similar to pp150, based on the comparable antigen distribution in UL96, UL32, or UL96/UL32 dual mutant virus-infected cells. pp150 associates with nuclear capsids prior to DNA encapsidation, whereas both pp150 and pUL96 associate with extracellular virus, suggesting that pUL96 is added after pp150. In the absence of pUL96, capsid egress from the nucleus continues; however, unlike wild-type virus infection, pp150 accumulates in the nuclear, as well as in the cytoplasmic, compartment. Ultrastructural evaluation of a UL96 conditional mutant revealed intact nuclear stages but aberrant nucleocapsids accumulating in the cytoplasm comparable to the known phenotype of UL32 mutant virus. In summary, pUL96 preserves the integrity of pp150-associated nucleocapsids during translocation from the nucleus to the cytoplasm.     

UL26-deficient human cytomegalovirus produces virions with hypophosphorylated pp28 tegument protein that is unstable within newly infected cells

The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.     

Impact of Sequence Variation in the UL128 Locus on Production of Human Cytomegalovirus in Fibroblast and Epithelial Cells

The human cytomegalovirus (HCMV) virion envelope contains a complex consisting of glycoproteins gH and gL plus proteins encoded by the UL128 locus (UL128L): pUL128, pUL130, and pUL131A. UL128L is necessary for efficient infection of myeloid, epithelial, and endothelial cells but limits replication in fibroblasts. Consequently, disrupting mutations in UL128L are rapidly selected when clinical isolates are cultured in fibroblasts. In contrast, bacterial artificial chromosome (BAC)-cloned strains TB40-BAC4, FIX, and TR do not contain overt disruptions in UL128L, yet no virus reconstituted from them has been reported to acquire mutations in UL128L in vitro. We performed BAC mutagenesis and reconstitution experiments to test the hypothesis that these strains contain subtle mutations in UL128L that were acquired during passage prior to BAC cloning. Compared to strain Merlin containing wild-type UL128L, all three strains produced higher yields of cell-free virus. Moreover, TB40-BAC4 and FIX spread cell to cell more rapidly than wild-type Merlin in fibroblasts but more slowly in epithelial cells. The differential growth properties of TB40-BAC4 and FIX (but not TR) were mapped to single-nucleotide substitutions in UL128L. The substitution in TB40-BAC4 reduced the splicing efficiency of UL128, and that in FIX resulted in an amino acid substitution in UL130. Introduction of these substitutions into Merlin dramatically increased yields of cell-free virus and increased cell-to-cell spread in fibroblasts but reduced the abundance of pUL128 in the virion and the efficiency of epithelial cell infection. These substitutions appear to represent mutations in UL128L that permit virus to be propagated in fibroblasts while retaining epithelial cell tropism.     

An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function

The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.     

The human cytomegalovirus-specific UL1 gene encodes a late-phase glycoprotein incorporated in the virion envelope

We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism.     

Human cytomegalovirus UL130 protein promotes endothelial cell infection through a producer cell modification of the virion

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in trans to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in trans the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC.     

Polo-Like Kinase 1 as a Target for Human Cytomegalovirus pp65 Lower Matrix Protein

Human cytomegalovirus (HCMV) pp65 protein is the major constituent of viral dense bodies but is dispensable for viral growth in vitro. pp65 copurifies with a S/T kinase activity and has been implicated in phosphorylation of HCMV IE1 immediate-early protein and its escape from major histocompatibility complex 1 presentation. Furthermore, the presence of pp65 correlates with a virion-associated kinase activity. To clarify the role of pp65, yeast two-hybrid system (THS) screening was performed to identify pp65 cellular partners. A total of 18 out of 48 yeast clones harboring cDNAs for putative pp65 binding proteins encoded the Polo-like kinase 1 (Plk1) C-terminal domain. Plk1 behaved as a bona fide pp65 partner in THS control crosses, and the interaction was confirmed by in vitro binding experiments. Endogenous Plk1 was coimmunoprecipitated with pp65 from transiently transfected COS7 cells. In infected fibroblasts, Plk1 was coimmunoprecipitated with pp65 at late infection stages. Furthermore, Plk1 was detected within wild-type HCMV particles but not within the particles of a pp65-negative mutant (RVAd65). The hydrophilic region of pp65 was phosphorylated in vitro by Plk1. These results suggest that one function of pp65 may be to capture a cell kinase, perhaps in order to alter its activity, nucleotide preference, substrate specificity, or subcellular localization to the advantage of HCMV.     

Deletion of the Human Cytomegalovirus US17 Gene Increases the Ratio of Genomes per Infectious Unit and Alters Regulation of Immune and Endoplasmic Reticulum Stress Response Genes at Early and Late Times after Infection

Human cytomegalovirus (HCMV) employs numerous strategies to combat, subvert, or co-opt host immunity. One evolutionary strategy for this involves capture of a host gene and then its successive duplication and divergence, forming a family of genes, many of which have immunomodulatory activities. The HCMV US12 family consists of 10 tandemly arranged sequence-related genes in the unique short (US) region of the HCMV genome (US12 to US21). Each gene encodes a protein possessing seven predicted transmembrane domains, patches of sequence similarity with cellular G-protein-coupled receptors, and the Bax inhibitor 1 family of antiapoptotic proteins. We show that one member, US17, plays an important role during virion maturation. Microarray analysis of cells infected with a recombinant HCMV isolate with a US17 deletion (the ΔUS17 mutant virus) revealed blunted host innate and interferon responses at early times after infection (12 h postinfection [hpi]), a pattern opposite that previously seen in the absence of the immunomodulatory tegument protein pp65 (pUL83). Although the ΔUS17 mutant virus produced numbers of infectious particles in fibroblasts equal to the numbers produced by the parental virus, it produced >3-fold more genome-containing noninfectious viral particles and delivered increased amounts of pp65 to newly infected cells. These results suggest that US17 has evolved to control virion composition, to elicit an appropriately balanced host immune response. At later time points (96 hpi), ΔUS17 mutant-infected cells displayed aberrant expression of several host endoplasmic reticulum stress response genes and chaperones, some of which are important for the final stages of virion assembly and egress. Our results suggest that US17 modulates host pathways to enable production of virions that elicit an appropriately balanced host immune response.     

Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism

Epithelial cells are one of the prominent cell types infected by human cytomegalovirus (HCMV) within its host. However, many cultured epithelial cells, such as ARPE-19 retinal pigmented epithelial cells, are poorly infected by laboratory-adapted strains in cell culture, and little is known about the viral factors that determine HCMV epithelial cell tropism. In this report, we demonstrate that the UL131 open reading frame (ORF), and likely the entire UL131-128 locus, is required for efficient infection of epithelial cells. Repair of the mutated UL131 gene in the AD169 laboratory strain of HCMV restored its ability to infect both epithelial and endothelial cells while compromising its ability to replicate in fibroblasts. ARPE-19 epithelial cells support replication of the repaired AD169 virus as well as clinical isolates of HCMV. Productive infection of cultured epithelial cells, endothelial cells, and fibroblasts with the repaired AD169 virus leads to extensive membrane fusion and syncytium formation, suggesting that the virus may spread through cell-cell fusion.     

Control of cytoplasmic maturation events by cytomegalovirus tegument protein pp150

Cytomegalovirus replication depends upon a betaherpesvirus-conserved 150-kDa tegument phosphoprotein (pp150; encoded by UL32) that supports the final steps in virion maturation at cytoplasmic assembly compartments. Amino acid substitutions were introduced into conserved region 1 (CR1) and CR2 of pp150, affecting a region that may interact with nucleocapsids. Two independent CR2 point mutants (N201A and G207A) failed to support viral replication in evaluations by a transient complementation assay or after reconstruction into recombinant viruses. An assembly compartment-like cytoplasmic inclusion developed in UL32 mutant virus-infected cells that was similar to that of wild-type virus-infected cells. The cellular localization of the trans-Golgi marker Golgin-97 suggested differences in the organization of the assembly compartment compared to that of wild-type virus-infected cells. Replication-defective CR2 point mutants exhibited the same phenotype as that of a virus carrying a complete deletion of the UL32 open reading frame in these assays. Electron micrographs of fibroblasts at 3 or 5 days postinfection with a deletion mutant (ΔUL32) grown on UL32-complementing cells showed a similar number and morphology of capsids in the nucleus, but the cytoplasmic region associated with virion assembly appeared highly vesiculated and contained few recognizable nucleocapsids or complete virus particles. These data demonstrate that the principle role of pp150 is to retain nucleocapsid organization through secondary envelopment at the assembly compartment.     

Sequence requirements for localization of human cytomegalovirus tegument protein pp28 to the virus assembly compartment and for assembly of infectious virus

The human cytomegalovirus UL99 open reading frame encodes a 190-amino-acid (aa) tegument protein, pp28, that is myristoylated and phosphorylated. pp28 is essential for assembly of infectious virus, and nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with a UL99 deletion. pp28 is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells, while in infected cells, it is localized together with other virion proteins in a juxtanuclear compartment termed the assembly compartment (AC). We investigated the sequence requirements for pp28 trafficking to the AC and assembly of infectious virus. Our studies indicated that the first 30 to 35 aa were required for localization of pp28 to the ERGIC in transfected cells. Mutant forms of pp28 containing only the first 35 aa localized with other virion structural proteins to cytoplasmic compartments early in infection, but localization to the AC at late times required a minimum of 50 aa. In agreement with previous reports, we demonstrated that the deletion of a cluster of acidic amino acids (aa 44 to 59) prevented wild-type trafficking of pp28 and recovery of infectious virus. A recombinant virus expressing only the first 50 aa was replication competent, and this mutant, pp28, localized to the AC in cells infected with this virus. These findings argued that localization of pp28 to the AC was essential for assembly of infectious virus and raised the possibility that amino acids in the amino terminus of pp28 have additional roles in the envelopment and assembly of the virion other than simply localizing pp28 to the AC.     

Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly

The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the trans-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.