Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site

ABSTRACT The initial events of viral infection at the primary mucosal entry site following horizontal person-to-person transmission have remained ill defined. Our limited understanding is further underscored by the absence of animal models in the case of human-restricted viruses, such as human cytomegalovirus (HCMV), a leading cause of congenital infection and a major pathogen in immunocompromised individuals. Here, we established a novel ex vivo model of HCMV infection in native human nasal turbinate tissues. Nasal turbinate tissue viability and physiological functionality were preserved for at least 7 days in culture. We found that nasal mucosal tissues were susceptible to HCMV infection, with predominant infection of ciliated respiratory epithelial cells. A limited viral spread was demonstrated, involving mainly stromal and vascular endothelial cells within the tissue. Importantly, functional antiviral and proleukocyte chemotactic signaling pathways were significantly upregulated in the nasal mucosa in response to infection. Conversely, HCMV downregulated the expression of nasal epithelial cell-related genes. We further revealed tissue-specific innate immune response patterns to HCMV, comparing infected human nasal mucosal and placental tissues, representing the viral entry and the maternal-to-fetal transmission sites, respectively. Taken together, our studies provide insights into the earliest stages of HCMV infection. Studies in this model could help evaluate new interventions against the horizontal transmission of HCMV. IMPORTANCE HCMV is a ubiquitous human pathogen causing neurodevelopmental disabilities in congenitally infected children and severe disease in immunocompromised patients. The earliest stages of HCMV infection in the human host have remained elusive in the absence of a model for the viral entry site. Here, we describe the establishment and use of a novel nasal turbinate organ culture to study the initial steps of viral infection and the consequent innate immune responses within the natural complexity and the full cellular repertoire of human nasal mucosal tissues. This model can be applied to examine new antiviral interventions against the horizontal transmission of HCMV and potentially that of other viruses.

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The IκB Kinases Restrict Human Cytomegalovirus Infection

Journal of Virology ◽

10.1128/jvi.02030-18 ◽

2019 ◽

Vol 93 (9) ◽

Cited By ~ 3

Author(s):

Christopher M. Goodwin ◽

Joshua Munger

Keyword(s):

Viral Replication ◽

Viral Infection ◽

Host Cell ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Innate Immune ◽

Immune Signaling ◽

Innate Immune Signaling ◽

Iκb Kinase ◽

Cell Spread

ABSTRACTHuman cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes disease in immunosuppressed populations. HCMV has a complex relationship with innate immune signaling pathways. Specifically, HCMV has been found to block some aspects of inflammatory signaling while benefiting from others. Through analysis of knockout cell lines targeting the NF-κB regulatory kinases IκB kinase α (IKKα) and IKKβ, we find that the IKKs are host restriction factors that contribute to cytokine-mediated resistance to viral infection, limit the initiation of HCMV infection, and attenuate viral cell-to-cell spread. The HCMV UL26 protein is a viral immune modulator important for HCMV infection that has been shown to inhibit host cell NF-κB signaling, yet it has remained unclear how UL26-mediated NF-κB modulation contributes to infection. Here, we find that UL26 modulation of NF-κB signaling is separable from its contribution to high-titer viral replication. However, we find that IKKβ is required for the induction of cytokine expression associated with ΔUL26 infection. Collectively, our data indicate that the IKKs restrict infection but HCMV targets their signaling to modulate the cellular inflammatory environment.IMPORTANCEInnate immune signaling is a critical defense against viral infection and represents a central host-virus interaction that frequently determines the outcomes of infections. NF-κB signaling is an essential component of innate immunity that is extensively modulated by HCMV, a significant cause of morbidity in neonates and immunosuppressed individuals. However, the roles that various facets of NF-κB signaling play during HCMV infection have remained elusive. We find that the two major regulatory kinases in this pathway, IKKα and IKKβ, limit the initiation of infection, viral replication, and cell-to-cell spread. In addition, our results indicate that these kinases contribute differently to the host cell response to infection in the absence of a virally encoded NF-κB inhibitor, UL26. Given the importance of NF-κB in viral infection, elucidating the contributions of various NF-κB constituents to infection is an essential first step toward the possibility of targeting this pathway therapeutically.

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Strategy of Human Cytomegalovirus To Escape Interferon Beta-Induced APOBEC3G Editing Activity

Journal of Virology ◽

10.1128/jvi.01224-18 ◽

2018 ◽

Vol 92 (19) ◽

Cited By ~ 9

Author(s):

Sara Pautasso ◽

Ganna Galitska ◽

Valentina Dell'Oste ◽

Matteo Biolatti ◽

Rachele Cagliani ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hot Spots ◽

Interferon Beta ◽

Hcmv Infection ◽

Viral Infections ◽

Innate Immune ◽

Mutational Robustness ◽

Wide Range ◽

Hcmv Replication ◽

Apobec3 Family

ABSTRACTThe apolipoprotein B editing enzyme catalytic subunit 3 (APOBEC3) is a family of DNA cytosine deaminases that mutate and inactivate viral genomes by single-strand DNA editing, thus providing an innate immune response against a wide range of DNA and RNA viruses. In particular, APOBEC3A (A3A), a member of the APOBEC3 family, is induced by human cytomegalovirus (HCMV) in decidual tissues where it efficiently restricts HCMV replication, thereby acting as an intrinsic innate immune effector at the maternal-fetal interface. However, the widespread incidence of congenital HCMV infection implies that HCMV has evolved to counteract APOBEC3-induced mutagenesis through mechanisms that still remain to be fully established. Here, we have assessed gene expression and deaminase activity of various APOBEC3 gene family members in HCMV-infected primary human foreskin fibroblasts (HFFs). Specifically, we show that APOBEC3G (A3G) gene products and, to a lesser degree, those of A3F but not of A3A, are upregulated in HCMV-infected HFFs. We also show that HCMV-mediated induction of A3G expression is mediated by interferon beta (IFN-β), which is produced early during HCMV infection. However, knockout or overexpression of A3G does not affect HCMV replication, indicating that A3G is not a restriction factor for HCMV. Finally, through a bioinformatics approach, we show that HCMV has evolved mutational robustness against IFN-β by limiting the presence of A3G hot spots in essential open reading frames (ORFs) of its genome. Overall, our findings uncover a novel immune evasion strategy by HCMV with profound implications for HCMV infections.IMPORTANCEAPOBEC3 family of proteins plays a pivotal role in intrinsic immunity defense mechanisms against multiple viral infections, including retroviruses, through the deamination activity. However, the currently available data on APOBEC3 editing mechanisms upon HCMV infection remain unclear. In the present study, we show that particularly the APOBEC3G (A3G) member of the deaminase family is strongly induced upon infection with HCMV in fibroblasts and that its upregulation is mediated by IFN-β. Furthermore, we were able to demonstrate that neither A3G knockout nor A3G overexpression appears to modulate HCMV replication, indicating that A3G does not inhibit HCMV replication. This may be explained by HCMV escape strategy from A3G activity through depletion of the preferred nucleotide motifs (hot spots) from its genome. The results may shed light on antiviral potential of APOBEC3 activity during HCMV infection, as well as the viral counteracting mechanisms under A3G-mediated selective pressure.

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Human Cytomegalovirus Infection Activates and Regulates the Unfolded Protein Response

Journal of Virology ◽

10.1128/jvi.79.11.6890-6899.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 6890-6899 ◽

Cited By ~ 203

Author(s):

Jennifer A. Isler ◽

Alison H. Skalet ◽

James C. Alwine

Keyword(s):

Endoplasmic Reticulum ◽

Transcription Factor ◽

Endoplasmic Reticulum Stress ◽

Viral Infection ◽

Unfolded Protein Response ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

ABSTRACT Viral infection causes stress to the endoplasmic reticulum. The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover by attenuating translation and upregulating the expression of chaperones, degradation factors, and factors that regulate the cell's metabolic and redox environment. Some consequences of the UPR (e.g., expression of chaperones and regulation of the metabolism and redox environment) may be advantageous to the viral infection; however, translational attenuation would not. Thus, viruses may induce mechanisms which modulate the UPR, maintaining beneficial aspects and suppressing deleterious aspects. We demonstrate that human cytomegalovirus (HCMV) infection induces the UPR but specifically regulates the three branches of UPR signaling, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE-1), to favor viral replication. HCMV infection activated the eIF2α kinase PERK; however, the amount of phosphorylated eIF2α was limited and translation attenuation did not occur. Interestingly, translation of select mRNAs, which is dependent on eIF2α phosphorylation, did occur, including the transcription factor ATF4, which activates genes which may benefit the infection. The endoplasmic reticulum stress-induced activation of the transcription factor ATF6 was suppressed in HCMV-infected cells; however, specific chaperone genes, normally activated by ATF6, were activated by a virus-induced, ATF6-independent mechanism. Lastly, HCMV infection activated the IRE-1 pathway, as indicated by splicing of Xbp-1 mRNA. However, transcriptional activation of the XBP-1 target gene EDEM (ER degradation-enhancing α-mannosidase-like protein, a protein degradation factor) was inhibited. These results suggest that, although HCMV infection induces the unfolded protein response, it modifies the outcome to benefit viral replication.

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The human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing

PLoS Pathogens ◽

10.1371/journal.ppat.1008807 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1008807

Author(s):

Einat Seidel ◽

Liat Dassa ◽

Corinna Schuler ◽

Esther Oiknine-Djian ◽

Dana G. Wolf ◽

...

Keyword(s):

Nk Cells ◽

Human Cytomegalovirus ◽

Cell Activation ◽

Hcmv Infection ◽

Nk Cell ◽

Innate Immune ◽

Target Cells ◽

Histocompatibility Complex ◽

Immunosuppressed Patients ◽

Infected Cells

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells without prior sensitization. One pivotal activating NK receptor is NKG2D, which binds a family of eight ligands, including the major histocompatibility complex (MHC) class I-related chain A (MICA). Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus causing morbidity and mortality in immunosuppressed patients and congenitally infected infants. HCMV encodes multiple antagonists of NK cell activation, including many mechanisms targeting MICA. However, only one of these mechanisms, the HCMV protein US9, counters the most prevalent MICA allele, MICA*008. Here, we discover that a hitherto uncharacterized HCMV protein, UL147A, specifically downregulates MICA*008. UL147A primarily induces MICA*008 maturation arrest, and additionally targets it to proteasomal degradation, acting additively with US9 during HCMV infection. Thus, UL147A hinders NKG2D-mediated elimination of HCMV-infected cells by NK cells. Mechanistic analyses disclose that the non-canonical GPI anchoring pathway of immature MICA*008 constitutes the determinant of UL147A specificity for this MICA allele. These findings advance our understanding of the complex and rapidly evolving HCMV immune evasion mechanisms, which may facilitate the development of antiviral drugs and vaccines.

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Human Cytomegalovirus Immediate Early 86-kDa Protein Blocks Transcription and Induces Degradation of the Immature Interleukin-1β Protein during Virion-Mediated Activation of the AIM2 Inflammasome

mBio ◽

10.1128/mbio.02510-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

Author(s):

Sara Botto ◽

Jinu Abraham ◽

Nobuyo Mizuno ◽

Kara Pryke ◽

Bryan Gall ◽

...

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Innate Immune ◽

Host Cells ◽

Interleukin 1Β ◽

Immediate Early ◽

Interferon Response ◽

Type I

ABSTRACTSecretion of interleukin-1β (IL-1β) represents a fundamental innate immune response to microbial infection that, at the molecular level, occurs following activation of proteolytic caspases that cleave the immature protein into a secretable form. Human cytomegalovirus (HCMV) is the archetypal betaherpesvirus that is invariably capable of lifelong infection through the activity of numerous virally encoded immune evasion phenotypes. Innate immune pathways responsive to cytoplasmic double-stranded DNA (dsDNA) are known to be activated in response to contact between HCMV and host cells. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) genome editing to demonstrate that the dsDNA receptorabsentinmelanoma 2 (AIM2) is required for secretion of IL-1β following HCMV infection. Furthermore, dsDNA-responsive innate signaling induced by HCMV infection that leads to activation of the type I interferon response is also shown, unexpectedly, to play a contributory role in IL-1β secretion. Importantly, we also show that rendering virus particles inactive by UV exposure leads to substantially increased IL-1β processing and secretion and that live HCMV can inhibit this, suggesting the virus encodes factors that confer an inhibitory effect on this response. Further examination revealed that ectopic expression of the immediate early (IE) 86-kDa protein (IE86) is actually associated with a block in transcription of the pro-IL-1β gene and, independently, diminishment of the immature protein. Overall, these results reveal two new and distinct phenotypes conferred by the HCMV IE86 protein, as well as an unusual circumstance in which a single herpesviral protein exhibits inhibitory effects on multiple molecular processes within the same innate immune response.IMPORTANCEPersistent infection with HCMV is associated with the operation of diverse evasion phenotypes directed at antiviral immunity. Obstruction of intrinsic and innate immune responses is typically conferred by viral proteins either associated with the viral particle or expressed immediately after entry. In line with this, numerous phenotypes are attributed to the HCMV IE86 protein that involve interference with innate immune processes via transcriptional and protein-directed mechanisms. We describe novel IE86-mediated phenotypes aimed at virus-induced secretion of IL-1β. Intriguingly, while many viruses target the function of the molecular scaffold required for IL-1β maturation to prevent this response, we find that HCMV and IE86 target the IL-1β protein specifically. Moreover, we show that IE86 impairs both the synthesis of the IL-1β transcript and the stability of the immature protein. This indicates an unusual phenomenon in which a single viral protein exhibits two molecularly separate evasion phenotypes directed at a single innate cytokine.

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HIV-1 Envelope Glycoprotein at the Interface of Host Restriction and Virus Evasion

Viruses ◽

10.3390/v11040311 ◽

2019 ◽

Vol 11 (4) ◽

pp. 311 ◽

Cited By ~ 3

Author(s):

Saina Beitari ◽

Yimeng Wang ◽

Shan-Lu Liu ◽

Chen Liang

Keyword(s):

Viral Infection ◽

Adaptive Immunity ◽

Viral Entry ◽

Envelope Protein ◽

Envelope Proteins ◽

Viral Envelope ◽

Innate Immune ◽

Host Restriction ◽

Hiv 1 ◽

Viral Envelope Proteins

Without viral envelope proteins, viruses cannot enter cells to start infection. As the major viral proteins present on the surface of virions, viral envelope proteins are a prominent target of the host immune system in preventing and ultimately eliminating viral infection. In addition to the well-appreciated adaptive immunity that produces envelope protein-specific antibodies and T cell responses, recent studies have begun to unveil a rich layer of host innate immune mechanisms restricting viral entry. This review focuses on the exciting progress that has been made in this new direction of research, by discussing various known examples of host restriction of viral entry, and diverse viral countering strategies, in particular, the emerging role of viral envelope proteins in evading host innate immune suppression. We will also highlight the effective cooperation between innate and adaptive immunity to achieve the synergistic control of viral infection by targeting viral envelope protein and checking viral escape. Given that many of the related findings were made with HIV-1, we will use HIV-1 as the model virus to illustrate the basic principles and molecular mechanisms on host restriction targeting HIV-1 envelope protein.

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Human Cytomegalovirus Stimulates the Synthesis of Select Akt-Dependent Antiapoptotic Proteins during Viral Entry To Promote Survival of Infected Monocytes

Journal of Virology ◽

10.1128/jvi.02879-15 ◽

2016 ◽

Vol 90 (6) ◽

pp. 3138-3147 ◽

Cited By ~ 24

Author(s):

Megan A. Peppenelli ◽

Kyle C. Arend ◽

Olesea Cojohari ◽

Nathaniel J. Moorman ◽

Gary C. Chan

Keyword(s):

Life Span ◽

Human Cytomegalovirus ◽

Viral Entry ◽

Peripheral Blood ◽

Primary Infection ◽

Hcmv Infection ◽

Blood Monocytes ◽

Peripheral Blood Monocytes ◽

Viral Dissemination ◽

Antiapoptotic Proteins

ABSTRACTPrimary peripheral blood monocytes are responsible for the hematogenous dissemination of human cytomegalovirus (HCMV) following a primary infection. To facilitate viral spread, we have previously shown HCMV to extend the short 48-h life span of monocytes. Mechanistically, HCMV upregulated two specific cellular antiapoptotic proteins, myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), to block the two proteolytic cleavages necessary for the formation of fully active caspase 3 and the subsequent initiation of apoptosis. We now show that HCMV more robustly upregulated Mcl-1 than normal myeloid growth factors and that Mcl-1 was the only myeloid survival factor to rapidly induce HSP27 prior to the 48-h cell fate checkpoint. We determined that HCMV glycoproteins gB and gH signal through the cellular epidermal growth factor receptor (EGFR) and αvβ3 integrin, respectively, during viral entry in order to drive the increase of Mcl-1 and HSP27 in an Akt-dependent manner. Although Akt is known to regulate protein stability and transcription, we found that gB- and gH-initiated signaling preferentially and cooperatively stimulated the synthesis of Mcl-1 and HSP27 through mTOR-mediated translation. Overall, these data suggest that the unique signaling network generated during the viral entry process stimulates the upregulation of select antiapoptotic proteins allowing for the differentiation of short-lived monocytes into long-lived macrophages, a key step in the viral dissemination strategy.IMPORTANCEHuman cytomegalovirus (HCMV) infection is endemic within the human population. Although primary infection is generally asymptomatic in immunocompetent individuals, HCMV is a significant cause of morbidity and mortality in the immunocompromised. The multiorgan inflammatory diseases associated with symptomatic HCMV infection are a direct consequence of the monocyte-mediated systemic spread of the virus. In order for peripheral blood monocytes to facilitate viral dissemination, HCMV subverts the short 48-h life span of monocytes by inducing the expression of cellular antiapoptotic proteins Mcl-1 and HSP27. Here, we demonstrate that the rapid and simultaneous upregulation of Mcl-1 and HSP27 is a distinctive feature of HCMV-induced monocyte survival. Moreover, we decipher the signaling pathways activated during viral entry needed for the robust synthesis of Mcl-1 and HSP27. Identifying the virus-specific mechanisms used to upregulate select cellular factors required for the survival of HCMV-infected monocytes is important to the development of new classes of anti-HCMV drugs.

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Intrinsic Immune Mechanisms Restricting Human Cytomegalovirus Replication

Viruses ◽

10.3390/v13020179 ◽

2021 ◽

Vol 13 (2) ◽

pp. 179

Author(s):

Eva-Maria Schilling ◽

Myriam Scherer ◽

Thomas Stamminger

Keyword(s):

Human Cytomegalovirus ◽

Viral Entry ◽

Hcmv Infection ◽

Current Knowledge ◽

Cytidine Deaminase ◽

Regulatory Protein ◽

Detailed Knowledge ◽

Restriction Factors ◽

Immune Mechanisms ◽

Early Protein

Cellular restriction factors (RFs) act as important constitutive innate immune barriers against viruses. In 2006, the promyelocytic leukemia protein was described as the first RF against human cytomegalovirus (HCMV) infection which is antagonized by the viral immediate early protein IE1. Since then, at least 15 additional RFs against HCMV have been identified, including the chromatin regulatory protein SPOC1, the cytidine deaminase APOBEC3A and the dNTP triphosphohydrolase SAMHD1. These RFs affect distinct steps of the viral replication cycle such as viral entry, gene expression, the synthesis of progeny DNA or egress. This review summarizes our current knowledge on intrinsic immune mechanisms restricting HCMV replication as well as on the viral strategies to counteract the inhibitory effects of RFs. Detailed knowledge on the interplay between host RFs and antagonizing viral factors will be fundamental to develop new approaches to combat HCMV infection.

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Human Cytomegalovirus Inhibits Granulopoiesis Via Direct Infection and Inducing Apoptosis.

Blood ◽

10.1182/blood.v106.11.3859.3859 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3859-3859

Author(s):

Qing Wen Wang ◽

Zheng Xian He ◽

Xiao Feng Li ◽

Wei Xiong ◽

Mei Lian Chen ◽

...

Keyword(s):

Bone Marrow ◽

Viral Infection ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Annexin V ◽

Pcr Analysis ◽

Control Group ◽

Dependent Manner ◽

Ad169 Strain ◽

Dose Dependent

Abstract Human cytomegalovirus (HCMV) infection can cause delayed leukocyte recovery after bone marrow transplantation and often associated with suppression of granulocyte/macrophage progenitor. Leukocyte lineage may be one of the major sites of HCMV infection. However, whether HCMV can interfere with CFU-GM formation and induce apoptosis in granulocyte/macrophage progenitor have not been well investigated. Human bone marrow mononuclear cells (Ficoll), promyelocyte cell line HL-60 and HCMV AD169 strain were co-cultured. Each bone marrow specimen was HCMV DNA and HCMV IgM negative by PCR and ELISA test. Our results showed that HCMV significantly inhibited the formation of CFU-GM as shown in two different concentrations of viral infection groups: 139.26 ± 5.42 (2×105pfu/ml), and 124.19 ± 8.82 (2×106pfu/ml) (colonies/2×105cells/ml, n=26). These were significant differences compared with the blank control group (P<0.01, P<0.05) respectively. HCMV also significantly inhibited the growth of HL-60 cells in three different concentrations of viral infection groups (101pfu/ml, 102pfu/ml, 103pfu/ml). After incubation for 7 days, viability cells in control group and each infection groups (n=20) were 96%, 83%, 73%, and 64%, showing that HCMV infection decreased the viability of HL-60 cells in a dose-dependent manner. The apoptotic effect was also investigated by Annexin V assay using flow cytometry. The percentage of Annexin V -positive cells were 19.60 ± 1.63 (in group of 101pfu/ml) (P<0.05), 28.70 ± 2.61 (102pfu/ml) (P<0.05), and 36.3 ± 2.57 (103pfu/ml) (P<0.01), compared with control group 3.96 ± 1.75 (n=8). The apoptotic cells were further confirmed by morphologic observation and DNA ladder formation. Furthermore, HCMV DNA was positive in HL-60 cells and cells of CFU-GM measured by PCR and IS-PCR analysis respectively. These were negative in blank and mock control groups. Our data demonstrated that: (1) HCMV AD169 strain inhibited the growth of HL-60 and CFU-GM formation in a dose-dependent manner; (2) HCMV transcription occurred in HL-60 and CFU-GM cells; and (3) This virus induced apoptosis in HL-60 cells. HCMV may have an inhibiting effect on granulopoiesis via direct infection and inducing apoptosis on myeloid progenitors.

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Expression of Oncogenic Alleles Induces Multiple Blocks to Human Cytomegalovirus Infection

Journal of Virology ◽

10.1128/jvi.00179-16 ◽

2016 ◽

Vol 90 (9) ◽

pp. 4346-4356 ◽

Cited By ~ 18

Author(s):

Shihao Xu ◽

Xenia Schafer ◽

Joshua Munger

Keyword(s):

Gene Expression ◽

Human Cytomegalovirus ◽

Viral Entry ◽

Hela Cells ◽

Hcmv Infection ◽

T Antigen ◽

Immediate Early ◽

Sv40 T Antigen ◽

Viral Dna ◽

Cancerous Cells

ABSTRACTIn contrast to many viruses, human cytomegalovirus (HCMV) is unable to productively infect most cancer-derived cell lines. The mechanisms of this restriction are unclear. To explore this issue, we tested whether defined oncogenic alleles, including the simian virus 40 (SV40) T antigen (TAg) and oncogenic H-Ras, inhibit HCMV infection. We found that expression of SV40 TAg blocks HCMV infection in human fibroblasts, whereas the replication of a related herpesvirus, herpes simplex virus 1 (HSV-1), was not impacted. The earliest restriction of HCMV infection involves a block of viral entry, as TAg expression prevented the nuclear delivery of viral DNA and pp65. Subsequently, we found that TAg expression reduces the abundance of platelet-derived growth factor receptor α (PDGFRα), a host protein important for HCMV entry. Viral entry into TAg-immortalized fibroblasts could largely be rescued by PDGFRα overexpression. Similarly, PDGFRα overexpression in HeLa cells markedly increased the levels of HCMV gene expression and DNA replication. However, the robust production of viral progeny was not restored by PDGFRα overexpression in either HeLa cells or TAg-immortalized fibroblasts, suggesting additional restrictions associated with transformation and TAg expression. In TAg-expressing fibroblasts, expression of the immediate early 2 (IE2) protein was not rescued to the same extent as that of the immediate early 1 (IE1) protein, suggesting that TAg expression impacts the accumulation of major immediate early (MIE) transcripts. Transduction of IE2 largely rescued HCMV gene expression in TAg-expressing fibroblasts but did not rescue the production of infectious virions. Collectively, our data indicate that oncogenic alleles induce multiple restrictions to HCMV replication.IMPORTANCEHCMV cannot replicate in most cancerous cells, yet the causes of this restriction are not clear. The mechanisms that restrict viral replication in cancerous cells represent viral vulnerabilities that can potentially be exploited therapeutically in other contexts. Here we found that SV40 T antigen-mediated transformation inhibits HCMV infection at multiple points in the viral life cycle, including through inhibition of proper viral entry, normal expression of immediate early genes, and viral DNA replication. Our results suggest that the SV40 T antigen could be a valuable tool to dissect cellular activities that are important for successful infection, thereby potentially informing novel antiviral development strategies. This is an important consideration, given that HCMV is a leading cause of birth defects and causes severe infection in immunocompromised individuals.

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