Potent Inhibition of Human Cytomegalovirus by Modulation of Cellular SNARE Syntaxin 5

ABSTRACT Formation of the cytoplasmic viral assembly compartment (cVAC) is an important step for efficient human cytomegalovirus (HCMV) assembly. To do this, the virus must alter and repurpose the normal cellular balance of membrane and protein flux, a process that is not well understood. Although a recent screen identified three viral proteins essential for cVAC formation, less is known about the contribution of cellular factors. We show that HCMV infection increases the protein level of a cellular trafficking factor, syntaxin 5 (STX5), a member of the syntaxin family of SNARE proteins. STX5 is recruited to the cVAC in infected cells and is required for the efficient production of infectious virions. We find that STX5 is important for normal cVAC morphology and the proper localization of viral proteins. A previously identified inhibitor of trafficking, Retro94, causes the mislocalization of STX5, an altered cVAC morphology, and dispersal of viral proteins. The presence of Retro94 results in severely impaired production of infectious virions, with a decrease as great as 5 logs. We show that this inhibition is conserved among different strains of HCMV and the various cell types that support infection, as well as for murine CMV. Thus, our data identify a key cellular trafficking factor important for supporting HCMV infection. IMPORTANCE Human cytomegalovirus (HCMV) infection causes severe disease and mortality in immunocompromised individuals, including organ transplant and AIDS patients. In addition, infection of a developing fetus may result in lifelong complications such as deafness and learning disabilities. Understanding in detail the processes involved in HCMV replication is important for developing novel treatments. One of these essential processes, assembly of infectious virions, takes places in the cytoplasmic viral assembly compartment. We identify a cellular protein, syntaxin 5, important for generating this compartment, and show that it is required for the efficient production of infectious virions. We also show that a small molecule that disrupts this protein also significantly reduces the amount of infectious virions that are generated. Thus, by pinpointing a cellular protein that is important in the replication cycle of HCMV, we identified a novel target that can be pursued for therapeutic intervention.

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Pattern of the Epitope-Specific IgG/IgM Response against Human Cytomegalovirus in Patients with Multiple Myeloma

Clinical and Vaccine Immunology ◽

10.1128/cvi.00317-13 ◽

2013 ◽

Vol 20 (8) ◽

pp. 1298-1304 ◽

Cited By ~ 3

Author(s):

Elke Bogner ◽

Gabriele Pecher

Keyword(s):

Multiple Myeloma ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Severe Disease ◽

Organ Transplant ◽

Clinical Diagnostics ◽

Enzyme Linked Immunosorbent Assay ◽

Recent Infection ◽

Early Therapy ◽

Igg Avidity

ABSTRACTHuman cytomegalovirus (HCMV) is a member of the herpesvirus family and represents a major human pathogen causing severe disease in newborns and immunocompromised patients, e.g., organ transplant recipients and patients with AIDS. One characteristic of herpesviruses is their ability to establish lifelong latency in their hosts; thus, reactivation during immunosuppression leads to recurrent episodes of disease. In several recent reports, it has been shown that HCMV infection may occur in patients with malignancy. This study focused on HCMV infection in patients with multiple myeloma (MM). In order to determine the IgM and IgG humoral immune response, sera from MM patients and healthy donors were analyzed with an accredited immunoblot test, and the IgM response was analyzed with an accredited enzyme-linked immunosorbent assay. A response against HCMV was detected in 80% of the MM patients. While the IgG pattern varied in each patient, the most prominent IgM response was against the tegument protein pp150 and two nonstructural proteins, the processivity factor (pUL44) and the single-stranded DNA binding protein (pUL57). An IgG avidity test revealed that 4 out of 20 MM patients had a fresh infection and 2 MM patients had a recent infection. The combination of IgG avidity and the IgM pattern will be a useful tool for reliable clinical diagnostics concerning HCMV and for application of early therapy for those MM patients suffering from a high viral load.

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Remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pUL50 and pUL53

Journal of General Virology ◽

10.1099/vir.0.83377-0 ◽

2008 ◽

Vol 89 (3) ◽

pp. 731-740 ◽

Cited By ~ 49

Author(s):

Daria Camozzi ◽

Sara Pignatelli ◽

Cecilia Valvo ◽

Giovanna Lattanzi ◽

Cristina Capanni ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Nuclear Lamina ◽

Viral Proteins ◽

Efficient Production ◽

Nuclear Lamins ◽

Viral Egress ◽

Infected Cells ◽

Quantitative Alteration ◽

Cellular Components

A fundamental step in the efficient production of human cytomegalovirus (HCMV) progeny is viral egress from the nucleus to the cytoplasm of infected cells. In the family Herpesviridae, this process involves alteration of nuclear lamina components by two highly conserved proteins, whose homologues in HCMV are named pUL50 and pUL53. This study showed that HCMV infection induced the mislocalization of nuclear lamins and that pUL50 and pUL53 play a role in this event. At late stages of infection, both lamin A/C and lamin B showed an irregular distribution on the nuclear rim, coincident with areas of pUL53 accumulation. No variations in the total amount of nuclear lamins could be detected, supporting the view that HCMV induces a qualitative, rather than a quantitative, alteration of these cellular components, as has been suggested previously for other herpesviruses. Interestingly, pUL53, in the absence of other viral products, localized diffusely in the nucleus, whilst the co-expression and interaction of pUL53 with its partner, pUL50, restored its nuclear rim localization in distinct patches, thus indicating that pUL50 is sufficient to induce the localization of pUL53 observed during virus infection. Importantly, analysis of the nuclear lamina in the presence of pUL50–pUL53 complexes at the nuclear boundary and in the absence of other viral products showed that the two viral proteins were sufficient to promote alterations of lamins, strongly resembling those observed during HCMV infection. These results suggest that pUL50 and pUL53 may play an important role in the exit of virions from the nucleus by inducing structural modifications of the nuclear lamina.

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Human Cytomegalovirus Drives WDR5 to the Virion Assembly Compartment to Facilitate Virion Assembly

10.1101/2020.08.03.235630 ◽

2020 ◽

Author(s):

Bo Yang ◽

YongXuan Yao ◽

Hui Wu ◽

Hong Yang ◽

Xue-Hui Ma ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Antiviral Treatment ◽

Critical Role ◽

Virion Assembly ◽

Potential Target ◽

Nuclear Egress ◽

Early Endosomes ◽

Hcmv Replication ◽

Assembly Compartment

AbstractWe previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WDR5 to facilitate capsid nuclear egress. Here, we further show that HCMV infection drives WDR5 to the perinuclear region by a mechanism that requires viral replication and intact microtubules. WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 interacted with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain the increasing WDR5 accumulation in the vAC during infection. WDR5 was then incorporated into HCMV virions and localized to the tegument layer, as demonstrated by fractionation and immune-gold electron microscopy. Thus, WDR5 is driven to the vAC and incorporated into virions, suggesting that WDR5 facilitates HCMV replication at later stage of virion assembly besides the capsid nuclear egress stage. These data highlight that WDR5 is a potential target for antiviral therapy.ImportanceHuman cytomegalovirus (HCMV) has a large (~235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are profoundly reconfigured to establish the virion assembly compartment (vAC), which is important for efficient assembly of progeny virions. We previously reported that WDR5 promotes HCMV nuclear egress. Here, we show that WDR5 is further driven to the vAC and incorporated into virions, perhaps to facilitate efficient virion maturation. This work identified potential roles for WDR5 in HCMV replication in the cytoplasmic stages of virion assembly. Taken together, WDR5 plays a critical role in HCMV capsid nuclear egress and is important for virion assembly, and thus is a potential target for antiviral treatment of HCMV-associated diseases.

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Human cytomegalovirus seroprevalence and titres in solid organ transplant recipients and transplant donors in Seoul, South Korea

BMC Infectious Diseases ◽

10.1186/s12879-019-4607-x ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 3

Author(s):

Yeonju La ◽

Da Eun Kwon ◽

Seul Gi Yoo ◽

Kyoung Hwa Lee ◽

Sang Hoon Han ◽

...

Keyword(s):

South Korea ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Organ Transplant ◽

Solid Organ Transplant ◽

Haematopoietic Stem Cell ◽

Solid Organ ◽

Transplant Recipients ◽

Seropositive Rate ◽

Healthy Donors

Abstract Background Human cytomegalovirus (HCMV) can cause poor outcomes in solid organ transplant (SOT) recipients; moreover, it is associated with cardiovascular diseases (CVD) in the general population. Accordingly, anti-HCMV immunoglobulin G (IgG) seroepidemiology may be useful in identifying the risk of post-SOT HCMV infection or disease as well as immunosenescence or CVD. However, HCMV seroprevalence and titre have not been fully evaluated with regard to age distribution or compared between SOT recipients and healthy individuals in South Korea. Methods We retrospectively retrieved all unduplicated anti-HCMV IgG results of individuals aged > 1 year evaluated between July 2006 and November 2017 at Severance Hospital in Seoul. The cohort, excluding haematopoietic stem cell transplant recipients and subjects with equivocal values, included 2184 SOT recipients and 3015 healthy transplant donors. All IgG results in the SOT recipients were measured during the pre-transplant period. Results The overall IgG seroprevalence and titres were significantly higher among SOT recipients than among healthy donors (98.7% vs. 88.6%, p < 0.001, and 64.7 ± 44.3 vs. 49.8 ± 20.6 arbitrary units/mL, p < 0.001, respectively). The lowest seropositive rate in the SOT group was observed in recipients aged between 11 and 15 years (70.6%). The frequency of seropositivity among adults aged ≥41 years increased to ≥90% in SOT recipients and healthy donors. Age was independently associated with higher HCMV seroprevalence (41–60 years, OR, 76.4, 95% CI, 24.5–238.9, p < 0.001; ≥ 61 years, OR, 4.4, 95% CI, 1.3–14.9, p < 0.001, compared to ≤40 years). The healthy donor group had an independently low HCMV seropositive rate (OR, 0.1, 95% CI, 0.1–0.2, p < 0.001). Conclusions HCMV seropositivity was the lowest among school-aged children and adolescents. IgG testing revealed an intermediate serostatus risk of post-transplant HCMV infection and disease for most adult SOT recipients in South Korea.

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Role of the Endoplasmic Reticulum Chaperone BiP, SUN Domain Proteins, and Dynein in Altering Nuclear Morphology during Human Cytomegalovirus Infection

Journal of Virology ◽

10.1128/jvi.00719-10 ◽

2010 ◽

Vol 84 (14) ◽

pp. 7005-7017 ◽

Cited By ~ 62

Author(s):

Nicholas J. Buchkovich ◽

Tobi G. Maguire ◽

James C. Alwine

Keyword(s):

Endoplasmic Reticulum ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Nuclear Periphery ◽

Nuclear Lamina ◽

Nuclear Morphology ◽

Morphological Alterations ◽

Endoplasmic Reticulum Protein ◽

Assembly Compartment ◽

Sun Domain

ABSTRACT The process of assembly and egress of human cytomegalovirus (HCMV) virions requires significant morphological alterations of the nuclear and cytoplasmic architecture. In the studies presented we show that the nuclear periphery is dramatically altered, especially near the cytoplasmic assembly compartment, where the nuclear lamina is specifically rearranged, the outer nuclear membrane is altered, and the nucleus becomes permeable to large molecules. In addition, the tethering of the inner and outer nuclear membranes is lost during infection due to a decrease in levels of the SUN domain proteins. We previously demonstrated that the endoplasmic reticulum protein BiP functions as a component of the assembly compartment and disruption of BiP causes the loss of assembly compartment integrity. In this study we show that the depletion of BiP, and the loss of assembly compartment integrity, results in the loss of virally induced lamina rearrangement and morphology of the nucleus that is characteristic of HCMV infection. BiP functions in lamina rearrangement through its ability to affect lamin phosphorylation. Depletion of BiP and disruption of the assembly compartment result in the loss of lamin phosphorylation. The dependency of lamin phosphorylation on BiP correlates with an interaction between BiP and UL50. Finally, we confirm previous data (S. V. Indran, M. E. Ballestas, and W. J. Britt, J. Virol. 84:3162-3177, 2010) suggesting an involvement of dynein in assembly compartment formation and extend this observation by showing that when dynein is inhibited, the nuclear morphology characteristic of an HCMV infection is lost. Our data suggest a highly integrated assembly-egress continuum.

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Human Cytomegalovirus Seroprevalence and Titres in Solid Organ Transplant Recipients and Transplant Donors in Seoul, South Korea

10.21203/rs.2.9312/v3 ◽

2019 ◽

Author(s):

Yeonju La ◽

Da Eun Kwon ◽

Seul Gi Yoo ◽

Kyoung Hwa Lee ◽

Sang Hoon Han ◽

...

Keyword(s):

South Korea ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Organ Transplant ◽

Solid Organ Transplant ◽

Haematopoietic Stem Cell ◽

Solid Organ ◽

Transplant Recipients ◽

Seropositive Rate ◽

Healthy Donors

Abstract Background: Human cytomegalovirus (HCMV) can cause poor outcomes in solid organ transplant (SOT) recipients; moreover, it is associated with cardiovascular diseases (CVD) in the general population. Accordingly, anti-HCMV immunoglobulin G (IgG) seroepidemiology may be useful in identifying the risk of post-SOT HCMV infection or disease as well as immunosenescence or CVD. However, HCMV seroprevalence and titre have not been fully evaluated with regard to age distribution or compared between SOT recipients and healthy individuals in South Korea. Methods: We retrospectively retrieved all unduplicated anti-HCMV IgG results of individuals aged > 1 year evaluated between July 2006 and November 2017 at Severance Hospital in Seoul. The cohort, excluding haematopoietic stem cell transplant recipients and subjects with equivocal values, included 2184 SOT recipients and 3015 healthy transplant donors. All IgG results in the SOT recipients were measured during the pre-transplant period. Results: The overall IgG seroprevalence and titres were significantly higher among SOT recipients than among healthy donors (98.7% vs. 88.6%, p < 0.001, and 64.7 ± 44.3 vs. 49.8 ± 20.6 arbitrary units/mL, p < 0.001, respectively). The lowest seropositive rate in the SOT group was observed in recipients aged between 11 and 15 years (70.6%). The frequency of seropositivity among adults aged ≥ 41 years increased to ≥ 90% in SOT recipients and healthy donors. Age was independently associated with higher HCMV seroprevalence (41–60 years, OR, 76.4, 95% CI, 24.5–238.9, p < 0.001; ≥ 61 years, OR, 4.4, 95% CI, 1.3–14.9, p < 0.001, compared to ≤ 40 years). The healthy donor group had an independently low HCMV seropositive rate (OR, 0.1, 95% CI, 0.1–0.2, p < 0.001). Conclusions: HCMV seropositivity was the lowest among school-aged children and adolescents. IgG testing revealed an intermediate serostatus risk of post-transplant HCMV infection and disease for most adult SOT recipients in South Korea.

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Human Cytomegalovirus Envelope Protein gpUL132 Regulates Infectious Virus Production through Formation of the Viral Assembly Compartment

mBio ◽

10.1128/mbio.02044-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Hui Wu ◽

Barbara Kropff ◽

Michael Mach ◽

William J. Britt

Keyword(s):

Human Cytomegalovirus ◽

Envelope Glycoprotein ◽

Envelope Protein ◽

Infectious Virus ◽

Virus Production ◽

Viral Assembly ◽

Mutant Virus ◽

Infected Cells ◽

Assembly Compartment ◽

Infectious Unit

ABSTRACT The human cytomegalovirus (HCMV) UL132 open reading frame encodes a 270-amino-acid type I envelope glycoprotein, gpUL132. The deletion of UL132 (ΔUL132) from the HCMV genome results in a pronounced deficit in virus yield, with an approximately 2-log decrease in the production of infectious virus compared to the wild-type (WT) virus. Characterization of the ΔUL132 mutant virus indicated that it was less infectious with a high particle-to-infectious unit ratio and an altered composition of virion proteins compared to the WT virus. In addition, the viral assembly compartment (AC) failed to form in cells infected with the ΔUL132 mutant virus. The expression of gpUL132 in trans rescued the defects in the morphogenesis of the AC in cells infected with the ΔUL132 mutant virus and in infectious virus production. Furthermore, using cell lines expressing chimeric proteins, we demonstrated that the cytosolic domain of gpUL132 was sufficient to rescue AC formation and WT levels of virus production. Progeny virions from ΔUL132-infected cells expressing the cytosolic domain of gpUL132 exhibited particle-to-infectious unit ratios similar to those of the WT virus. Together, our findings argue that gpUL132 is essential for HCMV AC formation and the efficient production of infectious particles, thus highlighting the importance of this envelope protein for the virus-induced reorganization of intracellular membranes and AC formation in the assembly of infectious virus. IMPORTANCE Following infection of permissive cells, human cytomegalovirus (HCMV) induces the reorganization of intracellular membranes resulting in the formation of a distinctive membranous compartment in the cytoplasm of infected cells. This compartment has been designated the viral assembly compartment (AC) and is thought to be a site for cytoplasmic virion assembly and envelopment. In this study, we have demonstrated that a single virion envelope glycoprotein is essential for AC formation in infected cells, and in its absence, there is a significant decrease in the production of infectious virions. These findings are consistent with those from other studies that have demonstrated the importance of host cell proteins in the formation of the AC and demonstrate a critical role of a single virion protein in AC formation and the efficient assembly of infectious virus.

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The SARS-CoV-2 nucleocapsid protein associates with the replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

10.1101/2021.06.15.448497 ◽

2021 ◽

Author(s):

Katharina M Scherer ◽

Luca Mascheroni ◽

George W Carnell ◽

Lucia C S Wunderlich ◽

Stanislaw Makarchuk ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Rna Replication ◽

Viral Proteins ◽

Viral Assembly ◽

Sequence Of Events ◽

Spatiotemporal Regulation ◽

Transport Vesicles ◽

Replication Foci ◽

Assembly Compartment

Despite being the target of extensive research efforts due to the COVID-19 pandemic, relatively little is known about the dynamics of SARS-CoV-2 replication within cells. We investigate and characterise the tightly orchestrated sequence of events during different stages of the infection cycle by visualising the spatiotemporal dynamics of the four structural proteins of SARS-CoV-2 at high resolution. The nucleoprotein is expressed first and accumulates around folded ER membranes in convoluted layers that connect to viral RNA replication foci. We find that of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ERGIC before the spike and envelope proteins. Relocation of the lysosome marker LAMP1 towards the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide new insights into the spatiotemporal regulation of SARS-CoV-2 assembly, and refine current understanding of SARS-CoV-2 replication.

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Human Cytomegalovirus Seroprevalence and Titres in Solid Organ Transplant Recipients and Transplant Donors in Seoul, South Korea

10.21203/rs.2.9312/v2 ◽

2019 ◽

Author(s):

Yeonju La ◽

Da Eun Kwon ◽

Seul Gi Yoo ◽

Kyoung Hwa Lee ◽

Sang Hoon Han ◽

...

Keyword(s):

South Korea ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Organ Transplant ◽

Solid Organ Transplant ◽

Haematopoietic Stem Cell ◽

Solid Organ ◽

Transplant Recipients ◽

Seropositive Rate ◽

Healthy Donors

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Restriction of Human Cytomegalovirus Infection by Galectin-9

Journal of Virology ◽

10.1128/jvi.01746-18 ◽

2018 ◽

Vol 93 (3) ◽

Cited By ~ 4

Author(s):

Emily A. Machala ◽

Selmir Avdic ◽

Lauren Stern ◽

Dirk M. Zajonc ◽

Chris A. Benedict ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Human Herpesvirus ◽

Temperature Shift ◽

Cell Types ◽

Cellular Protein ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Immune Systems ◽

Wide Range

ABSTRACTHuman cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. While HCMV infection is generally asymptomatic in the immunocompetent, it can have devastating consequences in those with compromised or underdeveloped immune systems, including transplant recipients and neonates. Galectins are a widely expressed protein family that have been demonstrated to modulate both antiviral immunity and regulate direct host-virus interactions. The potential for galectins to directly modulate HCMV infection has not previously been studied, and our results reveal that galectin-9 (Gal-9) can potently inhibit HCMV infection. Gal-9-mediated inhibition of HCMV was dependent upon its carbohydrate recognition domains and thus dependent on glycan interactions. Temperature shift studies revealed that Gal-9 specific inhibition was mediated primarily at the level of virus-cell fusion and not binding. Additionally, we found that during reactivation of HCMV in hematopoietic stem cell transplant (HSCT) patients soluble Gal-9 is upregulated. This study provides the first evidence for Gal-9 functioning as a potent antiviral defense effector molecule against HCMV infection and identifies it as a potential clinical candidate to restrict HCMV infections.IMPORTANCEHuman cytomegalovirus (HCMV) continues to cause serious and often life-threatening disease in those with impaired or underdeveloped immune systems. This virus is able to infect and replicate in a wide range of human cell types, which enables the virus to spread to other individuals in a number of settings. Current antiviral drugs are associated with a significant toxicity profile, and there is no vaccine; these factors highlight a need to identify additional targets for the development of anti-HCMV therapies. We demonstrate for the first time that secretion of a member of the galectin family of proteins, galectin-9 (Gal-9), is upregulated during natural HCMV-reactivated infection and that this soluble cellular protein possesses a potent capacity to block HCMV infection by inhibiting virus entry into the host cell. Our findings support the possibility of harnessing the antiviral properties of Gal-9 to prevent HCMV infection and disease.

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