A Phase 2 Clinical Trial to Evaluate the Safety, Reactogenicity, and Immunogenicity of Different Prime-Boost Vaccination Schedules of 2013 and 2017 A(H7N9) Inactivated Influenza Virus Vaccines Administered With and Without AS03 Adjuvant in Healthy US Adults.

INTRODUCTION: A surge of human influenza A(H7N9) cases began in 2016 in China from an antigenically distinct lineage. Data are needed about the safety and immunogenicity of 2013 and 2017 A(H7N9) inactivated influenza vaccines (IIVs) and the effects of AS03 adjuvant, prime-boost interval, and priming effects of 2013 and 2017 A(H7N9) IIVs. METHODS: Healthy adults (n = 180), ages 19-50 years, were enrolled into this partially blinded, randomized, multicenter phase 2 clinical trial. Participants were randomly assigned to 1 of 6 vaccination groups evaluating homologous versus heterologous prime-boost strategies with 2 different boost intervals (21 vs 120 days) and 2 dosages (3.75 or 15 µg of hemagglutinin) administered with or without AS03 adjuvant. Reactogenicity, safety, and immunogenicity measured by hemagglutination inhibition and neutralizing antibody titers were assessed. RESULTS: Two doses of A(H7N9) IIV were well tolerated, and no safety issues were identified. Although most participants had injection site and systemic reactogenicity, these symptoms were mostly mild to moderate in severity; injection site reactogenicity was greater in vaccination groups receiving adjuvant. Immune responses were greater after an adjuvanted second dose, and with a longer interval between prime and boost. The highest hemagglutination inhibition geometric mean titer (95% confidence interval) observed against the 2017 A(H7N9) strain was 133.4 (83.6-212.6) among participants who received homologous, adjuvanted 3.75 µg + AS03/2017 doses with delayed boost interval. CONCLUSIONS: Administering AS03 adjuvant with the second H7N9 IIV dose and extending the boost interval to 4 months resulted in higher peak antibody responses. These observations can broadly inform strategic approaches for pandemic preparedness. Clinical Trials Registration. NCT03589807.

Cytomegalovirus late transcription factor target sequence diversity orchestrates viral early to late transcription.

Beta- and gammaherpesviruses late transcription factors (LTFs) target viral promoters containing a TATT sequence to drive transcription after viral DNA replication has begun. Human cytomegalovirus (HCMV), a betaherpesvirus, uses the UL87 LTF to bind both TATT and host RNA polymerase II (Pol II), whereas the UL79 LTF has been suggested to drive productive elongation. Here we apply integrated functional genomics (dTag system, PRO-Seq, ChIP-Seq, and promoter function assays) to uncover the contribution of diversity in LTF target sequences in determining degree and scope to which LTFs drive viral transcription. We characterize the DNA sequence patterns in LTF-responsive and -unresponsive promoter populations, determine where and when Pol II initiates transcription, identify sites of LTF binding genome-wide, and quantify change in nascent transcripts from individual promoters in relation to core promoter sequences, LTF loss, stage of infection, and viral DNA replication. We find that HCMV UL79 and UL87 LTFs function concordantly to initiate transcription from over half of all active viral promoters in late infection, while not appreciably affecting host transcription. Both LTFs act on and bind to viral early-late and late kinetic-class promoters. Over one-third of these core promoters lack the TATT and instead have a TATAT, TGTT, or YRYT. The TATT and non-TATT motifs are part of a sequence block with a sequence code that correlates with promoter transcription level. LTF occupancy of a TATATA palindrome shared by back-to-back promoters is linked to bidirectional transcription. We conclude that diversity in LTF target sequences shapes the LTF-transformative program that drives the viral early-to-late transcription switch.

Herpes simplex virus dissemination with necrotizing hepatitis following Descemet membrane endothelial keratoplasty.

BACKGROUND: Corneal transplants are the most common type of transplant and increasing in frequency. Donor cornea tissues are a rare source of herpes simplex virus (HSV) transmission and not routinely tested for presence of HSV. Donor graft-to-recipient transmission typically causes graft failure and anterior uveitis, and extra-ocular HSV disease has not been previously reported. We present a case of HSV transmission from donor cornea tissue that nearly cost the corneal transplant recipient his life. CASE REPORT: An elderly immunocompetent man developed an acute illness 10 days after having donor corneal tissue implanted in a Descemet membrane endothelial keratoplasty (DMEK). He was found to have HSV necrotizing hepatitis per liver biopsy, trilineage cytopenia, rhabdomyolysis, acute kidney failure, altered mental status, early-stage hemophagocytic lymphohistiocytosis (HLH), and donor corneal tissue implant infection resulting in graft failure and anterior uveitis. HSV DNA was detected in cerebral spinal fluid, peripheral blood, explanted donor corneal tissue, and anterior chamber fluid (220 million HSV DNA copies per mL). HSV-1 seroconversion denoted a primary HSV infection, and the patient had no other risk factor for HSV acquisition. Early recognition of HSV dissemination prompting treatment with intravenous acyclovir, as well as a short course of HLH-directed therapy, resolved the systemic illness. Vision was restored to near normal by replacement of the infected corneal graft with new donor DMEK tissue in conjunction with intravitreal foscarnet treatment. CONCLUSION: Awareness of the potential risk of donor cornea tissue transmitting HSV and leading to life-threatening HSV disease is paramount to early diagnosis and treatment. The role of donor cornea tissue in HSV transmission and disease merits additional attention and investigation.

Human cytomegalovirus IE2 drives transcription initiation from a select subset of late infection viral promoters by host RNA polymerase II.

Herpesvirus late promoters activate gene expression after viral DNA synthesis has begun. Alphaherpesviruses utilize a viral immediate-early protein to do this, whereas beta- and gammaherpesviruses primarily use a 6-member set of viral late-acting transcription factors (LTF) that are drawn to a TATT sequence in the late promoter. The betaherpesvirus, human cytomegalovirus (HCMV), produces three immediate-early 2 protein isoforms, IE2-86, IE2-60, IE2-40, late in infection, but whether they activate late viral promoters is unknown. Here, we quickly degrade the IE2 proteins in late infection using dTag methodology and analyze effects on transcription using customized PRO-Seq and computational methods combined with multiple validation methods. We discover that the IE2 proteins selectively drive RNA Pol II transcription initiation at a subset of viral early-late and late promoters common to different HCMV strains, but do not substantially affect Pol II transcription of the 9,942 expressed host genes. Most of the IE2-activated viral late infection promoters lack the TATT sequence bound by the HCMV UL87-encoded LTF. The HCMV TATT-binding protein is not mechanistically involved in late RNA expression from the IE2-activated TATT-less UL83 (pp65) promoter, as it is for the TATT-containing UL82 (pp71) promoter. While antecedent viral DNA synthesis is necessary for transcription from the late infection viral promoters, continued viral DNA synthesis is unnecessary. We conclude that in late infection the IE2 proteins target a distinct subset of HCMV early-late and late promoters for transcription initiation by RNA Pol II. Commencement of viral DNA replication renders the HCMV genome late promoters susceptible to late-acting viral transcription factors.

Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome.

Oxidative stress has pervasive effects on cells but how they respond transcriptionally upon the initial insult is incompletely understood. We developed a nuclear walk-on assay that semi-globally quantifies nascent transcripts in promoter-proximal paused RNA polymerase II (Pol II). Using this assay in conjunction with ChIP-Seq, in vitro transcription, and a chromatin retention assay, we show that within a minute, hydrogen peroxide causes accumulation of Pol II near promoters and enhancers that can best be explained by a rapid decrease in termination. Some of the accumulated polymerases slowly move or 'creep' downstream. This second effect is correlated with and probably results from loss of NELF association and function. Notably, both effects were independent of DNA damage and ADP-ribosylation. Our results demonstrate the unexpected speed at which a global transcriptional response can occur. The findings provide strong support for the residence time of paused Pol II elongation complexes being much shorter than estimated from previous studies.

Variable Effects of Autophagy Induction by Trehalose on Herpesviruses Depending on Conditions of Infection.

Trehalose is a non-reducing sugar formed from two glucose units. Trehalose induces abundant autophagy in cultured cells and also reduces the rate of aggregation of the huntingtin protein in the animal model of Huntington disease, a chronic neurological disease in humans. The mechanism of this effect on autophagy is now known to be caused by starvation secondary to inhibition of a family of glucose transporters known as the solute carrier 2 or the glucose transporter family. Variable effects of trehalose treatment have been observed during infections with two herpesviruses-human cytomegalovirus and varicella-zoster virus. The reasons for differing results have now been delineated. These differences are caused by two variables in conditions of infection: timing of addition of trehalose and type of inoculum (cell-free virus vs. infected cells). When monolayers pretreated with trehalose were inoculated with cell-free virus, there was a decline in virus spread by as much as 93 percent when compared with untreated monolayers. However, when monolayers were inoculated with infected cells rather than cell-free virus, there was no decline in virus spread. These results demonstrated that the effect of trehalose was limited to monolayers that were starved when inoculated with cell-free virus. In contrast, sufficient virus was already present in infected cell inocula so as to minimize any inhibitory effect of a starved monolayer. These results also showed that trehalose did not specifically inhibit a herpesvirus; rather, addition of trehalose to cell culture media altered the intracellular environment.

Differentiation-Coupled Induction of Human Cytomegalovirus Replication by Union of the Major Enhancer Retinoic Acid, Cyclic AMP, and NF-κB Response Elements.

UNLABELLED: Triggers and regulatory pathways that effectively link human cytomegalovirus (HCMV) major immediate early (MIE) latent-lytic switch activation with progeny production are incompletely understood. In the quiescently infected human NTera2 cell model of primitive neural stem cells, we found that costimulation with vasoactive intestinal peptide (V) and phorbol ester (P) synergistically activated viral infection, but this effect waned over time. Coupling retinoic acid (R), an inducer of neuronal differentiation, to VP pulse stimulation attenuated the decline in viral activity and promoted the spread of the active infection through concentric layers of neighboring cells as cellular differentiation progressed. R stimulation alone was unable to activate the infection. The MIE enhancer cis-regulatory mechanisms responsible for this result were characterized by a strategy of combinatorial mutagenesis of five cis-acting element types (retinoic acid receptor binding elements [RARE], cyclic AMP [cAMP] response elements [CRE], NF-κB binding sites [kB], serum response element, and ETS/ELK-1 binding site) and multiple methods of assessment. We found that the CRE and kB combination sets the preinduction enhancer tone, is the major initiator and amplifier of RVP-induced MIE gene expression, and cooperates with RARE during cellular differentiation to enhance viral spread. In predifferentiated NTera2, we also found that the CRE-kB combination functions as initiator and amplifier of unstimulated HCMV MIE gene expression and cooperatively interacts with RARE to enhance viral spread. We conclude that RVP-stimulated signaling cascades and cellular differentiation operate through the enhancer CRE-kB-RARE core in strengthening induction of HCMV MIE gene expression in linkage with viral propagation. IMPORTANCE: Cytomegalovirus-seropositive persons commonly lack detectable levels of cytomegalovirus replication, even when profoundly immunocompromised. In a human NTera2 cell model of primitive neural stem cells carrying resting cytomegalovirus genomes, we show that costimulation of protein kinase A and C-delta signaling cascades in conjunction with retinoic acid-induced neuronal differentiation brings about progeny virus propagation. Iterated DNA binding sites for retinoic acid receptor, CREB, and NF-κB family members in the cytomegalovirus major enhancer are at the crux in the pathway to HCMV activation. The stimulated CREB and NF-κB binding site combination vigorously initiates and amplifies the active cytomegalovirus infection and cooperates with activated retinoic acid receptor binding sites to further promote viral proliferation and spread between differentiated cells. These results support a paradigm in which a specific combination of stimuli coupled with cellular differentiation satisfies a core cis-activating code that unlocks enhancer silence to repower the cycle of cytomegalovirus propagation.

Critical Role for the Human Cytomegalovirus Major Immediate Early Proteins in Recruitment of RNA Polymerase II and H3K27Ac To an Enhancer-Like Element in OriLyt.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects most of the population. The complex 236 kbp genome encodes more than 170 open reading frames, whose expression is temporally regulated by both viral transcriptional regulators and cellular factors that control chromatin and transcription. Here, we have used state of the art genomic technologies to investigate the viral transcriptome in conjunction with 2 key transcriptional regulators: Pol II and H3K27Ac. Although it is well known that the major immediate early (IE) proteins activate early gene expression through both direct and indirect interactions, and that histone modifications play an important role in regulating viral gene expression, the role of the IE proteins in modulating viral chromatin is not fully understood. To address this question, we have used a virus engineered for conditional expression of the IE proteins combined with RNA and Chromatin immunoprecipitation (ChIP) analyses to assess the role of these proteins in modulating both viral chromatin and gene expression. Our results show that (i) there is an enhancer-like element in OriLyt that is extraordinarily enriched in H3K27Ac; (ii) in addition to activation of viral gene expression, the IE proteins play a critical role in recruitment of Pol II and H3K27Ac to this element. IMPORTANCE HCMV is an important human pathogen associated with complications in transplant patients and birth defects. The complex program of viral gene expression is regulated by both viral proteins and host factors. Here, we have investigated the role of the immediate early proteins in regulating the viral epigenome. Our results show that the viral immediate early proteins bring about an enormous enrichment of H3K27Ac marks at the OriLyt RNA4.9 promoter, concomitant with an increase in RNA4.9 expression. This epigenetic characteristic adds importantly to the view that OriLyt has structural and functional characteristics of a strong enhancer that, we now discover, is regulated by IE proteins.

Acute Cytomegalovirus Illness in an Immunocompetent Adult Causing Intravascular Hemolysis and Suspected Hemophagocytic Lymphohistiocytosis.

Background: Primary cytomegalovirus (CMV) infection of the immunocompetent host usually produces little-to-no illness. Occasionally, the infection results in mononucleosis syndrome, protracted fever, hepatitis, tissue-invasive disease, or Guillain-Barré syndrome. Hemolytic anemia and hemophagocytic lymphohistiocytosis (HLH) are rare complications that have not been reported to co-occur. Having hemolytic anemia in conjunction with more common findings of fever and hepatitis complicates the diagnosis of HLH. Case Presentation. A 34-year-old male with previously good health presented with a prolonged febrile illness, jaundice, and anemia. An extensive work-up during hospitalization revealed intravascular hemolytic anemia, leukopenia, hepatosplenomegaly, and biopsy evidence of extensive lymphohistiocytic infiltration of the liver with microgranulomata and sinusoidal hemophagocytosis. Soluble CD25 level was mildly elevated at 1200.3 pg/mL and the HScore calculation (fever, bicytopenia, hepatosplenomegaly, aspartate aminotransaminase 99 IU/L, ferritin 1570 ng/mL, fibrinogen 488 mg/dL, and triglycerides 173 mg/dL) suggested a moderate probability of reactive HLH. Primary CMV infection was diagnosed based on CMV IgM positivity, low CMV IgG avidity index, and low-grade CMV DNAemia. The CMV antigen was not detected in the liver biopsy, and the bone marrow biopsy was unremarkable. The illness began to improve before he received oral valganciclovir for 5 days, and he was in good health 10 months later. Conclusion: Acute CMV illness in an immunocompetent adult can present with hemolytic anemia and clinicopathologic abnormalities consistent with a form fruste of HLH. The illness is likely due to an excessive or unbalanced immune response that may self-correct.

Human Cytomegalovirus IE2 Both Activates and Represses Initiation and Modulates Elongation in a Context-Dependent Manner.

Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional transcription factor that is essential for lytic HCMV infection. IE2 functions as an activator of viral early genes, negatively regulates its own promoter, and is required for viral replication. The mechanisms by which IE2 executes these distinct functions are incompletely understood. Using PRO-Seq, which profiles nascent transcripts, and a recently developed DFF-chromatin immunoprecipitation (DFF-ChIP; employs chromatin digestion by the endonuclease DNA fragmentation factor prior to IP) approach that resolves occupancy and local chromatin environment, we show that IE2 controls viral gene transcription in three distinct capacities during late HCMV infection and reveal mechanisms that involve direct binding of IE2 to viral DNA. IE2 represses a subset of viral promoters by binding within their core promoter regions and blocking the assembly of preinitiation complexes (PICs). Remarkably, IE2 forms a repressive complex at the major immediate-early promoter region involving direct association of IE2 with nucleosomes and TBP. IE2 stimulates transcription by binding nearby, but not within, core promoter regions. In addition, IE2 functions as a direct roadblock to transcription elongation. At one locus, this function of IE2 appears to be important for the synthesis of a spliced viral RNA. Consistent with the minimal observed effects of IE2 depletion on host gene transcription, IE2 does not functionally engage the host genome. Our results reveal mechanisms of transcriptional control by IE2, uncover a previously unknown function of IE2 as a Pol II elongation modulator, and demonstrate that DFF-ChIP is a useful tool for probing transcription factor occupancy and interactions between transcription factors and nucleosomes at high resolution. IMPORTANCE HCMV infects more than half of the world population and persists lifelong in its hosts. Although generally asymptomatic, HCMV infection can lead to life-threating disease in immunosuppressed individuals. Moreover, HCMV is the leading infectious cause of birth defects in the United States. As there are no vaccines effective against HCMV and antiviral drugs exhibit toxicity and are undermined by resistant HCMV variants, other vulnerabilities in HCMV must be explored. Here, we characterize the mechanism by which IE2 controls transcription during late HCMV infection. We demonstrate that IE2 engages numerous consensus sites across the HCMV genome and functions as an activator, repressor, or elongation modulator depending on the context of IE2 binding sites in relation to Pol II initiation and elongation complexes. Our findings have important implications for the ongoing exploration of IE2 as an antiviral drug target.

Human Cytomegalovirus Infection Elicits Global Changes in Host Transcription by RNA Polymerases I, II, and III.

How human cytomegalovirus (HCMV) infection impacts the transcription of the host genome remains incompletely understood. Here, we examine the global consequences of infection of primary human foreskin fibroblasts (HFFs) on transcription by RNA polymerase I, II, and III over the course of a lytic infection using PRO-Seq. The expected rapid induction of innate immune response genes is observed with specific subsets of genes exhibiting dissimilar expression kinetics. We find minimal effects on Pol II initiation, but increased rates of the release of paused Pol II into productive elongation are detected by 24 h postinfection and pronounced at late times postinfection. Pol I transcription increases during infection and we provide evidence for a potential Pol I elongation control mechanism. Pol III transcription of tRNA genes is dramatically altered, with many induced and some repressed. All effects are partially dependent on viral genome replication, suggesting a link to viral mRNA levels and/or a viral early-late or late gene product. Changes in tRNA transcription are connected to distinct alterations in the chromatin state around tRNA genes, which were probed with high-resolution DFF-ChIP. Additionally, evidence is provided that the Pol III PIC stably contacts an upstream -1 nucleosome. Finally, we compared and contrasted our HCMV data with results from published experiments with HSV-1, EBV, KSHV, and MHV68. We report disparate effects on Pol II transcription and potentially similar effects on Pol III transcription.

Differences in RNA polymerase II complexes and their interactions with surrounding chromatin on human and cytomegalovirus genomes.

Interactions of the RNA polymerase II (Pol II) preinitiation complex (PIC) and paused early elongation complexes with the first downstream (+1) nucleosome are thought to be functionally important. However, current methods are limited for investigating these relationships, both for cellular chromatin and the human cytomegalovirus (HCMV) genome. Digestion with human DNA fragmentation factor (DFF) before immunoprecipitation (DFF-ChIP) precisely revealed both similarities and major differences in PICs driven by TBP on the host genome in comparison with PICs driven by TBP or the viral-specific, late initiation factor UL87 on the viral genome. Host PICs and paused Pol II complexes are frequently found in contact with the +1 nucleosome and paused Pol II can also be found in a complex involved in the initial invasion of the +1 nucleosome. In contrast, viral transcription complexes have very limited nucleosomal interactions, reflecting a relative lack of chromatinization of transcriptionally active regions of HCMV genomes.

Phorbol ester-induced human cytomegalovirus major immediate-early (MIE) enhancer activation through PKC-delta, CREB, and NF-kappaB desilences MIE gene expression in quiescently infected human pluripotent NTera2 cells.

The ways in which human cytomegalovirus (HCMV) major immediate-early (MIE) gene expression breaks silence from latency to initiate the viral replicative cycle are poorly understood. A delineation of the signaling cascades that desilence the HCMV MIE genes during viral quiescence in the human pluripotent N-Tera2 (NT2) cell model provides insight into the molecular mechanisms underlying HCMV reactivation. In this model, we show that phorbol 12-myristate 13-acetate (PMA) immediately activates the expression of HCMV MIE RNA and protein and greatly increases the MIE-positive (MIE(+)) NT2 cell population density; levels of Oct4 (pluripotent cell marker) and HCMV genome penetration are unchanged. Decreasing PKC-delta activity (pharmacological, dominant-negative, or RNA interference [RNAi] method) attenuates PMA-activated MIE gene expression. MIE gene activation coincides with PKC-delta Thr505 phosphorylation. Mutations in MIE enhancer binding sites for either CREB (cyclic AMP [cAMP] response element [CRE]) or NF-kappaB (kappaB) partially block PMA-activated MIE gene expression; the ETS binding site is negligibly involved, and kappaB does not confer MIE gene activation by vasoactive intestinal peptide (VIP). The PMA response is also partially attenuated by the RNAi-mediated depletion of the CREB or NF-kappaB subunit RelA or p50; it is not diminished by TORC2 knockdown or accompanied by TORC2 dephosphorylation. Mutations in both CRE and kappaB fully abolish PMA-activated MIE gene expression. Thus, PMA stimulates a PKC-delta-dependent, TORC2-independent signaling cascade that acts through cellular CREB and NF-kappaB, as well as their cognate binding sites in the MIE enhancer, to immediately desilence HCMV MIE genes. This signaling cascade is distinctly different from that elicited by VIP.

Viral Acute Gastroenteritis in Special Populations.

Viral acute gastroenteritis (AGE) is common and afflicts people of all ages. Nonviral causes of AGE are less common. Norovirus is a leading cause of sporadic cases and outbreaks of AGE across all ages. Universal rotavirus vaccination of infants has reduced frequency and severity of rotavirus AGE cases in children and indirectly reduced cases in older adults. Severe illness is more likely in persons at age extremes or with immunocompromising conditions. Viral causes of AGE can lead to protracted diarrheal illness in immunocompromised persons. Nucleic acid amplification tests are changing diagnostic testing algorithms.

Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity.

Zinc protoporphyrin (ZnPP), a naturally occurring metalloprotoporphyrin (MPP), is currently under development as a chemotherapeutic agent although its mechanism is unclear. When tested against other MPPs, ZnPP was the most effective DNA synthesis and cellular proliferation inhibitor while promoting apoptosis in telomerase positive but not telomerase negative cells. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and cellular extracts with IC50 and EC50  values of ca 2.5 and 6 µM, respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.

Breaking human cytomegalovirus major immediate-early gene silence by vasoactive intestinal peptide stimulation of the protein kinase A-CREB-TORC2 signaling cascade in human pluripotent embryonal NTera2 cells.

The triggering mechanisms underlying reactivation of human cytomegalovirus (HCMV) in latently infected persons are unclear. During latency, HCMV major immediate-early (MIE) gene expression breaks silence to initiate viral reactivation. Using quiescently HCMV-infected human pluripotent embryonal NTera2 cells (NT2) to model HCMV reactivation, we show that vasoactive intestinal peptide (VIP), an immunomodulatory neuropeptide, immediately and dose-dependently (1 to 500 nM) activates HCMV MIE gene expression. This response requires the MIE enhancer cyclic AMP response elements (CRE). VIP quickly elevates CREB Ser133 and ATF-1 Ser63 phosphorylation levels, although the CREB Ser133 phosphorylation level is substantial at baseline. VIP does not change the level of HCMV genomes in nuclei, Oct4 (pluripotent cell marker), or hDaxx (cellular repressor of HCMV gene expression). VIP-activated MIE gene expression is mediated by cellular protein kinase A (PKA), CREB, and TORC2. VIP induces PKA-dependent TORC2 Ser171 dephosphorylation and nuclear entry, which likely enables MIE gene activation, as TORC2 S171A (devoid of Ser171 phosphorylation) exhibits enhanced nuclear entry and desilences the MIE genes in the absence of VIP stimulation. In conclusion, VIP stimulation of the PKA-CREB-TORC2 signaling cascade activates HCMV CRE-dependent MIE gene expression in quiescently infected NT2 cells. We speculate that neurohormonal stimulation via this signaling cascade is a possible means for reversing HCMV silence in vivo.

Comparisons of CD8+ T cells specific for human immunodeficiency virus, hepatitis C virus, and cytomegalovirus reveal differences in frequency, immunodominance, phenotype, and interleukin-2 responsiveness.

To better understand the components of an effective immune response to human immunodeficiency virus (HIV), the CD8(+) T-cell responses to HIV, hepatitis C virus (HCV), and cytomegalovirus (CMV) were compared with regard to frequency, immunodominance, phenotype, and interleukin-2 (IL-2) responsiveness. Responses were examined in rare patients exhibiting durable immune-mediated control over HIV, termed long-term nonprogressors (LTNP) or elite controllers, and patients with progressive HIV infection (progressors). The magnitude of the virus-specific CD8(+) T-cell response targeting HIV, CMV, and HCV was not significantly different between LTNP and progressors, even though their capacity to proliferate to HIV antigens was preserved only in LTNP. In contrast to HIV-specific CD8(+) T-cell responses of LTNP, HLA B5701-restricted responses within CMV pp65 were rare and did not dominate the total CMV-specific response. Virus-specific CD8(+) T cells were predominantly CD27(+)45RO(+) for HIV and CD27(-)45RA(+) for CMV; however, these phenotypes were highly variable and heavily influenced by the degree of viremia. Although IL-2 induced significant expansions of CMV-specific CD8(+) T cells in LTNP and progressors by increasing both the numbers of cells entering the proliferating pool and the number of divisions, the proliferative capacity of a significant proportion of HIV-specific CD8(+) T cells was not restored with exogenous IL-2. These results suggest that immunodominance by HLA B5701-restricted cells is specific to HIV infection in LTNP and is not a feature of responses to other chronic viral infections. They also suggest that poor responsiveness to IL-2 is a property of HIV-specific CD8(+) T cells of progressors that is not shared with responses to other viruses over which immunologic control is maintained.

Nucleotide Resolution Comparison of Transcription of Human Cytomegalovirus and Host Genomes Reveals Universal Use of RNA Polymerase II Elongation Control Driven by Dissimilar Core Promoter Elements.

The large genome of human cytomegalovirus (HCMV) is transcribed by RNA polymerase II (Pol II). However, it is not known how closely this betaherpesvirus follows host transcriptional paradigms. We applied PRO-Seq and PRO-Cap methods to profile and quantify transcription initiation and productive elongation across the host and virus genomes in late infection. A major similarity between host transcription and viral transcription is that treatment of cells with the P-TEFb inhibitor flavopiridol preempts virtually all productive elongation, which otherwise covers most of the HCMV genome. The deep, nucleotide resolution identification of transcription start sites (TSSs) enabled an extensive analysis of core promoter elements. An important difference between host and viral transcription is that initiation is much more pervasive on the HCMV genome. The sequence preferences in the initiator region around the TSS and the utilization of upstream T/A-rich elements are different. Upstream TATA positions the TSS and boosts initiation in both the host and the virus, but upstream TATT has a significant stimulatory impact only on the viral template. The major immediate early (MIE) promoter remained active during late infection and was accompanied by transcription of both strands of the MIE enhancer from promoters within the enhancer. Surprisingly, we found that the long noncoding RNA4.9 is intimately associated with the viral origin of replication (oriLyt) and was transcribed to a higher level than any other viral or host promoter. Finally, our results significantly contribute to the idea that late in infection, transcription takes place on viral genomes that are not highly chromatinized.IMPORTANCE Human cytomegalovirus infects more than half of humans, persists silently in virtually all tissues, and produces life-threatening disease in immunocompromised individuals. HCMV is also the most common infectious cause of birth defects and the leading nongenetic cause of sensorineural hearing loss in the United States. Because there is no vaccine and current drugs have problems with potency, toxicity, and antiviral drug resistance, alternative treatment strategies that target different points of viral control are needed. Our current study contributes to this goal by applying newly developed methods to examine transcription of the HCMV and host genomes at nucleotide resolution in an attempt to find targetable differences between the two. After a thorough analysis of productive elongation and of core promoter element usage, we found that some mechanisms of regulating transcription are shared between the host and HCMV but that others are distinctly different. This suggests that HCMV transcription may be a legitimate target for future antiviral therapies and this might translate to other herpesviruses.

Cytomegalovirus and the role of interferon in the expression of tumor necrosis factor-related apoptosis-inducing ligand in the placenta.

OBJECTIVE: Cytomegalovirus infection causes adverse outcomes during pregnancy. Our objective was to determine the role of cytomegalovirus in modulating tumor necrosis factor (TNF)-related apoptosis-inducing ligand expression in the placenta. STUDY DESIGN: TNF-related apoptosis-inducing ligand messenger RNA and protein were quantified in cytomegalovirus-infected placental fibroblasts by polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. Blocking antibodies against interferon and type I interferon receptor were applied to culture medium to characterize the role of type I interferon in cytomegalovirus-induced TNF-related apoptosis-inducing ligand upregulation. RESULTS: Expression of TNF-related apoptosis-inducing ligand messenger RNA and protein was increased in cytomegalovirus-infected placental fibroblasts, compared with uninfected controls. The cytomegalovirus-induced TNF-related apoptosis-inducing ligand messenger RNA upregulation was demonstrated across gestation, occurred in the absence of viral gene expression, and required cellular protein synthesis. TNF-related apoptosis-inducing ligand messenger RNA upregulation was markedly attenuated by inactivation of either type I interferon or its receptor. CONCLUSION: One mechanism by which cytomegalovirus infection causes unfavorable pregnancy outcomes may involve placental upregulation of TNF-related apoptosis-inducing ligand via an interferon-mediated pathway.