Viral Gene Products Research Articles

Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection.

Gammaherpesvirus (GHV) pathogenesis is a complex process that involves productive viral replication, dissemination to tissues that harbor lifelong latent infection, and reactivation from latency back into a productive replication cycle. Traditional loss-of-function mutagenesis approaches in mice using murine gammaherpesvirus 68 (MHV68), a model that allows for examination of GHV pathogenesis in vivo, have been invaluable for defining requirements for specific viral gene products in GHV infection. But these approaches are insufficient to fully reveal how viral gene products contribute when the encoded protein facilitates multiple processes in the infectious cycle and when these functions vary over time and from one host tissue to another. To address this complexity, we developed an MHV68 genetic platform that enables cell-type-specific and inducible viral gene deletion in vivo. We employed this system to re-evaluate functions of the MHV68 latency-associated nuclear antigen (mLANA), a protein with roles in both viral replication and latency. Cre-mediated deletion in mice of loxP-flanked ORF73 demonstrated the necessity of mLANA in B cells for MHV68 latency establishment. Impaired latency during the transition from draining lymph nodes to blood following mLANA deletion also was observed, supporting the hypothesis that B cells are a major conduit for viral dissemination. Ablation of mLANA in infected germinal center (GC) B cells severely impaired viral latency, indicating the importance of viral passage through the GC for latency establishment. Finally, induced ablation of mLANA during latency resulted in complete loss of affected viral genomes, indicating that mLANA is critically important for maintenance of viral genomes during stable latency. Collectively, these experiments provide new insights into LANA homolog functions in GHV colonization of the host and highlight the potential of a new MHV68 genetic platform to foster a more complete understanding of viral gene functions at discrete stages of GHV pathogenesis.

Proteomic Analysis of Secretomes of Oncolytic Herpes Simplex Virus-Infected Squamous Cell Carcinoma Cells.

Oncolytic herpes simplex virus type 1 (HSV-1) strain RH2 induced immunogenic cell death (ICD) with the release and surface exposure of damage-associated molecular patterns (DAMPs) in squamous cell carcinoma (SCC) SCCVII cells. The supernatants of RH2-infected SCCVII cells also exhibited antitumor ability by intratumoral administration in SCCVII tumor-bearing mice. The supernatants of RH2-infected cells and mock-infected cells were concentrated to produce Med24 and MedC for proteomic analyses. In Med24, the up- and down-regulated proteins were observed. Proteins including filamin, tubulin, t-complex protein 1 (TCP-1), and heat shock proteins (HSPs), were up-regulated, while extracellular matrix (ECM) proteins were markedly down-regulated. Viral proteins were detected in Med 24. These results indicate that HSV-1 RH2 infection increases the release of danger signal proteins and viral gene products, but decreases the release of ECM components. These changes may alter the tumor microenvironment (TME) and contribute to enhancement of anti-tumor immunity against SCC.

EBV-Encoded Latent Genes.

Epstein-Barr virus (EBV) is one of the most widespread human pathogens. EBV infection is usually asymptomatic, and it establishes life-long latent infection. EBV latent infection sometimes causes various tumorigenic diseases, such as EBV-related lymphoproliferative diseases, Burkitt lymphomas, Hodgkin lymphomas, NK/T-cell lymphomas, and epithelial carcinomas. EBV-encoded latent genes are set of viral genes that are expressed in latently infected cells. They include virally encoded proteins, noncoding RNAs, and microRNAs. Different latent gene expression patterns are noticed in different types of EBV-infected cells. Viral latent gene products contribute to EBV-mediated B cell transformation and likely contribute to lymphomagenesis and epithelial carcinogenesis as well. Many biological functions of viral latent gene products have been reported, making difficult to understand a whole view of EBV latency. In this review, we will focus on latent gene functions that have been verified by genetic experiments using EBV mutants. We will also summarize how viral latent genes contribute to EBV-mediated B cell transformation, Burkitt lymphomagenesis, and epithelial carcinogenesis.

Role of HTLV-1 Tax and HBZ in the Pathogenesis of HAM/TSP.

Human T cell lymphotropic virus type 1 (HTLV-1) infection can lead to development of adult T cell leukemia/lymphoma (ATL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. Understanding the interaction between host and HTLV-1 and the molecular mechanisms associated with disease pathogenesis is critical for development efficient therapies. Two HTLV-1 genes, tax and HTLV-1 basic leucine zipper factor (HBZ), have been demonstrated to play important roles in HTLV-1 infectivity and the growth and survival of leukemic cells. Increased HTLV-1 Tax expression induces the expression of various cellular genes such as IL-2 and IL-15, which directly contributes to lymphocyte activation and immunopathogenesis in HAM/TSP patients. However, little is known about the molecular and cellular mechanism of HBZ in development of HAM/TSP. It has been reported that HBZ mRNA expression was detected in HAM/TSP patients higher than in asymptomatic carriers and correlated with proviral load and disease severity. Unlike HTLV-1 tax, HBZ escapes efficient anti-viral immune responses and therefore these reactivities are difficult to detect. Thus, it is important to focus on understanding the function and the role of HTLV-1 tax and HBZ in disease development of HAM/TSP and discuss the potential use of these HTLV-1 viral gene products as biomarkers and therapeutic targets for HAM/TSP.

Simian T Lymphotropic Virus 1 Infection of Papio anubis: tax Sequence Heterogeneity and T Cell Recognition.

Baboons naturally infected with simian T lymphotropic virus (STLV) are a potentially useful model system for the study of vaccination against human T lymphotropic virus (HTLV). Here we expanded the number of available full-length baboon STLV-1 sequences from one to three and related the T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons, animals 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these T cell lines revealed them to be closely related but distinct from each other and from the baboon STLV-1 sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more gamma interferon and tumor necrosis factor alpha in response to Tax peptides corresponding to their own STLV-1 sequence than in response to Tax peptides corresponding to the reference baboon STLV-1 sequence. Thus, our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons, extremely low heterogeneity of STLV sequences within each baboon, no evidence for superinfection within each baboon, and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed, premature stop codons were observed in 7% and 56% of tax sequences from peripheral blood mononuclear cells from animals 12141 and 12752, respectively.IMPORTANCE It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventive vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons, the identity of the viral gene product that is the major target of cellular immune responses, the persistence of viral amino acid sequences that are the major targets of cellular immune responses, and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what are seen with HTLV infection of humans. These results justify the use of STLV-infected baboons as a model system for vaccine development efforts.

Comparison of Liver Detargeting Strategies for Systemic Therapy with Oncolytic Adenovirus Serotype 5.

Oncolytic viruses would ideally be of use for systemic therapy to treat disseminated cancer. To do this safely, this may require multiple layers of cancer specificity. The pharmacology and specificity of oncolytic adenoviruses can be modified by (1) physical retargeting, (2) physical detargeting, (3) chemical shielding, or (4) by modifying the ability of viral early gene products to selectively activate in cancer versus normal cells. We explored the utility of these approaches with oncolytic adenovirus serotype 5 (Ad5) in immunocompetent Syrian hamsters bearing subcutaneous HaK tumors. After a single intravenous injection to reach the distant tumors, the physically hepatocyte-detargeted virus Ad5-hexon-BAP was more effective than conditionally replicating Ad5-dl1101/07 with mutations in its E1A protein. When these control or Ad5 treated animals were treated a second time by intratumoral injection, prior exposure to Ad5 did not affect tumor growth, suggesting that anti-Ad immunity neither prevented treatment nor amplified anti-tumor immune responses. Ad5-dl1101/07 was next chemically shielded with polyethylene glycol (PEG). While 5 kDa of PEG blunted pro-inflammatory IL-6 production induced by Ad5-dl1101/07, this shielding reduced Ad oncolytic activity.

KSHV and the Role of Notch Receptor Dysregulation in Disease Progression

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of two human cancers, Kaposi’s Sarcoma (KS) and primary effusion lymphoma (PEL), and a lymphoproliferation, Multicentric Castleman’s Disease (MCD). Progression to tumor development in KS is dependent upon the reactivation of the virus from its latent state. We, and others, have shown that the Replication and transcriptional activator (Rta) protein is the only viral gene product that is necessary and sufficient for viral reactivation. To induce the reactivation and transcription of viral genes, Rta forms a complex with the cellular DNA binding component of the canonical Notch signaling pathway, recombination signal binding protein for Jk (RBP-Jk). Formation of this Rta:RBP-Jk complex is necessary for viral reactivation to occur. Expression of activated Notch has been shown to be dysregulated in KSHV infected cells and to be necessary for cell growth and disease progression. Studies into the involvement of activated Notch in viral reactivation have yielded varied results. In this paper, we review the current literature regarding Notch dysregulation by KSHV and its role in viral infection and cellular pathogenesis.

The HBx gene of hepatitis B virus can influence hepatic microenvironment via exosomes by transferring its mRNA and protein

The cellular secretory vesicles known as ‘exosomes’ have emerged as key player in intercellular transport and communication between different eukaryotic in order to maintain body homeostasis. Many pathogenic viruses utilize exosome pathway to efficiently transfer bioactive components from infected cells to naïve cells. Here, we show that HBx can tweak the exosome biogenesis machinery both by enhancing neutral sphingomyelinase2 activity as well as by interacting with exosomal biomarkers such as neutral sphingomyelinase2, CD9 and CD81. The nano particle tracking analysis revealed enhanced secretion of exosomes by the HBx-expressing cells while confocal studies confirmed the co-localization of HBx with CD9 and CD63. Importantly, we observed the encapsulation of HBx mRNA and protein in these exosomes besides some other qualitative changes. The exosomal cargo secreted by HBx-expressing cells had a profound effect on the recipient hepatic cells including creation of a milieu conducive for cellular-transformation. Thus, the present study unfolds a novel role of HBx in intercellular communication by facilitating horizontal transfer of viral gene products and other host factors via exosomes in order to support viral spread and pathogenesis.

PUM1 is a biphasic negative regulator of innate immunity genes by suppressing LGP2

PUM1 is an RNA binding protein shown to regulate the stability and function of mRNAs bearing a specific sequence. We report the following: (i) A key function of PUM1 is that of a repressor of key innate immunity genes by repressing the expression of LGP2. Thus, between 12 and 48 hours after transfection of human cells with siPUM1 RNA there was an initial (phase 1) upsurge of transcripts encoding LGP2, CXCL10, IL6, and PKR. This was followed 24 hours later (phase 2) by a significant accumulation of mRNAs encoding RIG-I, SP100, MDA5, IFIT1, PML, STING, and IFNβ. The genes that were not activated encoded HDAC4 and NF-κB1. (ii) Simultaneous depletion of PUM1 and LGP2, CXCL10, or IL6 revealed that up-regulation of phase 1 and phase 2 genes was the consequence of up-regulation of LGP2. (iii) IFNβ produced 48-72 hours after transfection of siPUM1 was effective in up-regulating LGP2 and phase 2 genes and reducing the replication of HSV-1 in untreated cells. (iv) Because only half of genes up-regulated in phase 1 and 2 encode mRNAs containing PUM1 binding sites, the upsurge in gene expression could not be attributed solely to stabilization of mRNAs in the absence of PUM1. (v) Lastly, depletion of PUM2 does not result in up-regulation of phase 1 or phase 2 genes. The results of the studies presented here indicate that PUM1 is a negative regulator of LGP2, a master regulator of innate immunity genes expressed in a cascade fashion.

Identification of immediate early gene products of bovine herpes virus 1 (BHV-1) as dominant antigens recognized by CD8 T cells in immune cattle.

In common with other herpes viruses, bovine herpes virus 1 (BHV-1) induces strong virus-specific CD8 T-cell responses. However, there is a paucity of information on the antigenic specificity of the responding T-cells. The development of a system to generate virus-specific CD8 T-cell lines from BHV-1-immune cattle, employing Theileria-transformed cell lines for antigen presentation, has enabled us to address this issue. Use of this system allowed the study to screen for CD8 T-cell antigens that are efficiently presented on the surface of virus-infected cells. Screening of a panel of 16 candidate viral gene products with CD8 T-cell lines from 3 BHV-1-immune cattle of defined MHC genotypes identified 4 antigens, including 3 immediate early (IE) gene products (ICP4, ICP22 and Circ) and a tegument protein (UL49). Identification of the MHC restriction specificities revealed that the antigens were presented by two or three class I MHC alleles in each animal. Six CD8 T-cell epitopes were identified in the three IE proteins by screening of synthetic peptides. Use of an algorithm (NetMHCpan) that predicts the peptide-binding characteristics of restricting MHC alleles confirmed and, in some cases refined, the identity of the epitopes. Analyses of the epitope specificity of the CD8 T-cell lines showed that a large component of the response is directed against these IE epitopes. The results indicate that these IE gene products are dominant targets of the CD8 T-cell response in BHV-I-immune cattle and hence are prime-candidate antigens for the generation of a subunit vaccine.

Ranaviruses and other members of the family Iridoviridae: Their place in the virosphere

Members of the family Iridoviridae, collectively referred to as iridovirids, are large, double-stranded DNA-containing viruses that infect invertebrates and cold-blooded (ectothermic) vertebrates. Infections in the former often lead to massive levels of virus replication resulting in iridescence of the infected animal and ultimately death. Among the latter, infections target a variety of organs and are capable of causing high levels of morbidity and mortality among commercially and ecologically important fish and amphibian species. The viral replication strategy has been elucidated primarily through the study of frog virus 3 (FV3) with additional input from other iridovirids of ecological or commercial importance. Replication occurs within both nuclear and cytoplasmic compartments and involves synthesis of genome length and concatemeric DNA, extensive methylation of the viral genome (among vertebrate viruses only), coordinate expression of three classes of viral gene products, and formation of icosahedral virions within cytoplasmic viral assembly sites. Phylogenetic analyses delineate five genera within the family and suggest that members of the families Iridoviridae, Ascoviridae, and Marseilleviridae compromise a monophyletic lineage in which ascoviruses are most closely related to invertebrate iridoviruses.

Chronic viral hepatitis and its association with liver cancer

Chronic infection with hepatitis viruses represents the major causative factor for end-stage liver diseases, including liver cirrhosis and primary liver cancer (hepatocellular carcinoma, HCC). In this review, we highlight the current understanding of the molecular mechanisms that drive the hepatocarcinogenesis associated with chronic hepatitis virus infections. While chronic inflammation (associated with a persistent, but impaired anti-viral immune response) plays a major role in HCC initiation and progression, hepatitis viruses can also directly drive liver cancer. The mechanisms by which hepatitis viruses induce HCC include: hepatitis B virus DNA integration into the host cell genome; metabolic reprogramming by virus infection; induction of the cellular stress response pathway by viral gene products; and interference with tumour suppressors. Finally, we summarise the limitations of hepatitis virus-associated HCC model systems and the development of new techniques to circumvent these shortcomings.

Metabolic Profiles in Cell Lines Infected with Classical Swine Fever Virus.

Viruses require energy and biosynthetic precursors from host cells for replication. An understanding of the metabolic interplay between classical swine fever virus (CSFV) and host cells is important for exploring the complex pathological mechanisms of classical swine fever (CSF). In the current study, and for the first time, we utilized an approach involving gas chromatography coupled with mass spectrometry (GC-MS) to examine the metabolic profiles within PK-15 and 3D4/2 cells infected with CSFV. The differential metabolites of PK-15 cells caused by CSFV infection mainly included the decreased levels of glucose 6-phosphate [fold change (FC) = −1.94)] and glyceraldehyde-3-phosphate (FC = −1.83) during glycolysis, ribulose 5-phosphate (FC = −1.51) in the pentose phosphate pathway, guanosine (FC = −1.24) and inosine (FC = −1.16) during purine biosynthesis, but the increased levels of 2-ketoisovaleric acid (FC = 0.63) during the citrate cycle, and ornithine (FC = 0.56) and proline (FC = 0.62) during arginine and proline metabolism. However, metabolite changes caused by CSFV infection in 3D4/2 cells included the reduced glyceraldehyde-3-phosphate (FC = −0.77) and pyruvic acid (FC = −1.42) during glycolysis, 2-ketoglutaric acid (FC = −1.52) in the citrate cycle, and the elevated cytosine (FC = 2.15) during pyrimidine metabolism. Our data showed that CSFV might rebuild cellular metabolic programs, thus aiding viral replication. These findings may be important in developing targets for new biomarkers for the diagnosis and identification of enzyme inhibitors or metabolites as antiviral drugs, or screening viral gene products as vaccines.

Expression of the ERBB Family of Ligands and Receptors in Gastric Cancer

Objective: Gastric cancer (GC) is the second most common cancer and the third leading cause of cancer-related death in Korea. Alterations in the ERBB (homology to the erythroblastoma viral gene product, v-erbB) receptor family and ERBB-related signaling pathways are frequently observed in GC. However, the roles of the ERBB receptors and their ligands in GC are not well established. Methods: We evaluated the expression levels of various ERBB receptor ligands (i.e., heparin-binding epidermal growth factor-like growth factor [HBEGF], transforming growth factor-α [TGFA], amphiregulin [AREG], epiregulin [EREG], epidermal growth factor [EGF], and betacellulin [BTC]) and 3 ERBB family receptors (i.e., epidermal growth factor receptor [EGFR], human EGFR2 [HER2], and ERBB3) in 313 cases of GC using immunohistochemistry, fluorescence in situ hybridization, and mRNA in situ hybridization. Results: A high expression of EGFR, HER2, and ERBB3 was observed in 30, 32, and 27 cases, respectively. A high expression of HBEGF, TGFA, AREG, EREG, EGF, and BTC was observed in 91, 97, 151, 74, 26, and 37 cases, respectively. A high expression of TGFA was associated with better survival, while a high expression of BTC was associated with worse survival. These results were confirmed using Cox proportional hazards analysis. HBEGF, TGFA, AREG, tumor-node-metastasis classification, Lauren's classification, and ERBB3 were significant survival parameters in multivariate analysis. Conclusion: Among the ERBB family receptors and ligands examined, 3 ligands (i.e., TGFA, HBEGF, and AREG) and ERBB3 had a prognostic impact.

Synthetic lethal mutations in the cyclin A interface of human cytomegalovirus

Generally, the antagonism between host restriction factors and viral countermeasures decides on cellular permissiveness or resistance to virus infection. Human cytomegalovirus (HCMV) has evolved an additional level of self-imposed restriction by the viral tegument protein pp150. Depending on a cyclin A-binding motif, pp150 prevents the onset of viral gene expression in the S/G2 cell cycle phase of otherwise fully permissive cells. Here we address the physiological relevance of this restriction during productive HCMV infection by employing a cyclin A-binding deficient pp150 mutant virus. One consequence of unrestricted viral gene expression in S/G2 was the induction of a G2/M arrest. G2-arrested but not mitotic cells supported viral replication. Cyclin A destabilization by the viral gene product pUL21a was required to maintain the virus-permissive G2-arrest. An HCMV double-point mutant where both pp150 and pUL21a are disabled in cyclin A interaction forced mitotic entry of the majority of infected cells, with a severe negative impact on cell viability and virus growth. Thus, pp150 and pUL21a functionally cooperate, together building a cell cycle synchronization strategy of cyclin A targeting and avoidance that is essential for productive HCMV infection.

Nanoparticle exposure reactivates latent herpesvirus and restores a signature of acute infection

BackgroundInhalation of environmental (nano) particles (NP) as well as persistent herpesvirus-infection are potentially associated with chronic lung disease and as both are omnipresent in human society a coincidence of these two factors is highly likely. We hypothesized that NP-exposure of persistently herpesvirus-infected cells as a second hit might disrupt immune control of viral latency, provoke reactivation of latent virus and eventually lead to an inflammatory response and tissue damage.ResultsTo test this hypothesis, we applied different NP to cells or mice latently infected with murine gammaherpesvirus 68 (MHV-68) which provides a small animal model for the study of gammaherpesvirus-pathogenesis in vitro and in vivo. In vitro, NP-exposure induced expression of the typically lytic viral gene ORF50 and production of lytic virus. In vivo, lytic viral proteins in the lung increased after intratracheal instillation with NP and elevated expression of the viral gene ORF50 could be detected in cells from bronchoalveolar lavage. Gene expression and metabolome analysis of whole lung tissue revealed patterns with striking similarities to acute infection. Likewise, NP-exposure of human cells latently infected with Epstein-Barr-Virus also induced virus production.ConclusionsOur results indicate that NP-exposure of persistently herpesvirus-infected cells – murine or human – restores molecular signatures found in acute virus infection, boosts production of lytic viral proteins, and induces an inflammatory response in the lung – a combination which might finally result in tissue damage and pathological alterations.

Conditioned medium from persistently RSV-infected macrophages alters transcriptional profile and inflammatory response of non-infected macrophages

Cells susceptible to persistent viral infections undergo important changes in their biological functions as a consequence of the expression of viral gene products that are capable of altering the gene expression profile of the host cell. Previously, we reported that persistence of the RSV genome in a mouse macrophage cell line induces important alterations in cell homeostasis, including constitutive expression of IFN-β and other pro-inflammatory cytokines. Here, we postulated that changes in the homeostasis of non-infected macrophages could be induced by soluble factors secreted by persistently RSV- infected macrophages. To test this hypothesis, non-infected mouse macrophages were treated with conditioned medium (CM) collected from cultures of persistently RSV-infected macrophages. Total RNA was extracted and a microarray-based gene expression analysis was performed. Non-infected macrophages, treated under similar conditions with CM obtained from cultures of non-infected macrophages, were used as a control to establish differential gene expression between the two conditions. Results showed that CM from the persistently RSV-infected cultures altered expression of a total of 95 genes in non-infected macrophages, resulting in an antiviral gene-transcription profile along with inhibition of the inflammatory response, since some inflammatory genes were down-regulated, including Nlrp3 and Il-1 β, both related to the inflammasome pathway. However, down-regulation of Nlrp3 and Il-1 β was reversible upon acute RSV infection. Additionally, we observed that the inflammatory response, evaluated by secreted IL-1 β, a final product of the inflammasome activity, was enhanced during acute RSV infection in macrophages treated with CM from persistently RSV-infected cultures, compared to that in macrophages treated with the control CM. This suggests that soluble factors secreted during RSV persistence may induce an exacerbated inflammatory response in non-infected cells.

Herpesvirus Nuclear Egress.

Herpesviruses assemble and package their genomes into capsids in the nucleus, but complete final assembly of the mature virion in the cell cytoplasm. This requires passage of the genome-containing capsid across the double-membrane nuclear envelope. Herpesviruses have evolved a mechanism that relies on a pair of conserved viral gene products to shuttle the capsids from the nucleus to the cytoplasm by way of envelopment and de-envelopment at the inner and outer nuclear membranes, respectively. This complex process requires orchestration of the activities of viral and cellular factors to alter the architecture of the nuclear membrane, select capsids at the appropriate stage for egress, and accomplish efficient membrane budding and fusion events. The last few years have seen major advances in our understanding of the membrane budding mechanism and helped clarify the roles of viral and cellular proteins in the other, more mysterious steps. Here, we summarize and place into context this recent research and, hopefully, clarify both the major advances and major gaps in our understanding.

Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential.

Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions.

AMC-053: Pilot Study of an Oncolytic Viral Strategy Using Bortezomib with ICE +/- Rituximab for Relapsed/Refractory HIV+ Lymphomas

Introduction:Both HIV+ Hodgkin and non-Hodgkin lymphomas have higher rates of latent infection by the gamma-herpesviruses (GHVs), Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpes virus (KSHV), than corresponding lymphomas in the HIV-seronegative population. Bortezomib, a proteasome inhibitor, induces lytic activation of both EBV and KSHV. Lytic activation of GHVs latently infecting lymphoma cells is hypothesized to beneficial both for direct tumor cell lysis as well as increased cytotoxic immune response due to viral lytic gene products. Furthermore, preclinical studies found proteasome inhibition impairs HIV infectivity via preservation of human anti-retroviral APOBEC3G, suggesting a novel therapeutic strategy to control HIV. Given that therapy of relapsed or refractory HIV-associated lymphoma (R/R-HAL) results in modest rates of remission, we sought to capitalize on the high viral association within HAL using an oncolytic strategy with bortezomib.Objectives:The primary objective of this study was to evaluate safety and overall response rate (ORR) of R/R-HAL to bortezomib combined with ifosfamide, carboplatin, etoposide +/- rituximab (ICE/R).The secondary objectives of this study were to estimate the impact of bortezomib on lytic activation of EBV and KSHV, using peripheral blood mononuclear cell (PBMC) viral loads, and on HIV using single copy plasma viral loads; to report overall survival at 1 year (1yr-OS); and to correlate EBV and KSHV viral load changes with lymphoma response.Methods:A 3+3 dose escalation design with a 7-day lead-in period of bortezomib alone prior to bortezomib + ICE/R allowed for assessment of early effects of bortezomib on viral loads. Bortezomib was given intravenously on day 1 and 8 of each cycle at one of 4 dose levels: 0.7, 1, 1.3 or 1.5 mg/m2. Standard dose ICE +/- R began day 8 of cycle 1 (28-day cycle); ICE +/- R began day 1 of all subsequent cycles (21-day cycle). Rituximab was included in the regimen only for CD20+ lymphoma.Binomial proportions were used to estimate ORR. The product-limit (Kaplan-Meier) method was used to estimate 1yr-OS. The Wilcoxon signed rank test was used to evaluate changes in viral loads.Results:Twenty-three subjects were enrolled from 7 sites within the AIDS Malignancy Consortium (AMC). More than 90% of enrolled subjects were men and half were minorities; at baseline, 20/23 were on antiretroviral therapy, median CD4 count was315/µL and median HIV viral load was undetectable. Mean age was 50 years. Over half of subjects had stage IV HAL; the majority had diffuse large B-cell lymphoma (DLBCL) (n=15), 2 had primary effusion (PEL), 3 had plasmablastic and 2 had Hodgkin lymphoma.Figure 1 summarizes grade 3-4 toxicities of the 22 subjects evaluable for adverse events during the dose-limiting toxicity period (cycles 1+2). The maximum tolerated dose was not reached at the highest dose cohort studied (bortezomib 1.5mg/m2).Responses occurred in 14/22 subjects initiating protocol therapy: 5 complete and 9 partial responses (PR). Of the 20 subjects who completed 2 or more cycles, the ORR was 80%. Nine of the responders underwent auto-hematopoietic stem cell transplant after protocol therapy. 1yr-OS was 55%.After bortezomib alone, median values of EBV PBMC viral load measured on day 8 were 2x greater than baseline. However, paired analysis did not confirm significant change in this small sample (n=16 evaluable), and there was no correlation found between change in EBV viral load and response. The 2 subjects with known KSHV+ lymphoma (PEL) each had more than a 1-log increase in day 8 KSHV viral load compared with baseline. Both of these subjects attained a PR from protocol therapy.Conclusions:Addition of bortezomib to ICE/R in R/R HAL is feasible with ORR (80%) and 1yr-OS (55%) comparing very favorably with a prior AMC retrospective report of ICE/R in R/R HAL (n=31, ORR 32%, 1yr-OS 38%, Bayraktar 2012). Evaluation of data collected from individual subjects suggests GHV lytic activation may occur with bortezomib alone. However, the lower bortezomib dose levels used to treat the bulk of study subjects and the limited study sample size limit our power to confirm this conclusion. We plan to further explore the effects of proteasome inhibition on GHVs. Evaluation of the impact of bortezomib on HIV replication is pending and will be presented at the meeting. [Display omitted] DisclosuresReid:Millennium: Research Funding. Sparano:Takeda: Research Funding.