Immediate-early Research Articles

Multiple functions of the herpesvirus UL14 gene product in viral infection

Herpesviruses are a family of double-stranded DNA viruses with a tegument structure and a genome composed of a single sequence and terminal repeat (TR) sequences. The herpesvirus UL14 gene encodes the protein UL14 (pUL14), which has various subcellular localizations and plays a vital role in regulating immediate–early (IE) gene transcription and expression, influences the intracellular localization patterns of several proteins belonging to the capsid and the DNA packaging machinery, participates in secondary envelopment, and influences viral particle release. Additionally, pUL14 has roles in maintaining cellular homeostasis and preventing apoptosis. This review discusses how pUL14 engages in the life cycle of herpesviruses and provides new ideas for further research on pUL14’s function in viral infection.

Virus-Specific Nanobody-Chimeras Degrade the Human Cytomegalovirus US28 Protein in CD34+ Cells.

After primary infection, human cytomegalovirus (HCMV) establishes lifelong persistence, underpinned by latent carriage of the virus with spontaneous reactivation events. In the immune-competent, primary infection or reactivation from latency rarely causes disease. However, HCMV can cause significant disease in immune-compromised individuals such as immune-suppressed transplant patients. Latency, where the viral genome is carried in the absence of the production of infectious virions, can be established in undifferentiated cells of the myeloid lineage. A number of stimuli can cause virus reactivation from latency to occur, beginning with the induction of viral immediate-early (IE) lytic gene expression. The suppression of viral IE gene expression to establish and maintain latent infection is known to result from a balance of viral and cellular factors. One key viral factor involved in this is the G protein-coupled receptor US28. Recently, we have shown that US28 is targeted for degradation by a modified nanobody (PCTD-Vun100bv) based on the novel PACTAC (PCSK9-antibody clearance-targeting chimeras) approach for targeted protein degradation. Furthermore, we have shown that this PCTD-Vun100bv-induced degradation of US28 results in IE gene expression in experimentally latently infected CD14+ monocytes. However, HCMV also establishes latency in CD34+ bone marrow cells, the progenitors of CD14+ cells. Here, we show that PCTD-Vun100bv also causes US28 degradation in these CD34+ primary cells, again resulting in the induction of viral IE gene expression. Additionally, we show that PCTD-Vun100bv can target US28 in naturally latently infected CD14+ monocytes from an HCMV-seropositive donor, allowing these latently infected cells to be killed by HCMV-specific cytotoxic T cells from that same donor. These observations support the view that targeting US28 for degradation during natural latency could be a tractable 'shock-and-kill' strategy to target the latent HCMV reservoir in myeloid cells.

The Pentamer glycoprotein complex inhibits viral Immediate Early transcription during Human Cytomegalovirus infections

The Pentamer complex of Human Cytomegalovirus (HCMV) consists of the viral glycoproteins gH, gL, UL128, UL130, and UL131 and is incorporated into infectious virions. HCMV strains propagated extensively in vitro in fibroblasts carry UL128, UL130, or UL131 alleles that do not make a functional complex and thus lack Pentamer function. Adding functional Pentamer to such strains decreases virus growth in fibroblasts. Here, we show that the Pentamer inhibits productive HCMV replication in fibroblasts by repressing viral Immediate Early (IE) transcription. We show that ectopic expression of the viral IE1 protein, a target of Pentamer-mediated transcriptional repression, complements the growth defect of a Pentamer-positive virus. Furthermore, we show that the Pentamer also represses viral IE transcription in cell types where HCMV in vitro latency is studied. Finally, we identify UL130 as a functional subunit of the Pentamer for IE transcriptional repression and demonstrate that cyclic AMP Response Element (CRE) and NFkB sites within the Major Immediate Early Promoter that drives IE1 transcription contribute to this repression. We conclude that the HCMV Pentamer represses viral IE transcription.

Cyprinid herpesvirus 3 replication is inhibited via decreased ORF3 promoter activity mediated by multiple cellular transcription factors

Cyprinid herpesvirus 3 (CyHV-3) is an enveloped and double-stranded DNA virus that primarily infects common carp and its variants. The transcriptional regulation program of the viral immediate early (IE) genes plays a crucial role in virus replication. However, the transcriptional regulatory mechanism of CyHV-3 IE genes remains largely unclear. In this study, we found that the IE gene ORF3 promoter, which exhibited the highest activity among the eight CyHV-3 IE genes, was inhibited in the host common carp brain (CCB) cells. Deletions of the ORF3 promoter revealed the presence of multiple regulatory elements. Site-directed deletions of multiple transcription factors binding sites (TFBS), including Yin Yang-1(YY1), TATA-binding protein (TBP), sex-determining region Y-box2 (SOX2), and cyclin-dependent kinase 8 (CDK8) binding sites, markedly inhibited the ORF3 promoter activity. Moreover, overexpression of these transcription factors in CCB cells also led to a noticeable reduction in ORF3 promoter activity. Importantly, YY1, TBP, SOX2, and CDK8 played antiviral roles against CyHV-3 infection, as evidenced by decreased viral transcripts (ORF55, ORF81, and ORF92) and genome copies after overexpression of these TFs followed by CyHV-3 infection. Collectively, CyHV-3 replication was markedly inhibited by reducing the promoter activity of ORF3 mediated by multiple cellular TFs. These findings enhance our understanding of the role of IE gene promoters in CyHV-3 and lays a foundation for in-depth studies on viral gene transcriptional regulation mechanisms.

Daxx mediated histone H3.3 deposition on HSV-1 DNA restricts genome decompaction and the progression of immediate-early transcription.

Herpesviruses are ubiquitous pathogens that cause a wide range of disease. Upon nuclear entry, their genomes associate with histones and chromatin modifying enzymes that regulate the progression of viral transcription and outcome of infection. While the composition and modification of viral chromatin has been extensively studied on bulk populations of infected cells by chromatin immunoprecipitation, this key regulatory process remains poorly defined at single-genome resolution. Here we use high-resolution quantitative imaging to investigate the spatial proximity of canonical and variant histones at individual Herpes Simplex Virus 1 (HSV-1) genomes within the first 90 minutes of infection. We identify significant population heterogeneity in the stable enrichment and spatial proximity of canonical histones (H2A, H2B, H3.1) at viral DNA (vDNA) relative to established promyelocytic leukaemia nuclear body (PML-NB) host factors that are actively recruited to viral genomes upon nuclear entry. We show the replication-independent histone H3.3/H4 chaperone Daxx to cooperate with PML to mediate the enrichment and spatial localization of variant histone H3.3 at vDNA that limits the rate of HSV-1 genome decompaction to restrict the progress of immediate-early (IE) transcription. This host response is counteracted by the viral ubiquitin ligase ICP0, which degrades PML to disperse Daxx and variant histone H3.3 from vDNA to stimulate the progression of viral genome expansion, IE transcription, and onset of HSV-1 replication. Our data support a model of intermediate and sequential histone assembly initiated by Daxx that limits the rate of HSV-1 genome decompaction independently of the stable enrichment of histones H2A and H2B at vDNA required to facilitate canonical nucleosome assembly. We identify HSV-1 genome decompaction upon nuclear infection to play a key role in the initiation and functional outcome of HSV-1 lytic infection, findings pertinent to the transcriptional regulation of many nuclear replicating herpesvirus pathogens.

Identification of the Promoter Antisense Transcript Enhancing the Transcription of the Equine Herpesvirus-1 Immediate-Early Gene.

Equine herpesvirus-1 (EHV-1) causes respiratory diseases, abortion, and encephalomyelitis in horses. The EHV-1 immediate-early (IE) protein, essential for viral replication, is transactivated by the binding of a multiprotein complex including the open reading frame 12 (ORF12) and some host factors to the IE promoter region. Promoter-associated non-coding RNAs (pancRNAs), which are transcribed from bidirectional promoters, regulate the transcription of neighboring genes in mammals and pathogens. In this study, we identified a novel pancRNA transcribed from across the areas of the 5'-untranslated region and a promoter of EHV-1 IE and named it IE pancRNA. IE pancRNA and mRNA were simultaneously expressed in EHV-1-infected RN33B-A68B2M cells. This pancRNA was also transcribed in RK13 and E. Derm cells, which are highly susceptible to EHV-1 infection. Furthermore, IE pancRNA upregulated IE gene expression in the presence of ORF12, and stable expression of IE pancRNA increased the number of EHV-1-infected RN33B-A68B2M cells. These results suggest that IE pancRNAs facilitate EHV-1 proliferation by promoting IE gene expression.

CGAS-STING-TBK1 Signaling Promotes Valproic Acid-Responsive Human Cytomegalovirus Immediate-Early Transcription during Infection of Incompletely Differentiated Myeloid Cells.

Repression of human cytomegalovirus (HCMV) immediate-early (IE) gene expression is a key regulatory step in the establishment and maintenance of latent reservoirs. Viral IE transcription and protein accumulation can be elevated during latency by treatment with histone deacetylase inhibitors such as valproic acid (VPA), rendering infected cells visible to adaptive immune responses. However, the latency-associated viral protein UL138 inhibits the ability of VPA to enhance IE gene expression during infection of incompletely differentiated myeloid cells that support latency. UL138 also limits the accumulation of IFNβ transcripts by inhibiting the cGAS-STING-TBK1 DNA-sensing pathway. Here, we show that, in the absence of UL138, the cGAS-STING-TBK1 pathway promotes both IFNβ accumulation and VPA-responsive IE gene expression in incompletely differentiated myeloid cells. Inactivation of this pathway by either genetic or pharmacological inhibition phenocopied UL138 expression and reduced VPA-responsive IE transcript and protein accumulation. This work reveals a link between cytoplasmic pathogen sensing and epigenetic control of viral lytic phase transcription and suggests that manipulation of pattern recognition receptor signaling pathways could aid in the refinement of MIEP regulatory strategies to target latent viral reservoirs.

Characterization of the Ictalurid herpesvirus 1 immediate-early gene ORF24 and its potential role in transcriptional regulation in yeast.

Herpesviruses adhere to a precise temporal expression model in which immediate-early (IE) genes play a crucial role in regulating the viral life cycle. However, there is a lack of functional research on the IE genes in Ictalurid herpesvirus 1 (IcHV-1). In this study, we identified the IcHV-1 ORF24 as an IE gene via a metabolic inhibition assay, and subcellular analysis indicated its predominant localisation in the nucleus. To investigate its function, we performed yeast reporter assays using an ORF24 fusion protein containing the Gal4-BD domain and found that BD-ORF24 was able to activate HIS3/lacZ reporter genes without the Gal4-AD domain. Our findings provide concrete evidence that ORF24 is indeed an IE gene that likely functions as a transcriptional regulator during IcHV-1 infection. This work contributes to our understanding of the molecular mechanisms underlying fish herpesvirus IE gene expression.

A dual fluorescent herpes simplex virus type 1 recombinant reveals divergent outcomes of neuronal infection.

Critical stages of lytic herpes simplex virus type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 can also persist in neuronal cells via a non-replicative, transcriptionally repressed infection called latency. The regulation of lytic and latent transcriptional profiles is critical to HSV-1 pathogenesis and persistence. We sought a fluorescence-based approach to observe the outcome of neuronal HSV-1 infection at the single-cell level. To achieve this goal, we constructed and characterized a novel HSV-1 recombinant that enables discrimination between lytic and latent infection. The dual reporter HSV-1 encodes a human cytomegalovirus-immediate early (hCMV-IE) promoter-driven enhanced yellow fluorescent protein (eYFP) to visualize the establishment of infection and an endogenous mCherry-VP26 fusion to report lytic replication. We confirmed that viral gene expression, replication, and spread of infection are not altered by the incorporation of the fluorescent reporters, and fluorescent protein (FP) detection virtuously reports the progression of lytic replication. We demonstrate that the outcome of HSV-1 infection of compartmentalized primary neurons is determined by viral inoculating dose: high-dose axonal inoculation proceeds to lytic replication, whereas low-dose axonal inoculation establishes a latent HSV-1 infection. Interfering with low-dose axonal inoculation via small molecule drugs reports divergent phenotypes of eYFP and mCherry reporter detection, correlating with altered states of viral gene expression. We report that the transcriptional state of neuronal HSV-1 infection is variable in response to changes in the intracellular neuronal environment.IMPORTANCEHerpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global human population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by the regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We used a recombinant HSV-1 to evaluate viral gene expression during infection of primary neurons. Manipulation of cell signaling pathways impacts the establishment and transcriptional state of HSV-1 latency in neurons. The work here provides critical insight into the cellular and viral factors contributing to the establishment of latent HSV-1 infection.

Discovery of a Novel Antiviral Effect of the Restriction Factor SPOC1 against Human Cytomegalovirus.

The chromatin-remodeler SPOC1 (PHF13) is a transcriptional co-regulator and has been identified as a restriction factor against various viruses, including human cytomegalovirus (HCMV). For HCMV, SPOC1 was shown to block the onset of immediate-early (IE) gene expression under low multiplicities of infection (MOI). Here, we demonstrate that SPOC1-mediated restriction of IE expression is neutralized by increasing viral titers. Interestingly, our study reveals that SPOC1 exerts an additional antiviral function beyond the IE phase of HCMV replication. Expression of SPOC1 under conditions of high MOI resulted in severely impaired viral DNA replication and viral particle release, which may be attributed to inefficient viral transcription. With the use of click chemistry, the localization of viral DNA was investigated at late time points after infection. Intriguingly, we detected a co-localization of SPOC1, RNA polymerase II S5P and polycomb repressor complex 2 (PRC2) components in close proximity to viral DNA in areas that are hypothesized to harbor viral transcription sites. We further identified the N-terminal domain of SPOC1 to be responsible for interaction with EZH2, a subunit of the PRC2 complex. With this study, we report a novel and potent antiviral function of SPOC1 against HCMV that is efficient even with unrestricted IE gene expression.

Sp1 facilitates continued HSV-1 gene expression in the absence of key viral transactivators.

Productive replication of herpes simplex virus (HSV) relies upon a well-ordered transcriptional cascade flowing from immediate-early (IE) to early (E) to late (L) gene products. While several virus-encoded transcriptional activators are involved in this process, IE and E gene promoters also contain multiple binding sites for the ubiquitously expressed cellular transcription factor Sp1. Sp1 has been previously implicated in activating HSV-1 gene transcription downstream of these sites, but why Sp1-binding sites are maintained in the promoters of genes activated by virus-encoded activators remains unclear. We hypothesized that Sp1 enables continued HSV-1 transcription and replication when viral transactivators are limited. We used a depletion-based approach in human foreskin fibroblasts to investigate the specific contribution of Sp1 to the initiation and progression of the HSV-1 lytic gene cascade. We found that Sp1 increased viral transcript levels, protein expression, and replication following infection with VP16- or ICP0-deficient viruses but had little to no effect on rescued viruses or during wild-type (WT) HSV-1 infection. Moreover, Sp1 promoted WT virus transcription and replication following interferon treatment of fibroblasts and thus may contribute to viral immune evasion. Interestingly, we observed reduced expression of Sp1 and Sp1-family transcription factors in differentiated sensory neurons compared to undifferentiated cells, suggesting that reduced Sp1 levels may also contribute to HSV-1 latent infection. Overall, these findings indicate that Sp1 can promote HSV-1 gene expression in the absence of key viral transactivators; thus, HSV-1 may use Sp1 to maintain its gene expression and replication under adverse conditions.IMPORTANCEHerpes simplex virus (HSV) is a common human pathogen that actively replicates in the epithelia but can persist for the lifetime of the infected host via a stable, latent infection in neurons. A key feature of the HSV replication cycle is a complex transcriptional program in which virus and host-cell factors coordinate to regulate expression of the viral gene products necessary for continued viral replication. Multiple binding sites for the cellular transcription factor Sp1 are located in the promoters of HSV-1 genes, but how Sp1 binding contributes to transcription and replication of wild-type virus is not fully understood. In this study, we identified a specific role for Sp1 in maintaining HSV-1 gene transcription under adverse conditions, as when virus-encoded transcriptional activators were absent or limited. Preservation of Sp1-binding sites in HSV-1 gene promoters may thus benefit the virus as it navigates diverse cell types and host-cell conditions during infection.

A Revision of Herpes Simplex Virus Type 1 Transcription: First, Repress; Then, Express.

The herpes virus genome bears more than 80 strong transcriptional promoters. Upon entry into the host cell nucleus, these genes are transcribed in an orderly manner, producing five immediate-early (IE) gene products, including ICP0, ICP4, and ICP22, while non-IE genes are mostly silent. The IE gene products are necessary for the transcription of temporal classes following sequentially as early, leaky late, and true late. A recent analysis using precision nuclear run-on followed by deep sequencing (PRO-seq) has revealed an important step preceding all HSV-1 transcription. Specifically, the immediate-early proteins ICP4 and ICP0 enter the cell with the incoming genome to help preclude the nascent antisense, intergenic, and sense transcription of all viral genes. VP16, which is also delivered into the nucleus upon entry, almost immediately reverses this repression on IE genes. The resulting de novo expression of ICP4 and ICP22 further repress antisense, intergenic, and early and late viral gene transcription through different mechanisms before the sequential de-repression of these gene classes later in infection. This early repression, termed transient immediate-early protein-mediated repression (TIEMR), precludes unproductive, antisense, intergenic, and late gene transcription early in infection to ensure the efficient and orderly progression of the viral cascade.

Early detection, reactivation of cytomegalovirus DNA & immediate early (IE)-mRNA expression in hematopoietic stem cell-transplant patients

BackgroundHuman cytomegalovirus (HCMV) reactivation is a major cause of morbidity and mortality among stem cell transplant recipients post-transplantation. AimHCMV immediate-early messenger RNA (IE-mRNA) was evaluated as marker of post-transplant HCMV reactivation in bone marrow transplant recipients. Methodology: An in-house real-time reverse transcriptase PCR targeting IE-mRNA was developed to estimate HCMV mRNA levels post-transplantation. Blood samples collected in K2-EDTA tubes from patients (n = 162) admitted with Department of Clinical Hematology were transported in cold condition for routine HCMV DNA screening. For HCMV IE-mRNA quantification, peripheral blood mononuclear cells (PBMCs) were separated from whole blood and stored in RNA later at −70 °C until testing. Samples were collected weekly once for first 3 weeks post-transplantation and thereafter from week 4–12, samples were collected twice weekly. A total of 2467 samples were collected from 162 study participants. ResultsThirty five patients (21.6 %) had post-transplant HCMV reactivation. Twenty five patients with complete follow-up were selected for monitoring HCMV DNA. HCMV IE-mRNA PCR was performed for 15 patients and 7(46.6 %) patients had detectable mRNA levels. HCMV IE-mRNA was detected in all patients with increasing HCMV DNA levels except for one patient in whom IE-mRNA appeared 3 days before HCMV DNA was detected. One patient had detectable HCMV IE-mRNA during declining HCMV DNA level. However the patient showed an increased HCMV DNA one week later, indicating the importance of HCMV mRNA in predicting HCMV replication. ConclusionQuantification of HCMV IE-mRNA may be a valuable tool to predict progression of HCMV infection post-transplantation, with further prospective studies needed for validation.

MODL-43. SOX2 DOWNREGULATION OF PML PROMOTES HCMV GENE EXPRESSION AND GROWTH OF GLIOMA CELLS

Abstract The presence of human cytomegalovirus (HCMV) in glioblastoma (GBM) and improved outcomes of GBM patients receiving therapies targeting the virus have implicated HCMV in GBM progression. However, a unifying mechanism that accounts for the contribution of HCMV to the malignant phenotype of GBM remains incompletely defined. Here we have identified SOX2, a marker of glioma stem cells (GSCs), as a key determinant of HCMV gene expression in gliomas. Our studies demonstrated that SOX2 downregulated promyelocytic leukemia (PML) and Sp100 and consequently facilitated viral gene expression by decreasing the amount of PML nuclear bodies in HCMV-infected glioma cells. Conversely, the expression of PML antagonized the effects of SOX2 on HCMV gene expression. Furthermore, this regulation of SOX2 on HCMV infection was demonstrated in a neurosphere assay of GSCs and in a murine xenograft model utilizing xenografts from patient-derived glioma tissue. In both cases, SOX2 overexpression enhanced HCMV gene expression and facilitated the growth of neurospheres and xenografts implanted in immunodeficient mice. Lastly, the expression of SOX2 and HCMV immediate early 1 (IE1) protein could be correlated in tissues from glioma patients, and interestingly, elevated levels of SOX2 and IE1 were predictive of a worse clinical outcome. These studies argue that HCMV gene expression in gliomas is regulated by SOX2 through its regulation of PML expression and that targeting molecules in this SOX2-PML pathway could identify therapies for glioma treatment.

Herpes simplex virus 1 immediate early transcription initiation, pause-release, elongation, and termination in the presence and absence of ICP4.

Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.

WSV403 suppresses JAK/STAT signaling to impair antiviral resistance in Pacific white shrimp Penaeus vannamei

Viral immediate early (IE) genes are key regulators of host antiviral defenses. White spot syndrome virus (WSSV) IE gene WSV403 encodes an E3 ligase which regulates the latency state of WSSV. However, the role of WSV403 in host immune response remains unclear. Here, we generated a transgenic Drosophila line that overexpresses WSV403 and found that WSV403 suppresses the JAK/STAT pathway and its downstream genes, leading to increased susceptibility and viral load upon Drosophila C virus infection. We further showed that WSV403 suppresses Stat92E expression without affecting its phosphorylation or degradation, suggesting a novel mechanism of JAK/STAT inhibition. Moreover, we demonstrated that knockdown of WSV403 in the Pacific white shrimp Penaeus vannamei reduces WSSV replication and enhances transcript levels of JAK/STAT pathway components. Our results revealed the immune modulatory function of WSV403 and uncovered a new mechanism by which WSSV inhibits host JAK/STAT signaling to impair antiviral resistance against WSSV infection.

Single-Cell RNA Sequencing Transcriptomics Revealed HCMV IE2-Related Microglia Responses in Alzheimer's-Like Disease in Transgenic Mice.

Although multiple factors are known to concur with Alzheimer's disease (AD), the relationship between human cytomegalovirus (HCMV) and AD-like disease is unclear. Here, we propose a hypothesis that HCMV immediate-early 2 (IE2) protein promotes microglia activation and thus leads to AD-like disease. We successfully constructed IE2 transgenic mice expressing IE2 in the hippocampus. Single-cell sequencing analysis revealed that IE2 promoted the activation of microglia and upregulated the expression of disease-associated microglia genes. Differentially expressed gene analysis and pathway enrichment revealed that IE2 upregulated immune and nervous system disease-related genes. Immunohistochemical analysis showed that the expressions of both amyloid precursor protein (APP) and p-Tau were significantly upregulated in the brains of IE2 mice and were markers of AD. Taken together, these findings provide useful insights into AD-like disease activated by HCMV IE2.

Impact of Baseline and Week 2 and Week 4 Posttransplant CMV Cell-Mediated Immunity on Risk of CMV Infections and Mortality in Recipients of Allogeneic Hematopoietic Cell Transplant.

Cytomegalovirus (CMV) infection is a common opportunistic infection after allogeneic hematopoietic cell transplant (alloHCT). We explored whether a change in CMV cell-mediated immunity during the first month after transplant predicts the risk of development of CMV infection and all-cause mortality. This follow-up analysis is based on data from the REACT study, a multicenter prospective observational study of recipients of alloHCT who were CMV-seropositive. Production of interferon γ following ex vivo stimulation with CMV antigens IE1 (immediate early 1) and pp65 (phosphoprotein 65) was assessed by CMV ELISPOT assay at baseline and 2 and 4 weeks after transplant. Clinically significant CMV infection (CS-CMVi) was defined as CMV viremia and/or disease necessitating antiviral therapy. We evaluated the impact of CMV CMI changes on the risk of CS-CMVi and post transplant mortality. The analysis included 226 recipients of alloHCT with CMV cell-mediated immunity data at baseline and 2 and/or 4 weeks after transplant. CS-CMVi occurred in 64 patients (28%). On Cox regression analyses, independent predictors of CS-CMVi included a negative Δ change from baseline to week 2 of pp65 spot counts (hazard ratio, 3.65 [95% CI, 1.65-8.04]; P = .001) to week 4 of IE1 spot counts (hazard ratio, 2.79 [95% CI, 1.46-5.35]; P = .002), anti-thymocyte globulin conditioning regimen, type of transplant, female sex, and corticosteroid use. Kaplan-Meir analysis showed a significant association of a negative IE1 change from baseline to week 4 and increased all-cause mortality after transplant (log rank test = 0.041). A decrease in CMV-specific T-cell responses during the first month after transplant may predict CS-CMVi and is associated with all-cause mortality in recipients of alloHCT.

ICP4-Associated Activation of Rap1b Facilitates Herpes Simplex Virus Type I (HSV-1) Infection in Human Corneal Epithelial Cells.

The strong contribution of RAS-related protein 1b (Rap1b) to cytoskeleton remodeling determines intracellular and extracellular physiological activities, including the successful infection of viruses in permissive cells, but its role in the HSV-1 life cycle is still unclear. Here, we demonstrated that the HSV-1 immediate early (IE) gene ICP4 inhibits protein kinase A (PKA) phosphorylation to induce Rap1b-activation-mediated viral infection. Rap1b activation and membrane enrichment begin at the early stage of HSV-1 infection and remain active during the proliferation period of the virus. Treating the cells with Rap1b small interfering RNA (siRNA) showed a dose-dependent decrease in viral infection levels, but no dose-dependent increase was observed after Rap1b overexpression. Further investigation indicated that the suppression of Rap1b activation derives from phosphorylated PKA and Rap1b mutants with partial or complete prenylation instead of phosphorylation, which promoted viral infection in a dose-dependent manner. Furthermore, the PKA agonist Forskolin disturbed Rap1b activation in a dose-dependent manner, accompanied by a decreasing trend in viral infection. Moreover, the HSV-1 IE gene ICP4 induced PKA dephosphorylation, leading to continuous Rap1b activation, followed by cytoskeleton rearrangement induced by cell division control protein 42 (CDC42) and Ras-related C3 botulinum toxin substrate 1 (RAC1). These further stimulated membrane-triggered physiological processes favoring virus infection. Altogether, we show the significance of Rap1b during HSV-1 infection and uncover the viral infection mechanism determined by the posttranslational regulation of the viral ICP4 gene and Rap1b host protein.

Herpes Simplex Virus-1 ICP27 Nuclear Export Signal Mutants Exhibit Cell Type-Dependent Deficits in Replication and ICP4 Expression.

Herpes simplex virus type-1 (HSV-1) protein ICP27 is an essential immediate early (IE) protein that promotes the expression of viral early (E) and late (L) genes via multiple mechanisms. Our understanding of this complex regulatory protein has been greatly enhanced by the characterization of HSV-1 mutants bearing engineered alterations in the ICP27 gene. However, much of this analysis has been performed in interferon-deficient Vero monkey cells. Here, we assessed the replication of a panel of ICP27 mutants in several other cell types. Our analysis shows that mutants lacking ICP27's amino (N)-terminal nuclear export signal (NES) display a striking cell type-dependent growth phenotype, i.e., they grow semi-permissively in Vero and some other cells but are tightly blocked for replication in primary human fibroblasts and multiple human cell lines. This tight growth defect correlates with a failure of these mutants to replicate viral DNA. We also report that HSV-1 NES mutants are deficient in expressing the IE protein ICP4 at early times postinfection. Analysis of viral RNA levels suggests that this phenotype is due, at least in part, to a defect in the export of ICP4 mRNA to the cytoplasm. In combination, our results (i) show that ICP27's NES is critically important for HSV-1 replication in many human cells, and (ii) suggest that ICP27 plays a heretofore unappreciated role in the expression of ICP4. IMPORTANCE HSV-1 IE proteins drive productive HSV-1 replication. The major paradigm of IE gene induction, developed over many years, involves the parallel activation of the five IE genes by the viral tegument protein VP16, which recruits the host RNA polymerase II (RNAP II) to the IE gene promoters. Here, we provide evidence that ICP27 can enhance ICP4 expression early in infection. Because ICP4 is required for transcription of viral E and L genes, this finding may be relevant to understanding how HSV-1 enters and exits the latent state in neurons.