Human Cytomegalovirus (HCMV) infection induces several metabolic activities that are essential for viral replication. Despite the important role that this metabolic modulation plays during infection, the viral mechanisms involved are largely unclear. We find that the HCMV UL38 protein is responsible for many aspects of HCMV-mediated metabolic activation, with UL38 being necessary and sufficient to drive glycolytic activation and induce the catabolism of specific amino acids. UL38’s metabolic reprogramming role is dependent on its interaction with TSC2, a tumor suppressor that inhibits mTOR signaling. Further, shRNA-mediated knockdown of TSC2 recapitulates the metabolic phenotypes associated with UL38 expression. Notably, we find that in many cases the metabolic flux activation associated with UL38 expression is largely independent of mTOR activity, as broad spectrum mTOR inhibition does not impact UL38-mediated induction of glycolysis, glutamine consumption, or the secretion of proline or alanine. In contrast, the induction of metabolite concentrations observed with UL38 expression are largely dependent on active mTOR. Collectively, our results indicate that the HCMV UL38 protein induces a pro-viral metabolic environment via inhibition of TSC2.

Influenza B viruses (IBVs) cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated morbidity and mortality. Studying influenza viruses requires the use of secondary methodologies to identify virus-infected cells. To this end, replication-competent influenza A viruses (IAVs) expressing easily traceable fluorescent proteins have been recently developed. In contrast, similar approaches for IBV are mostly lacking. In this report, we describe the generation and characterization of replication-competent influenza B/Brisbane/60/2008 viruses expressing fluorescent mCherry or GFP fused to the C-terminal of the viral non-structural 1 (NS1) protein. Fluorescent-expressing IBVs display similar growth kinetics and plaque phenotype to wild-type IBV, while fluorescent protein expression allows for the easy identification of virus-infected cells. Without the need of secondary approaches to monitor viral infection, fluorescent-expressing IBVs represent an ideal approach to study the biology of IBV and an excellent platform for the rapid identification and characterization of antiviral therapeutics or neutralizing antibodies using high-throughput screening approaches. Lastly, fluorescent-expressing IBVs can be combined with the recently described reporter-expressing IAVs for the identification of novel therapeutics to combat these two important human respiratory pathogens.

While combinatory antiretroviral therapy (cART) can effectively reduce HIV-1 viremia, it cannot eliminate HIV-1 infection. In the presence of cART, viral reservoirs remain latent, impeding the cure of HIV-1/AIDS. Recently, latency-reversing agents (LRAs) have been developed with the intent of purging latent HIV-1, providing an intriguing strategy for the eradication of the residual viral reservoirs. Our earlier studies show that the first-generation, methyl-triazolo bromodomain, and extra-terminal domain inhibitor (BETi), JQ1, facilitates the reversal of HIV-1 latency. BETis have emerged as a new class of compounds that are promising for this HIV-1 “shock and kill” eradication approach. However, when used as a single drug, JQ1 only modestly reverses HIV-1 latency, which complicates studying the underlining mechanisms. Meanwhile, it has been widely discussed that the induction of latent proviruses is stochastic (Ho et al., 2013). Thus, new BETis are currently under active development with focus on improving potency, ease of synthesis and structural diversity. Using fluorous-tagged multicomponent reactions, we developed a novel second-generation, 3,5-dimethylisoxazole BETi based on an imidazo[1,2-a] pyrazine scaffold, UMB-32. Furthermore, we screened 37 UMB-32 derivatives and identified that one, UMB-136, reactivates HIV-1 in multiple cell models of HIV-1 latency with better efficiency than either JQ1 or UMB-32. UMB-136 enhances HIV-1 transcription and increases viral production through the release of P-TEFb. Importantly, UMB-136 enhances the latency-reversing effects of PKC agonists (prostratin, bryostatin-1) in CD8-depleted PBMCs containing latent viral reservoirs. Our results illustrate that structurally improved BETis, such as UMB-136, may be useful as promising LRAs for HIV-1 eradication.

Viruses are parasites that depend on the host cell’s metabolic resources to provide the energy and molecular building blocks necessary for the production of viral progeny. It has become increasingly clear that viruses extensively modulate the cellular metabolic network to support productive infection. Here, we review the numerous ways through which human cytomegalovirus (HCMV) modulates cellular metabolism, highlighting known mechanisms of HCMV-mediated metabolic manipulation and identifying key outstanding questions that remain to be addressed.

Viruses depend on cellular metabolic resources to supply the energy and biomolecular building blocks necessary for their replication. Human cytomegalovirus (HCMV), a leading cause of birth defects and morbidity in immunosuppressed individuals, induces numerous metabolic activities that are important for productive infection. However, many of the mechanisms through which these metabolic activities are induced and how they contribute to infection are unclear. We find that HCMV infection of fibroblasts induces a neuronal gene signature as well as the expression of several metabolic enzyme isoforms that are typically expressed in other tissue types. Of these, the most substantially induced glycolytic gene was the neuron-specific isoform of enolase 2 (ENO2). Induction of ENO2 expression is important for HCMV-mediated glycolytic activation as well as for the virally induced remodeling of pyrimidine-sugar metabolism, which provides the glycosyl subunits necessary for protein glycosylation. Inhibition of ENO2 expression or activity reduced uridine diphosphate (UDP)-sugar pools, attenuated the accumulation of viral glycoproteins, and induced the accumulation of noninfectious viral particles. In addition, our data indicate that the induction of ENO2 expression depends on the HCMV U L 38 protein. Collectively, our data indicate that HCMV infection induces a tissue atypical neuronal glycolytic enzyme to activate glycolysis and UDP-sugar metabolism, increase the accumulation of glycosyl building blocks, and enable the expression of an essential viral glycoprotein and the production of infectious virions.

Human Cytomegalovirus (HCMV) infection modulates cellular metabolism to support viral replication. Calcium/calmodulin-dependent kinase kinase (CaMKK) and AMP-activated protein kinase (AMPK) regulate metabolic activation and have been found to be important for successful HCMV infection. Here, we explored the contributions that specific CaMKK isoforms and AMPK subunit isoforms make toward HCMV infection. Our results indicate that various CaMKK and AMPK isoforms contribute to infection in unique ways. For example, CaMKK1 is important for HCMV infection at a low multiplicity of infection, but is dispensable for AMPK activation at the earliest times of infection, which our data suggest is more reliant on CaMKK2. Our results also indicate that HCMV specifically induces the expression of the non-ubiquitous AMPKa2 catalytic subunit, found to be important for both HCMV-mediated glycolytic activation and high titer infection. Further, we find that AMPK-mediated glycolytic activation is important for infection, as overexpression of GLUT4, the high capacity glucose transporter, partially rescues viral replication in the face of AMPK inhibition. Collectively, our data indicate that HCMV infection selectively induces the expression of specific metabolic regulatory kinases, relying on their activity to support glycolytic activation and productive infection. IMPORTANCE Viruses are obligate parasites that depend on the host cell to provide the energy and molecular building blocks to mass produce infectious viral progeny. The processes that govern viral modulation of cellular resources have emerged as critical for successful infection. Here, we find that HCMV depends on two kinase isoforms to support infection, CaMKK1 and AMPKa2. We find that HCMV specifically induces expression of the AMPKa2 subunit to induce metabolic activation and drive robust viral replication. These results suggest that HCMV has evolved mechanisms to target specific metabolic regulatory kinase subunits to support productive infection, thereby providing insight into how HCMV hijacks cellular metabolism for its replication, and sheds light on potential viral therapeutic vulnerabilities.

Adoptive immunotherapy with transplant donor derived virus specific T cells is an effective strategy for the treatment of CMV viremia and disease arising after an allogeneic hematopoietic stem cell (HSCT). This approach is not readily applicable if the donor is seronegative or not available to provide lymphocytes for in vitro expansion for CMV specific cytotoxic T lymphocytes (CMV-CTL) lines or if the CMV CTL lines derived from non-identical donors are restricted by non-shared HLA alleles. We and others have previously presented results indicating that treatment with in vitro expanded CMV-CTLs derived from an HLA partially matched third party donors can effectively treat CMV infections in the post-transplant setting. However, the patient population most likely to benefit from this approach has not been well defined. Patients at especially high risk of succumbing to CMV infection include those who acquire resistance to antiviral therapy. We now present results of treatment with banked off-the-shelf third party CMVpp65 specific CTLs in patients with genetically defined mutations in the UL54 DNA polymerase and UL97 kinase CMV genes predicting resistance to antiviral agents. Fifteen recipients of HSCT with mutations defined prior to the start of cell therapy were treated. All 15 had mutations associated with resistance to ganciclovir. Overall 5 had resistance to ganciclovir, foscarnet and cidofovir, 5 had resistance to ganciclovir and foscarnet, one had resistance to ganciclovir and cidofovir and 4 had resistance to ganciclovir alone. Third party CMV-CTLs were selected on the basis of HLA matching at high resolution at a minimum of 2/8 recipient alleles and HLA restriction of the T cells by one or more HLA alleles present in the patient. CMV-CTLS were selected from a bank of 132 lines generated under GMP conditions from normal HSCT donors specifically consented for use of their T cells in patients other than the designated transplant recipient. These 15 patients had a median age of 60.5 (7.4-70.1) years and started therapy with CMV-CTLs a median of 157 (70-564) days after reactivation of CMV. Four of these patients had CMV disease while 11 were treated for viremia alone. Prior therapy in this cohort included foscarnet (N=15) ganciclovir and/or valganciclovir (N=14) and cidofovir and/or brincidofovir (N=9). Each cycle of CMV-CTLs consisted of 3 weekly infusions of 1x106 T-cells/kg/infusion. These 15 patients received a median of 2 (1-3) cycles of CMV-CTLs. CMV-CTLs were well tolerated and there were limited toxicities. Six patients experienced AEs of which one grade 3 and one grade 4 AE were deemed possibly related to infusions with CMV-CTLs. Overall 11/15 (73.3%) patients responded to CMV-CTL therapy including 2 patients with disease with 6 CRs and 5 PRs. Overall survival at 6 months in the 11 responders and 4 non-responders was 72.7% and 25.0% respectively. Within the 6 month follow-up one of the 11 responding patients died of CMV while 3 of the 4 non-responding patients died of CMV. These results indicate that third party donor derived "off-the-shelf" CMV-CTLs can effectively treat CMV viremia or disease in patients not responding to antiviral therapy with demonstrated genetic resistance to antiviral agents. Figure 1. Figure 1. Disclosures Doubrovina: Atara Biotherapeutics: Consultancy, Research Funding. Hasan:Atara Biotherapeutics: Consultancy, Research Funding. Koehne:Atara Biotherapeutics: Consultancy.

Adoptive immunotherapy with transplant donor-derived virus-specific T-cells is effective in the treatment of CMV viremia and disease complicating allogeneic hematopoietic stem cell transplant (HCT), but is not available if the donor is seronegative or unavailable to provide lymphocytes. In addition, CMV-specific T-cell lines (CMV-CTLs) from non-identical donors may be restricted by HLA alleles not shared by the patient, rendering them ineffective. This limitation has become more problematic with increased use of haploidentical HCT donors. We treated 50 transplant recipients with third party donor-derived CMVpp65-specific T-cells between 10/14/11 and 11/28/16, evaluable for response assessment as of 6/20/17. Patients had received an unmodified (n=11) or T-cell depleted HCT (n=33) or a cord blood (n=6), transplant. Fifteen were treated for overt CMV disease involving CNS (N=6) GI (N=10) and Lung (N=2) and 35 for CMV viremia persisting despite >2 weeks of induction therapy with 1-3 antiviral agents. Treatment with CMVpp65-CTLs was initiated at a median of 151 (29-4940) days post transplant and 128 (7-564) days after CMV reactivation. One patient was treated for CMV colitis developing more than 10 years after transplant due to immune suppression for chronic graft versus host disease. Patients had received a median of 3 (1-6) prior antiviral treatments. Third party CMVpp65-CTLs were selected from a bank of 186 lines generated under GMP conditions from normal HCT donors who specifically consented to use of their T cells in patients other than their designated transplant recipient. Selection was made on the basis of HLA restriction by at least one HLA allele shared by the patient and HCT donor, and matching for > 2/10 recipient alleles. If such a line was not available, a patient could be treated with a line matched at only one HLA allele as long as the restriction was through that matched allele. Patients received 3 weekly infusions of approximately 1x106 CMVpp65-CTL/kg/infusion. Patients were sequentially evaluated for clinical and radiographic changes, quantifications of CMV DNA by PCR and IFN+ CMVpp65-specific T-cells in the blood. Responses were assessed 28-42 days after the first of each cycle of CMVpp65-CTLs. Response in patients with CMV disease was considered complete (CR) if all sites were cleared of virus by biopsy and blood sampling and partial (PR) if symptoms resolved and viremia met criteria of PR. In patients treated for persistent viremia, responses were complete if CMV DNA was cleared in repeated testing, and partial if the level of CMV fell based on the testing method by >50% (N=2) or by 2log10 (N=12). Of the 50 patients 18 had a complete and 14 a partial response for an overall response rate of 64%. Response rates in patients with disease (5CR+4PR/15) were similar to those of patients with persistent viremia (13CR+10PR/35). In patients treated for viremia alone, survival at 6 months was 65.7% and in those with disease 60.0% (a). More extensively pretreated patients who received CMVpp65 CTLs > 100 days post CMV initial detection fared as well as those treated earlier (62.1% vs. 66.7% OS) (b). Patients who responded to CMVpp65-CTL therapy (CR or PR) had an improved survival with 6 month overall survival of 81.3% (b) and 12 month overall survival of 62.1% (c); only 1 of these 32 patients died of CMV. In contrast 7 of 18 non-responding patients died of CMV; overall survival in this cohort was 33.3% at 6 months. By 12 months, 8 non-responding patients had died of CMV and overall survival had decreased to 22.2%. Toxicities associated with CMVpp65-CTL infusions in this cohort are limited with 5 patients experiencing adverse events of > grade 3 severity deemed possibly related to CMVpp65-CTL therapy. Two of these patients died, one due to sepsis and one due to progression of CMV. This study demonstrates a high response rate among patients with otherwise refractory CMV viremia and disease. The bank of CMVpp65-CTLs can provide an immediate source of HLA partially-matched appropriately restricted T cells for adoptive immunotherapy to treat persistent CMV viremia and CMV disease, including disease isolated to the CNS. The availability of 3rd party CMVpp65-CTLs enables treatment early in the course of disease and may thereby improve response rates while minimizing toxicity from anti-viral therapy. Figure 1 Figure 1. Disclosures Doubrovina: Atara: Consultancy, Research Funding. Hasan: Atara Biotherapeutics: Consultancy, Other: During time of this study, Research Funding; Merck: Employment. Kernan: Gentium: Other: Received grants from Gentium during the conduct of the study and research was supported by The National Cancer Institute of the National Institutes of Health under award number P30 CA 008748, Research Funding. Koehne: Atara: Consultancy, Patents & Royalties. O'Reilly: Atara: Patents & Royalties, Research Funding.