A Tyrosine-Based Trafficking Motif of the Tegument Protein pUL71 Is Crucial for Human Cytomegalovirus Secondary Envelopment

Abstract

The human cytomegalovirus (HCMV) tegument protein pUL71 is required for efficient secondary envelopment and accumulates at the Golgi compartment-derived viral assembly complex (vAC) during infection. Analysis of various C-terminally truncated pUL71 proteins fused to enhanced green fluorescent protein (eGFP) identified amino acids 23 to 34 as important determinants for its Golgi complex localization. Sequence analysis and mutational verification revealed the presence of an N-terminal tyrosine-based trafficking motif (YXXΦ) in pUL71. This led us to hypothesize a requirement of the YXXΦ motif for the function of pUL71 in infection. Mutation of both the tyrosine residue and the entire YXXΦ motif resulted in an altered distribution of mutant pUL71 at the plasma membrane and in the cytoplasm during infection. Both YXXΦ mutant viruses exhibited similarly decreased focal growth and reduced virus yields in supernatants. Ultrastructurally, mutant-virus-infected cells exhibited impaired secondary envelopment manifested by accumulations of capsids undergoing an envelopment process. Additionally, clusters of capsid accumulations surrounding the vAC were observed, similar to the ultrastructural phenotype of a UL71-deficient mutant. The importance of endocytosis and thus the YXXΦ motif for targeting pUL71 to the Golgi complex was further demonstrated when clathrin-mediated endocytosis was inhibited either by coexpression of the C-terminal part of cellular AP180 (AP180-C) or by treatment with methyl-β-cyclodextrin. Both conditions resulted in a plasma membrane accumulation of pUL71. Altogether, these data reveal the presence of a functional N-terminal endocytosis motif that is an important determinant for intracellular localization of pUL71 and that is furthermore required for the function of pUL71 during secondary envelopment of HCMV capsids at the vAC.IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of birth defects among congenital virus infections and can lead to life-threatening infections in immunocompromised hosts. Current antiviral treatments target viral genome replication and are increasingly overcome by viral mutations. Therefore, identifying new targets for antiviral therapy is important for future development of novel treatment options. A detailed molecular understanding of the complex virus morphogenesis will identify potential viral as well as cellular targets for antiviral intervention. Secondary envelopment is an important viral process through which infectious virus particles are generated and which involves the action of several viral proteins, such as tegument protein pUL71. Targeting of pUL71 to the site of secondary envelopment appears to be crucial for its function during this process and is regulated by utilizing host trafficking mechanisms that are commonly exploited by viral glycoproteins. Thus, intracellular trafficking, if targeted, might present a novel target for antiviral therapy.

Keywords: HCMV; cytomegalovirus; endocytosis; intracellular trafficking; morphogenesis; secondary envelopment; tegument; trafficking signal.

FIG 1

Generation and intracellular localization of pUL71 truncations. (A) Schematic of pUL71 fragments fused to eGFP expressed from the indicated expression plasmids. The sequence in bold indicates the YXXΦ motif in pUL71 fragments as well as the mutated motif in pUL71eGFP_23–26A, pUL71eGFP_Y23A, and pUL71eGFP_Y23F. HeLa cells transiently expressing full-length pUL71eGFP and the two pUL71 fragments, pUL71eGFP_1–34 and pUL71eGFP_1–22 (B) as well as YXXΦ motif mutants, pUL71eGFP_23–26A, pUL71eGFP_Y23A, and pUL71eGFP_Y23F (C), were stained for TGN46 and cell nuclei with DAPI at 20 h posttransfection. Scale bar, 20 μm.

FIG 2

Amino acid sequence alignment of HCMV pUL71 homologues. The N-terminal amino acid sequence of residues 1 to 34 of HCMV pUL71 was aligned to pUL71 homologues of other herpesviruses using the online tool MUSCLE (69). Homologous proteins (square brackets) from human herpesviruses of all three subfamilies, Alpha-, Beta-, and Gammaherpesvirinae, show a highly conserved N-terminal YXXΦ trafficking signal (bold), where X stands for any amino acid and Φ stands for an amino acid with a bulky hydrophobic side chain. HHV, human herpesvirus; EBV, Epstein-Barr virus; VSV, vesicular stomatitis virus.

FIG 3

Generation and growth analysis of YXXΦ mutant viruses. (A) Shown are the genomic organization down- and upstream of the UL71 gene and the amino acid sequence of the pUL71 N terminus of the wild-type virus, YXXΦ mutant viruses, and the revertant virus, respectively. The YXXΦ motif as well as the introduced mutations within this motif is shown in bold. (B) Focus expansion assay of the indicated viruses in HFF under methylcellulose overlay. HCMV-infected cells were detected by indirect immunofluorescence staining for IE1/2 antigen at 7 dpi. Each data point represents the number of IE1/2-positive cell nuclei per focus. Shown is the mean value of 50 foci for each virus for one of three experiments or, in the case of the TB-UL71mutY23F and TB-UL71stop viruses, for one of two experiments. Significance was determined by applying a two-tailed Student t test (***, P < 0,0001; n.s., not significant). (C) Images of representative foci of the indicated viruses at 7 dpi under methylcellulose overlay. IE1/2-positive cells were detected by an anti-IE1/2 antibody and visualized by an Alex Fluor 488-conjugated secondary antibody. Cell nuclei were stained with DAPI. Scale bar, 100 μm. (D) Multistep growth kinetics analyses of the indicated viruses were performed by infecting HFF at an MOI of 0.02 by coseeding of infected with uninfected cells. Virus yields in the supernatants of infected cells were determined at the indicated times by titration on HFF. Growth curves show the mean virus yields and standard deviations of three independent virus supernatants. Virus yields at time zero represent the starting infection rates determined at 24 h postcoseeding.

FIG 4

Subcellular localization of pUL71 and pp28 in wild-type, TB-UL71mut23–26A, TB-UL71mutY23A, TB-UL71mutY23F, and TB-UL71revY23A virus-infected HFF at 120 hpi. Proteins were detected by antibodies directed against pUL71 (green) and against pp28 (red). Cell nuclei were marked with DAPI. Arrows indicate accumulations of pp28 signals, which are also positive for DAPI, at the area of the vAC. Scale bar, 20 μm. Boxed areas of the TB-UL71mut23–26A and TB-UL71mutY23A infections are shown at higher magnification in the insets.

FIG 5

Ultrastructural analysis of wild-type, TB-UL71mut23–26A, and TB-UL71mutY23A virus-infected HFF at 120 hpi. (A) Electron micrographs of the area of vACs in the cytoplasm (cy) of infected cells with parts of the nucleus (nu). (B) Higher magnifications with close-ups of selected areas show fully enveloped particles for the wild-type-infected cell and multiple budding events and incompletely enveloped particles for TB-UL71mut23–26A and TB-UL71mutY23A virus-infected cells.

FIG 6

Inhibition of endocytosis by methyl-β-cyclodextrin (MβCD). (A) HeLa cells transiently expressing pUL71eGFP and pUL71eGFP_Y23A were incubated with 5 μg/ml human transferrin conjugated with Alexa Fluor 568 for 30 min at 37°C. Afterwards, cells were fixed, stained with DAPI, and examined using a fluorescence microscope. (B) Endocytosis of HeLa cells transiently expressing the indicated pUL71 proteins was inhibited by incubation in serum-free medium supplemented with 30 mM MβCD for 45 min. At 30 min prior to fixation, endocytosis inhibition was controlled by adding 5 μg/ml human transferrin conjugated with Alexa Fluor 568. Following fixation, cell nuclei were stained with DAPI and analyzed by fluorescence microscopy. Scale bar, 20 μm.

FIG 7

Inhibition of clathrin-mediated endocytosis by coexpression of AP180-C. (A) Inhibition of endocytosis in AP180-C-expressing HeLa cells was tested by transferrin uptake at 20 h posttransfection (control). After uptake of Alexa Fluor 568-conjugated human transferrin for 30 min, cells were fixed. AP180-C was detected with a primary antibody against its myc tag and a secondary antibody coupled to Alexa Fluor 647 (white). (B) HeLa cells coexpressing the indicated pUL71eGFP proteins (green) and AP180-C were tested for endocytosis inhibition as described for control cells. Cell nuclei were stained with DAPI. AP180-C-positive cells are indicated with an asterisk. Scale bar, 20 μm.

FIG 8

Model of intracellular trafficking of pUL71 in transfected and infected cells. Indicated is a potential trafficking route from the Golgi apparatus, delivering membrane-associated pUL71 to the plasma membrane (PM). At the plasma membrane, where pUL71 is localized at the cytosolic phase, recognition of the N-terminal YXXΦ motif recruits pUL71 to endocytic vesicles; pUL71 then gets recycled back by yet unknown mechanisms (question mark) to the Golgi apparatus or vAC in infection. Endocytosis from the plasma membrane can be blocked by coexpression of AP180-C and cholesterol depletion using methyl-β-cyclodextrin (MβCD). Cy, cytoplasm.