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. 2016 Oct 4;7(5):e01554-16.
doi: 10.1128/mBio.01554-16.

Phosphorylation of Golgi Peripheral Membrane Protein Grasp65 Is an Integral Step in the Formation of the Human Cytomegalovirus Cytoplasmic Assembly Compartment

G Michael Rebmann  1 , Robert Grabski  2 , Veronica Sanchez  2 , William J Britt  3
Affiliations

Phosphorylation of Golgi Peripheral Membrane Protein Grasp65 Is an Integral Step in the Formation of the Human Cytomegalovirus Cytoplasmic Assembly Compartment

G Michael Rebmann et al. mBio. .

Abstract

Human cytomegalovirus (HCMV) is the largest member of the Herpesviridae and represents a significant cause of disease. During virus replication, HCMV alters cellular functions to facilitate its replication, including significant reorganization of the secretory and endocytic pathways of the infected cell. A defining morphologic change of the infected cell is the formation of a membranous structure in the cytoplasm that is designated the virion assembly compartment (AC), which consists of virion structural proteins surrounded by cellular membranes. The loss of normal Golgi compartment morphology and its relocalization from a juxtanuclear ribbonlike structure to a series of concentric rings on the periphery of the AC represents a readily recognized reorganization of cellular membranes in the HCMV-infected cell. Although trafficking of viral proteins to this compartment is required for the assembly of infectious virions, the functional significance of the reorganization of intracellular membranes like the Golgi membranes into the AC in the assembly of infectious virus remains understudied. In this study, we determined that Golgi membrane ribbon fragmentation increased during the early cytoplasmic phase of virion assembly and that Golgi membrane fragmentation in infected cells was dependent on the phosphorylation of an integral cis-Golgi protein, Grasp65. Inhibition of Golgi membrane fragmentation and of its reorganization into the AC resulted in decreased production of infectious particles and alteration of the incorporation of an essential protein into the envelope of the mature virion. These results demonstrated the complexity of the virus-host cell interactions required for efficient assembly of this large DNA virus.

Importance: The human cytomegalovirus (HCMV)-induced reorganization of intracellular membranes that is required for the formation of the viral assembly compartment (AC) has been an area of study over the last 20 years. The significance of this virus-induced structure has been evinced by the results of several studies which showed that relocalization of viral proteins to the AC was required for efficient assembly of infectious virus. In this study, we have identified a mechanism for the fragmentation of the Golgi ribbon in the infected cell en route to AC morphogenesis. Identification of this fundamental process during HCMV replication allowed us to propose that the functional role of Golgi membrane reorganization during HCMV infection was the concentration of viral structural proteins and subviral structures into a single intracellular compartment in order to facilitate efficient protein-protein interactions and the virion protein trafficking required for the assembly of this large and structurally complex virus.

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Figures

FIG 1
FIG 1
Golgi apparatus-derived membranes form the outer boundary of the HCMV cytoplasmic assembly compartment. HF cells were plated on glass coverslips and allowed to grow to confluence, followed by a 3-day incubation in serum-free medium. Cells were then infected at an MOI of 1. Coverslips were collected at 4 days p.i. and analyzed as described in Materials and Methods. Top, mock-infected cells were stained with antibodies reactive with cis-Golgi marker GM130 (merge: green), trans-Golgi marker golgin-245 (merge: red), and DAPI (merge: blue). Middle, HCMV AD 169-infected cells were stained with antibodies directed against GM130 (merge: green), golgin-245 (merge: red), pp65 (merge: blue), and DAPI (merge: gray). Bottom, HCMV AD 169-infected cells were stained with antibodies reactive with GM130 (merge: green), golgin-245 (merge: red), gM (merge: blue), and DAPI (merge: gray). Representative images are shown.
FIG 2
FIG 2
Kinetics of Golgi membrane fragmentation during HCMV infection. HF cells were plated on glass coverslips and infected as described in the legend to Fig. 1. (A) Coverslips were collected on days 1 to 4 p.i. and stained with antibodies reactive with GM130 (green), Grasp65 (red), pp65 (blue), and DAPI (gray). Representative images demonstrate intact Golgi membrane ribbon (days 1 and 2), fragmented Golgi membrane ribbons (day 3), and the AC (day 4). Scale bar is equal to 10 µm. (B) The length of cis-Golgi tubules is dynamically altered during HCMV infection. GM130+ membrane length on the indicated day postinfection was measured using the trace feature in FluoView software 10.1. Median values are plotted, and error bars show standard deviations. The significance of differences between groups was determined using the Kruskal-Wallis test (*, P < 0.05; ***, P < 0.001). The number of cells analyzed each day was as follows: day 1, n = 100; day 2, n = 120; day 3, n = 80; day 4, n = 75.
FIG 3
FIG 3
Fragmentation of the Golgi membrane during HCMV infection correlates temporally with pp65 cytoplasmic accumulation, AC morphogenesis, and virus production. Confocal microscopy was used to score the number of infected cells displaying the phenotypes illustrated in Fig. 2A. (A) Percentages of infected cells with fragmented Golgi membrane ribbon, presence of fully formed AC, nuclear pp65 localization, and cytoplasmic pp65 localization at the indicated time points. Percentages were calculated based on the total number of infected cells counted at each time point; the numbers of cells counted and percentages are listed in Table 1. (B) The detection of the cytoplasmic AC correlated with the logarithmic increase in virus production. Viral titers were determined as described in Materials and Methods. Data are representative of three independent experiments. The results from one experiment are shown. Error bars show standard deviations.
FIG 4
FIG 4
Treatment of HCMV-infected cells with the antiviral cidofovir inhibits Golgi membrane fragmentation and AC formation. HF cells were plated on glass coverslips and infected as described in the legend to Fig. 1. The inoculum was removed and replaced with medium or medium containing 30 µM cidofovir. Coverslips were fixed in PFA on day 4 p.i., and cells were analyzed for viral protein expression and changes in Golgi membrane morphology by confocal microscopy. (A) Intact Golgi membrane morphology was retained when HCMV-infected cells were treated with cidofovir. Cells were stained with antibodies reactive with IE1 (merge: green), pp65 (merge: blue), GM130 (merge: red), and DAPI (merge: gray). Top, medium-treated cells (media); bottom, cidofovir-treated cells (CDV). (B) Immunoblot of IE1 expression and early and late virus-encoded proteins in CDV-treated and untreated HCMV-infected cells. Cultures of infected cells were treated with 30 µM CDV or left untreated and harvested on the indicated days postinfection. Lysates were subjected to immunoblotting and probed with a mixture of anti-IE1 (exon 4), anti-UL57, anti-UL86 (MCP), and anti-UL44 MAbs. Note the lack of expression of early and late proteins in CDV-treated cells. Numbers to the left show molecular weight (in thousands). (C) Cidofovir treatment inhibits Golgi membrane fragmentation and AC morphogenesis. Confocal microscopy was used to score the numbers of infected cells displaying the phenotypes illustrated in panel A. The number of cells analyzed is listed above each bar. Cells with fragmented Golgi membranes or AC were not identified in the CDV-treated cultures. Experiments were repeated 2 times, and data shown are from one representative experiment.
FIG 5
FIG 5
Grasp65 is phosphorylated during HCMV infection. HF cells were plated and grown to confluence prior to serum starvation for 72 h to synchronize cells. Cultures were infected with HCMV AD 169 at an MOI of 1. (A) Steady-state levels of Grasp55 are increased by HCMV infection compared to the level in an uninfected, asynchronous population (cycling) or uninfected, noncycling cells (confluent). (B) Grasp65 levels increase during infection, and higher-molecular-weight forms can be detected in cycling and infected cells. (C) HCMV infection induces higher-molecular-weight forms of Grasp65 that are eliminated with calf intestinal phosphatase (CIP) treatment but not by incubation in a cocktail of phosphatase inhibitors (PIC). Numbers to the left of each gel show molecular weight (in thousands).
FIG 6
FIG 6
Construction of the Grasp65 phosphorylation-negative mutant. Grasp65wt was cloned into pEGFP-N1 to generate Grasp65-GFP. Overlapping mutagenic primers were used to generate alanine mutations at serines and threonines located in mitotic kinase target sequences in the S/P domain. The resulting mutant was designated Grasp65-7A. To generate lentiviral constructs, wt and mutant Grasp65-7A–GFP constructs were subcloned into the pLVX-puro vector.
FIG 7
FIG 7
Grasp65-7A–GFP mutant is hypophosphorylated compared to Grasp65wt-GFP in HCMV-infected cells. HF cells were transduced with recombinant lentiviruses expressing either Grasp65wt-GFP or Grasp65-7A–GFP and synchronized prior to infection with HCMV AD 169 at an MOI of 1. Cultures were harvested on day 3 and 4 postinfection, and lysates analyzed for Grasp65wt or Grasp65-7A GFP-tagged protein as described in the legend to Fig. 5. Treatment of infected cell lysates with calf intestinal phosphatase (CIP +) increased the migration of higher-molecular-weight species of Grasp65wt-GFP but did not alter the migration of Grasp65-7A–GFP, suggesting that the Grasp65-7A–GFP mutant is hypophosphorylated during infection compared to Grasp65wt-GFP.
FIG 8
FIG 8
Expression of Grasp65-7A–GFP reduces fragmentation of the Golgi membrane ribbon in HCMV-infected cells. HF cells were transduced with recombinant lentiviruses expressing either Grasp65wt-GFP or Grasp65-7A–GFP, plated on coverslips, and synchronized prior to infection with HCMV AD 169 at an MOI of 1. Coverslips were collected on days 3 and 4 p.i. and then stained with antibodies to detect GM130 (merge: red), pp65 (merge: blue), and DAPI (merge: gray). (A) The phosphorylation-negative Grasp65-7A mutant inhibits Golgi membrane fragmentation. Panels show representative images of Golgi membrane morphology in cells expressing Grasp65wt-GFP (top) or Grasp65-7A–GFP (bottom) at day 3 p.i. (B) Confocal microscopy was used to score the number of infected cells displaying fragmented Golgi membrane on days 3 and 4 postinfection. Percentages were calculated based on the total number of infected cells (Grasp65wt, n = 225; Grasp65-7A, n = 238) counted at each time point. The experiment was repeated 2 times, and the data shown are from a single experiment. Error bars show standard deviations. (C) GM130+ membrane tubules are longer in cells expressing Grasp65-7A–GFP than in cells expressing Grasp65wt-GFP. The Freehand Trace feature in the FluoView 10.1 software (Olympus Corporation) was used to measure the length of individual GM130+ membrane tubules in a subset of infected cells displaying the fragmented Golgi membrane phenotype (Grasp65wt-GFP, n = 16; Grasp65-7A–GFP, n = 13). Individual membrane lengths are plotted, and median values and standard deviations are shown. Differences between groups were analyzed using the Mann-Whitney test (*, P < 0.05; ***, P < 0.001). The experiments were repeated twice, and the data shown are from one experiment.
FIG 9
FIG 9
Expression of the Grasp65-7A–GFP mutant inhibits AC morphogenesis. HF cell lines expressing either Grasp65wt-GFP or Grasp65-7A–GFP were plated on glass coverslips and allowed to grow until confluent prior to infection with HCMV AD 169 at an MOI of 1. Coverslips were collected 3 and 4 days p.i. and fixed with PFA. Coverslips were stained with antibodies to viral proteins pp65 (merge: blue), the envelope protein gM (merge: red), and DAPI (merge: gray). (A) The phospho-negative Grasp65-7A mutant inhibits AC formation. Panels show representative images of the AC in cells expressing Grasp65wt-GFP (top) or Grasp65-7A–GFP (bottom) at day 4 p.i. (B) Confocal microscopy was used to score the number of GFP-positive, HCMV-infected cells displaying a fully formed AC as shown in panel A. Percentages were calculated based on the total numbers of infected cells (Grasp65wt, n = 205; Grasp65-7A, n = 219) counted at each time point. The experiment was repeated 2 times, and the data shown are from a single experiment. Error bars show standard deviations.
FIG 10
FIG 10
Expression of Grasp65-7A–GFP inhibits assembly of infectious HCMV virus particles. HF cells expressing either Grasp65wt-GFP or Grasp65-7A–GFP were plated and grown to confluence prior to infection with HCMV AD 169 at an MOI of 1. Viral supernatant was collected every 24 h and analyzed for the production of viral progeny in a multicycle virus yield assay. (A) Expression of Grasp65-7A reduces virus yield compared to the yield when Grasp65wt is expressed. At the times indicated, supernatants were collected from HCMV-infected cultures expressing Grasp65wt or mutant Grasp65-7A protein, and infectious virus production was determined as described in Materials and Methods. (B) The release of virus particles containing DNA was not significantly altered by the expression of mutant Grasp65-7A. Quantification of HCMV DNA released into supernatant shows that release of viral DNA is similar in transduced cells. (C) Expression of Grasp65-7A increases the proportion of defective viral particles compared to the proportion in cultures expressing Grasp65wt. The particle/infectious unit ratio was calculated using the data shown in panels A and B. Results are expressed as mean values ± standard deviations.
FIG 11
FIG 11
Expression of Grasp65-7A–GFP alters the envelope protein composition of extracellular virions. Transduced cells expressing either Grasp65wt-GFP or Grasp65-7A–GFP were plated and allowed to grow to confluence prior to infection with HCMV AD 169 at an MOI of 1. Extracellular virions were pelleted on day 4 p.i. by high-speed centrifugation through a 25% sorbitol cushion and analyzed by immunoblotting. (A) Expression of mutant Grasp65-7A alters virion composition. Panels show representative blots of envelope proteins (gB and gH), tegument proteins (pp150, pp65, and pp28), and minor capsid protein (pUL85) in extracellular particles collected from cells expressing Grasp65wt or Grasp65-7A. (B) Expression of Grasp65-7A leads to reduced incorporation of gH into extracellular particles compared to that in control samples. Plots show the densitometric ratio of the respective protein bands from Western blots of virions pelleted from infected cells expressing Grasp65-7A or Grasp65wt. Densitometry of the respective bands was accomplished by generating multiple exposures to create a linear range of densities and calculating density using Image Studio Lite version 5.2 software (Li-Cor). Ratios are expressed as mean values ± standard deviations. (C) The genome copy numbers were similar for pelleted virions prepared from supernatants of infected cell cultures expressing Grasp65wt (8.67log) and Grasp-7A (8.53log).
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References

    1. Britt W. 2008. Manifestations of human cytomegalovirus infection: proposed mechanisms of acute and chronic disease. Curr Top Microbiol Immunol 325:417–470. doi:10.1007/978-3-540-77349-8_23. - DOI - PubMed
    1. Yamamoto AY, Mussi-Pinhata MM, Isaac MD, Amaral FR, Carvalheiro CG, Aragon DC, Manfredi AK, Boppana SB, Britt WJ. 2011. Congenital cytomegalovirus infection as a cause of sensorineural hearing loss in a highly immune population. Pediatr Infect Dis J 30:1043–1046. doi:10.1097/INF.0b013e31822d9640. - DOI - PMC - PubMed
    1. Griffiths P, Baraniak I, Reeves M. 2015. The pathogenesis of human cytomegalovirus. J Pathol 235:288–297. doi:10.1002/path.4437. - DOI - PubMed
    1. Zhang A, Hildreth RL, Colberg-Poley AM. 2013. Human cytomegalovirus inhibits apoptosis by proteasome-mediated degradation of Bax at endoplasmic reticulum-mitochondrion contacts. J Virol 87:5657–5668. doi:10.1128/JVI.00145-13. - DOI - PMC - PubMed
    1. Brune W. 2011. Inhibition of programmed cell death by cytomegaloviruses. Virus Res 157:144–150. doi:10.1016/j.virusres.2010.10.012. - DOI - PubMed

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