Virus Production Research Articles

Toxic small alarmone synthetase FaRel2 inhibits translation by pyrophosphorylating tRNAGly and tRNAThr.

Translation-targeting toxic small alarmone synthetases (toxSAS) are effectors of bacterial toxin-antitoxin systems that pyrophosphorylate the 3'-CCA end of transfer RNA (tRNA) to prevent aminoacylation. toxSAS are implicated in antiphage immunity: Phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNAGly and tRNAThr. The first, second, fourth, and fifth base pairs of the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRelSJ46 differ in tRNA specificity from FaRel2 and rationalize this through structural modeling: While the universal 3'-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defenses, we hypothesize that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

Whole-blood model reveals granulocytes as key sites of dengue virus propagation, expanding understanding of disease pathogenesis.

Dengue virus (DENV) infection poses a significant global health threat, yet our understanding of its immunopathogenesis remains incomplete due to limitations of existing models. Here, we establish an in vitro whole-blood model using hirudin, an anticoagulant that preserves complement activity and cellular interactions, to study DENV infection. Our model reveals the susceptibility of all major leukocyte populations to DENV infection, with monocytes and granulocytes demonstrating high permissiveness and production of infectious virus progeny. Notably, granulocytes emerge as previously unrecognized targets of DENV infection, highlighting the importance of studying viral tropism within a physiologically relevant context. We also observed efficient DENV binding to B cells, but limited production of infectious virus, suggesting a potential role in viral sequestration or immune dysregulation. Interestingly, both NK and T cells, while less permissive, were also found to be susceptible to DENV infection. Our ex vivo analysis of whole blood from DENV-infected patients confirms the susceptibility of granulocytes, monocytes, B cells, natural killer cells, and T cells to infection, further validating the clinical relevance of our model. Additionally, we observed dynamic changes in circulating blood cell populations during acute dengue, potentially reflecting both direct virus-mediated effects and immune responses. This whole-blood model offers a valuable tool for investigating the complex interplay between DENV and host factors, facilitating a deeper understanding of dengue pathogenesis and ultimately contributing to the development of novel therapeutic strategies.IMPORTANCEDengue virus (DENV) infection is a significant global health threat, with increasing incidence in endemic regions and expanding geographic range due to factors like global warming. Current models for studying DENV pathogenesis often lack the complexity of the human immune system, hindering the development of effective therapies and vaccines. To address this, we have established the first in vitro whole-blood model using hirudin, preserving critical immune components and cellular interactions. This model reveals granulocytes as previously unrecognized targets of productive DENV infection, challenging existing paradigms of viral tropism. Our ex vivo analysis of patient blood samples confirms the clinical relevance of this finding and validates our model's utility. This unique model offers a powerful platform for future studies to dissect the complex interactions between DENV and the host immune system, including the roles of different leukocyte populations, ultimately informing the development of novel therapeutic strategies to combat this devastating disease.

NS1-mediated DNMT1 degradation regulates human bocavirus 1 replication and RNA processing.

Methylation of the DNA genome plays an important role in viral gene inactivation. However, the role of DNA methylation in human bocavirus (HBoV) remains unclear. In this study, the HBoV1 genomic DNA was found extensively methylated at the CHG and CHH sites. Inhibiting DNA methylation with 5-aza-2'-deoxycytidine (DAC) altered the methylation status and reduced viral DNA production, while enhanced the RNA splicing at D1 and D3 sites and the polyadenylation at the proximal polyadenylation site, (pA)p. Knockdown of DNA methyltransferase 1 (DNMT1) had the same effect on viral DNA synthesis and RNA processing as the DAC treatment, indicating that DNMT1 is the major host methyltransferase involved in viral DNA methylation. In addition, the nonstructural protein NS1 promoted DNMT1 degradation through the ubiquitin-proteasome pathway to regulate viral replication and RNA processing. Collectively, the results suggest that DNA methylation and DNMT1 facilitate HBoV replication and are essential for appropriate NS1 localization in the nucleus. DNMT1 degradation through NS1 promotes the virus RNA processing, leading to viral protein expression.

Prokaryote- and Eukaryote-Based Expression Systems: Advances in Post-Pandemic Viral Antigen Production for Vaccines

This review addresses the ongoing global challenge posed by emerging and evolving viral diseases, underscoring the need for innovative vaccine development strategies. It focuses on the modern approaches to creating vaccines based on recombinant proteins produced in different expression systems, including bacteria, yeast, plants, insects, and mammals. This review analyses the advantages, limitations, and applications of these expression systems for producing vaccine antigens, as well as strategies for designing safer, more effective, and potentially ‘universal’ antigens. The review discusses the development of vaccines for a range of viral diseases, excluding SARS-CoV-2, which has already been extensively studied. The authors present these findings with the aim of contributing to ongoing research and advancing the development of antiviral vaccines.

Pilot-scale process development for recombinant adeno-associated virus (rAAV) production based on high-density Sf9 cell culture

BackgroundIn recent years, gene therapy drugs have been widely marketed, and their effectiveness and potential have been confirmed. Thus, increasing their production on an industrial scale is critical. Recombinant adeno-associated viruses (rAAVs) are optimal vectors for gene therapy applications, and the baculovirus expression vector system (BEVS), which is based on Sf9 cell culture, is a common tool for rAAV production.MethodsIn this work, an Sf9 cell fed-batch process was developed using shake flasks. In the laboratory-scale bioreactor, four processes were selected as the key factors when carrying out the orthogonal experiment. On the basis of the equal P/V principle and considering the problem posed by air bubbles, a pilot-scale level bioreactor process was established.ResultsHere, we describe a method in which a BEVS was used to produce rAAV vectors, with the cell density increasing to 22.8 × 106 cells/mL and the rAAV titre increasing to 20 × 1011 VG/mL upon adding feed material. By resolving the problems associated with high-density cell culture and air bubbles, this process was successfully scaled to a 50 L pilot-scale level.ConclusionsThis successful experiment not only provides a technological basis for further scale-up but also guarantees product capacity. We hope that this development process can provide reference data for studying cell culture-based drug production.

Production of Oncolytic Measles Virus in Vero Cells: Impact of Culture Medium and Multiplicity of Infection

Oncolytic measles virus (MeV) is a promising anti-cancer treatment. However, the production of high titers of infectious MeV (typically 107–109 TCID50 per dose) is challenging because the virus is unstable under typical production conditions. The objective of this study was to investigate how the multiplicity of infection (MOI) and different media—a serum-containing medium (SCM), a serum-free medium (SFM) and two chemically defined media (CDM)—affect MeV production. We infected Vero cells at MOIs of 0.02, 0.2 or 2 TCID50 mL−1 and the lowest MOI resulted in the largest number of infected cells towards the end of the production period. However, this did not equate to higher maximum MeV titers, which were similar for all the MOIs. The medium had a moderate effect, generating maximum titers of 0.89–2.17 × 106, 1.08–1.25 × 106 and 4.58–9.90 × 105 TCID50 mL−1 for the SCM, SFM and CDM, respectively. Infection at a low MOI often required longer process times to reach maximum yields. On the other hand, a high MOI requires a large amount of MeV stock. We would therefore recommend a mid-range MOI of 0.2 TCID50 mL−1 for MeV production. Our findings show that SCM, SFM and CDM are equally suitable for MeV production in terms of yield and process time. This will allow MeV production in serum-free conditions, addressing the safety risks and ethical concerns associated with the use of serum.

FoxK1 and FoxK2 cooperate with ORF45 to promote late lytic replication of Kaposi's sarcoma-associated herpesvirus.

Lytic replication is essential for persistent infection of Kaposi's sarcoma-associated herpesvirus (KSHV) and the pathogenesis of related diseases, and many cellular pathways are hijacked by KSHV proteins to initiate and control the lytic replication of this virus. However, the mechanism involved in KSHV lytic replication from the early to the late phases remains largely undetermined. We previously revealed that KSHV open reading frame 45 (ORF45) plays important roles in late transcription and translation. In the present study, we revealed that the Forkhead box proteins FoxK1 and FoxK2 are ORF45-binding proteins and are essential for KSHV lytic gene expression and virion production, and that depletion of FoxK1 or FoxK2 significantly suppresses the expression of many late viral genes. FoxK1 and FoxK2 directly bind to the promoters of several late viral genes, ORF45 augments the promoter binding and transcriptional activity of FoxK1 and FoxK2, and then FoxK1 or FoxK2 cooperates with ORF45 to promote late viral gene expression. Our findings suggest that ORF45 interacts with FoxK1 and FoxK2 and promotes their occupancy on a cluster of late viral promoters and their subsequent transcriptional activity; consequently, FoxK1 and FoxK2 promote late viral gene expression to facilitate KSHV lytic replication.IMPORTANCEThe forkhead box proteins FoxK1 and FoxK2 can act as transcriptional inhibitors or activators to regulate several important processes, including aerobic glycolysis, metabolism, autophagy, and antiviral responses. However, the subversion and functions of FoxK1 and FoxK2 during KSHV infection and the pathogenesis of related diseases remain unknown. Here, we revealed that ORF45 binds to FoxK1 and FoxK2 and increases their transcriptional activity during KSHV lytic replication; consequently, FoxK1 and FoxK2 bind to late viral promoters and cooperate with ORF45 to promote late lytic gene expression. Our findings reveal two new ORF45 partners and a new function of ORF45 in which it utilizes FoxK1 and FoxK2 to promote transcription during late KSHV lytic replication.

Curcumin derivative C210 induces Epstein–Barr virus lytic cycle and inhibits virion production by disrupting Hsp90 function

Lytic induction therapy was devised to selectively combat malignancies associated with Epstein–Barr virus (EBV) by triggering viral reactivation from latency. At present, the major challenges of lytic induction therapy are to maximize reactivating efficiencies and meanwhile minimize infectious virion production. C210, a novel curcumin derivative with potent Hsp90 inhibitory activity, was explored for EBV-reactivating and virion-producing effects in EBV-positive nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC) cell lines. And the molecular mechanisms underlying these effects were determined. Follow C210 treatment, EBV lytic RNAs and proteins were upregulated, but infectious virions were not produced. Knockdown of heat shock protein 90 (Hsp90) induced expression of lytic RNAs and proteins, and diminished C210-driven EBV lytic induction. Pretreatment with an X box binding protein 1 (XBP1) inhibitor reduced C210-induced EBV lytic RNA. Furthermore, we demonstrated that C210 inhibited the binding of Hsp90 with its clients, signal transducer and activator of transcription 3 (STAT3) and xeroderma pigmentosum group B-complementing protein (XPB), which subsequently promoted their proteasomal degradation. Degradation of STAT3 by C210 enhanced the EBV-reactivating and anticancer capacity of suberoylanilide hydroxamic acid (SAHA). Depletion of XPB blocked SAHA-induced expression of late viral genes and production of infectious virions. These results elucidate a novel Hsp90 inhibitor targeting EBV lytic phase and extend the research on lytic induction strategy, which may offer reference value in the treatment of EBV-positive malignancies.

Fish Snx27 promotes viral products by modulating the innate immune response and exosomal machinery.

Viral nervous necrosis caused by the nervous necrosis virus (NNV) poses a significant threat to the global aquaculture industry. Developing preventive methods to minimize economic losses due to NNV infections is crucial. This study explored the role of the sorting nexin 27 (Snx27) gene, encoded by the orange-spotted grouper (Epinephelus coioides) and referred to as EcSnx27, as an immune regulator affecting red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro. Our findings revealed that EcSnx27 negatively regulates interferon (IFN)-related cytokines and the promoter activities of fish ISRE and NF-κB. Furthermore, we identified the SNX-FERM and SNX-FERM-like domains as responsible for the interaction between EcSnx27 and RGNNV coat protein. Through the detection of viable virions associated with EcSnx27-containing exosomes, we propose that EcSnx27 may contribute to the release process of RGNNV by influencing the apoptosis-linked gene 2-interacting protein X (ALIX)-associated exosomal pathway. Consequently, our study suggests that EcSnx27 promotes RGNNV replication by inhibiting the IFN immune response and facilitating virus production and release through ALIX-mediated exosomal machinery.IMPORTANCERed grouper nervous necrosis virus (RGNNV), a member of the Nodaviridae family, has emerged as a significant cause of fish diseases worldwide, leading to high morbidity and mortality rates. This study investigated the sorting nexin 27 (Snx27) gene encoded by the orange-spotted grouper (Epinephelus coioides) on RGNNV infection in grouper kidney cells. Our findings revealed that EcSnx27 negatively regulated the interferon pathway, resulting in the promotion of RGNNV replication. Additionally, we observed that EcSnx27 could interact with apoptosis-linked gene 2-interacting protein X (ALIX) and the RGNNV coat protein, suggesting its potential involvement in viral release processes through modulation of the exosomal pathway. Our study identified EcSnx27 as a key target that RGNNV exploits to enhance viral production. This finding offers valuable insights into the immune evasion and viral release mechanisms of non-enveloped RNA viruses.

Rapid production of recombinant rotaviruses by overexpression of NSP2 and NSP5 genes with modified nucleotide sequences.

Reverse genetics systems for rotaviruses (RV) facilitate the generation of genetically engineered RVs by transfection of 11 plasmids encoding 11 genomic viral RNA segments. In addition to viral genome expression, overexpression of NSP2 and NSP5 has been used to increase the rescue efficiency of recombinant RVs. Here, we showed that the overexpression of nucleotide sequence-modified NSP2 and NSP5 enabled the rapid and efficient production of recombinant RVs. Using improved reverse genetics, we established a reverse genetics system for human and bovine RV clinical isolates, as well as laboratory strains of bovine RV (NCDV and UK) and porcine RV (Gottfried). In addition, we rescued low-replicating recombinant RVs carrying a mutant NSP4 lacking the double-layered particle-binding domain, which was deficient in the efficient production of mature virions. These advancements in reverse genetics enabled the generation of molecular clones of RV clinical isolates and recombinant RVs harboring critical amino acid mutations, offering a versatile platform for investigating RV biology and pathogenesis.IMPORTANCERecombinant rotavirus (RV) synthesis via reverse genetics relies on both the viral propagation capacity and the efficiency of the experimental system. Since the establishment of our reverse genetics system, several enhancements have been implemented to augment the rescue efficiency. Nevertheless, challenges persist in generating RV clinical strains and recombinant viruses with low replication capacities. Notably, this improved reverse genetics system successfully facilitated the establishment of molecular clones of human and bovine RV clinical isolates. Fecal samples from patients with RV typically harbor quasi-species or, occasionally, multiple genotypes of RV. In the present study, we performed the genetic sequencing of clinical viral strains during the early propagation stages in cultured cells. Subsequently, infectious viruses were synthesized, allowing the characterization of circulating viruses in nature. This approach provides valuable insights into the genetic diversity and dynamics of RV populations and contributes to a more comprehensive understanding of viral pathogenesis and evolution.

Development of an optimized SEC method for characterization of genome DNA leakage from adeno-associated virus products.

Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes due to their superior safety, relatively low immunogenicity, and ability to target diverse tissues. AAV gene therapy products are typically formulated as frozen liquid and stored below - 60 °C, and therefore are subjected to multiple freeze/thaw cycles during manufacturing and administration. Recent studies have shown that genome DNA leakage could be induced by freeze/thaw stress. DNA leakage from AAV capsids has been reported to potentially impact product stability, induce immune responses, and compromise product efficacy. Thus, further characterization to improve the understanding of genome DNA leakage is necessary for mitigating the risks associated with genome DNA leakage during AAV product development. In this work, we developed an optimized size-exclusion chromatography (SEC) method for quantifying the leakage of genome DNA across multiple different AAV serotypes and demonstrated satisfactory assay performance in sensitivity, precision, and linearity. Furthermore, we showed that this method could also be applied to quantifying additional quality attributes of AAV, including the percentage of full capsids and quantification of AAV dimers. By using this optimized SEC method, we demonstrated that significantly increased free DNA was observed with increasing freeze/thaw cycles or at a temperature approaching the onset temperature for genome DNA ejection, which was effectively mitigated by the addition of 1.5% w/v sucrose in the AAV formulation. Thus, this optimized SEC method can serve as an invaluable tool for AAV formulation, product, and process development in ensuring the quality and stability of AAV gene therapy products.

A Nanobody-based TRIM-away targets the intracellular protein degradation of African swine fever virus

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a hemorrhagic illness with high fatality rates in domestic pigs that has resulted in a substantial socio-economic loss and threatens the global pork industry. Very few safe and efficient vaccines or compounds against ASF are commercially available, thus developing new antiviral strategies is urgently required. Targeted protein degradation (TPD) has emerged as one of the most innovative strategies for drug discovery. In this study, we generate Nanobody-based TRIM-aways specifically binding with and targeting ASFV-encoded structural proteins p30, p54, and p72 for degradation. Furthermore, nanobody-based trim-aways exhibit robust viral structural protein degradation capabilities in ASFV-infected iPAM and MA104 cells through both proteasomal and lysosomal pathways, concurrently demonstrating potent anti-ASFV activity with less viral production. Our study highlights the Nanobody-based TRIM-away targeting viral protein degradation as a potential candidate for the development of a novel antiviral strategy against ASF.

Molecular Mechanisms of Kaposi Sarcoma-Associated Herpesvirus (HHV8)-Related Lymphomagenesis.

Approximately 15-20% of cancers are caused by viruses. Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is an oncogenic virus that is the etiologic agent of not only Kaposi sarcoma but also the lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). KSHV can infect a broad tropism of cells, including B lymphocytes, wherein KSHV encodes specific viral proteins that can transform the cell. KSHV infection precedes the progression of PEL and MCD. KSHV establishes lifelong infection and has two phases of its lifecycle: latent and lytic. During the latent phase, viral genomes are maintained episomally with limited gene expression. Upon sporadic reactivation, the virus enters its replicative lytic phase to produce infectious virions. KSHV relies on its viral products to modulate host factors to evade immune detection or to co-opt their function for KSHV persistence. These manipulations dysregulate normal cell pathways to ensure cell survival and inhibit antiviral immune responses, which in turn, contribute to KSHV-associated malignancies. Here, we highlight the known molecular mechanisms of KSHV that promote lymphomagenesis and how these findings identify potential therapeutic targets for KSHV-associated lymphomas.

Modulators of the Hop-HSP90 Protein-Protein Interaction Disrupt KSHV Lytic Replication.

The central role of the chaperome in maintaining cellular proteostasis has seen numerous viral families evolve to parasitically exploit host chaperones in their life cycle. The HSP90 chaperone protein and its cochaperone Hop have both individually been shown to be essential factors for Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication. Given the fundamental regulatory role that protein-protein interactions (PPIs) play in cellular biology, we reasoned that disrupting the Hop-HSP90 PPI may provide a new host-based target for inhibiting KSHV lytic replication. This study expands upon a previous report of non-natural peptides, which were found to disrupt the association between the HopTPR2A domain and its interacting HSP90CTD. Here, in addition to providing insight into the structure-activity relationships of PPI inhibition, we show disruption of the full-length Hop-HSP90 PPI. The inhibitory peptides selectively engaged the HopTPR2A domain in cell lysates and when tethered to a cell-penetrating peptide acted as noncytotoxic inhibitors of KSHV lytic replication by lowering the viral load, preventing the production of infectious virions, and reducing the expression of KSHV lytic genes. In addition to tentative evidence of Hop-HSP90 PPI as a much-needed target for KSHV drug discovery, this study represents an important step in understanding viral interactions with the host proteostasis machinery.

The Low-Density Lipoprotein Receptor-Related Protein-1 Is Essential for Dengue Virus Infection

Dengue virus (DENV) causes the most prevalent and rapidly spreading arboviral disease of humans. It enters human cells by receptor-mediated endocytosis. Numerous cell-surface proteins were proposed as DENV entry factors. Among these, the phosphatidylserine receptor TIM-1 is the only one known to mediate virus internalization. However, several cellular models lacking TIM-1 are permissive to DENV infection, suggesting that other receptors exist. Here, we show that the low-density lipoprotein receptor-related protein-1 (LRP1) binds DENV virions by interacting with the DIII of the viral envelope glycoprotein. DENV infection is effectively inhibited by the purified receptor at 5 × 10−8 mol/L, and the interaction of the envelope protein with LRP1 is also blocked by a natural ligand of LRP1. The depletion of LRP1 causes 100-fold lower production of infectious virus than controls. Our results indicate that LRP1 is another DENV receptor, thus becoming an attractive target to evaluate for the development of effective antiviral drugs against DENV.

HIV-1 latency reversal agent boosting is not limited by opioid use.

Opioid use may impact the HIV-1 reservoir and its reversal from latency. We studied forty-seven virally suppressed people with HIV (PWH) and observed that lower concentration of HIV-1 latency reversal agents (LRA), used in combination with small molecules that did not reverse latency, synergistically increased the magnitude of HIV-1 re-activation ex vivo, regardless of opioid use. This LRA boosting, which combined a Smac mimetic or low-dose protein kinase C agonist with histone deacetylase inhibitors, generated significantly more unspliced HIV-1 transcription than phorbol 12-myristate 13-acetate (PMA) with ionomycin (PMAi), the maximal known HIV-1 reactivator. LRA boosting associated with greater histone acetylation, modulated surface activation-induced markers, and altered T cell production of TNFα, IL-2, and IFNγ. HIV-1 reservoirs in PWH contained unspliced and polyadenylated (polyA) virus mRNA, the ratios of which were greater in resting than total CD4+ T cells and correct to 1:1 with PMAi exposure. We characterized treated suppressed HIV-1 infection as a period of inefficient, not absent, virus transcription. Multiply spliced HIV-1 transcripts and virion production did not consistently increase with LRA boosting, suggesting the presence of a persistent post-transcriptional block. LRA boosting can be leveraged to probe mechanisms of an effective cellular HIV-1 latency reversal program.

Microbial distribution in Mudbank regions off Alappuzha, South-West coast of India.

The coastal waters of Kerala, in the South Eastern Arabian Sea (SEAS), are unique during the Southwest monsoon season due to the concurrent occurrence of two physical processes, the upwelling and Mudbanks. However, little is known about the viral ecology and activity in a system where upwelling and Mudbanks coexist, though it is generally recognized that microbial assemblages play a vital role in the food web dynamics of marine systems, particularly in upwelling. Water samples were taken from monsoon and pre-monsoon period from three locations, (M1, M2, and M3) off Alappuzha, on the southwest coast of India to examine the viral activity and distribution. The dissolved oxygen levels showed the incursion of hypoxic waters in all the stations during the peak upwelling period. Upwelling signals were prominent in all the stations, but Mudbank and upwelling co-occurred at M2 alone during monsoon. The abundance of viruses ranged from 0.86 to 15.68 × 106 Viral-like Particles (VLPs mL-1) and prokaryotic abundance ranged from 2.73-16.26 × 105 cells mL-1. Viral and prokaryotic abundance was significantly higher in the monsoon compared to pre and late-monsoon. Based on Transmission electron microscopy (TEM) results, the non-tailed viruses constituted the major (43%) proportion of the total viruses in this study region. However, the viral production rates and viral-mediated bacterial mortality were high in the pre-monsoon compared to the monsoon and late-monsoon periods. There was no obvious effect of Mudbanks on viral dynamics and the observed variations in virological and hydrological features were governed mainly by coastal upwelling.

Identifying the amino acid domains of ORF59 responsible for interactions with ORF57 and PAN RNA during KSHV lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor, ORF59, is a lytic protein essential for viral DNA synthesis as part of the core replication complex. The multifunctional nature of ORF59 has prompted the investigation into its various functional domains. Prior studies of ORF59 have identified dimerization, DNA interaction, and polymerase interaction domains. The regions of ORF59 responsible for the interaction with the viral mRNA transport accumulation protein (MTA/ORF57) and the viral long non-coding polyadenylated nuclear (PAN) RNA have not been explored. Using a series of previously characterized ORF59 deletion KSHV BACmid mutants, we identified the domains of ORF59 that interact with ORF57 and PAN RNA. Interestingly, amino acids 51-100 were essential for interacting with both ORF57 and PAN RNA. Using this information, we generated a plasmid that expresses a DsRed-tagged polypeptide spanning amino acids 30-100 of ORF59. When the 30-100 aa DsRed-tagged polypeptide expression plasmid was transfected into KSHV wild-type iSLK cells prior to lytic reactivation, a dominant-negative inhibition of virus replication was observed, resulting in a decrease of infectious virus production. Our data suggest that interactions between ORF59 with ORF57 and PAN RNA are important to successful lytic replication.IMPORTANCETo better understand the Kaposi's sarcoma-associated herpesvirus (KSHV) DNA polymerase processivity factor ORF59, we investigated the interaction of ORF59 with ORF57 and polyadenylated nuclear (PAN) RNA. We used a previously characterized KSHV BACmid containing internal deletions of ORF59 to identify the domains of ORF59 that interact with ORF57 and PAN RNA. Our study revealed multiple domains of ORF59 that are essential for its association with PAN RNA. These domains span amino acids 51-100, 251-300, and 351-396. Additional experiments confirmed amino acids 51-100 are critical for the interaction between ORF59 and ORF57. Using this information, we generated an expression plasmid encompassing the ORF57 and PAN RNA interaction domains of ORF59. The ORF59 polypeptide expression plasmid of amino acids 30-100 functioned as a dominant negative inhibitor during viral reactivation and caused a decrease in virus production. These findings provide valuable insights into the key domains of ORF59, essential for its functionality, and ultimately the production of infectious viruses.

Regulation of expression of unintegrated and integrated HIV-1 DNA: keeping the wolves at bay

The unintegrated HIV-1 DNAs formed by reverse transcription in the early hours after infection are subject to profound transcriptional silencing. The repression of expression of foreign DNA, as an aspect of the innate immune system, serves to restrict the activity of many invading pathogens. Newly formed retroviral DNAs are rapidly loaded with histones upon entry into the nucleus, and the repression of their expression is mediated by an array of host proteins that introduce histone modifications characteristic of heterochromatin, including histone methylation and histone deacetylation. Knockout or knockdown of expression or inhibition of these host factors can relieve the silencing, allowing for viral gene expression even in settings where HIV-1 DNA integration is blocked. When viral DNA integration is allowed, forming the integrated provirus, the silencing in most cases is dramatically relieved, leading to high levels of expression and formation of progeny virus. In some settings and cell types, silencing of the integrated DNA is maintained, or re-established, such that the infected cells retain a silent copy of the viral DNA without production of progeny virus. The basis for the typical switch from silent DNA to actively expressed DNA upon integration is not yet fully clear. This review will summarize the current understanding of the regulation of expression of unintegrated HIV-1 DNAs and the nature of the chromatin that is formed on the viral DNA, and will especially focus on the host machinery that establishes repressive heterochromatin-like structures on the unintegrated DNA. The activation of expression that normally occurs upon integration, and the special circumstances when viral DNA expression is not activated, will also be discussed. These cases can result in the formation of populations of infected cells carrying silent proviruses, which persist for decades in infected individuals in spite of antiviral therapy. This pool of latently infected cells can be stochastically reactivated to give rise to spreading virus whenever antiviral drugs are withdrawn, and constitute the barrier to a true “cure” of AIDS. The hope is that a deeper understanding of the regulation of expression of viral DNAs will lead to new means to prevent or control viremia and disease.

Lytic promoter activity during herpes simplex virus latency is dependent on genome location.

Herpes simplex virus 1 (HSV-1) is a significant pathogen that establishes lifelong latent infections with intermittent episodes of resumed disease. In mouse models of HSV infection, sporadic low-level lytic gene expression has been detected during latency in the absence of reactivation events that lead to production of new viruses. This viral activity during latency has been reported using a sensitive Cre-marking model for several lytic gene promoters placed in one location in the HSV-1 genome. Here, we extend these findings in the same model by examining first, the activity of an ectopic lytic gene promoter in several places in the genome and second, whether any promoters might be active in their natural context. We found that Cre expression was detected during latency from ectopic and native promoters, but only in locations near the ends of the unique long genome segment. This location is significant because it is in close proximity to the region from which latency-associated transcripts (LATs) are derived. These results show that native HSV-1 lytic gene promoters can produce protein products during latency, but that this activity is only detectable when they are located close to the LAT locus.IMPORTANCEHSV is a significant human pathogen and the best studied model of mammalian virus latency. Traditionally, the active (lytic) and inactive (latent) phases of infection were considered to be distinct, but the notion of latency being entirely quiescent is evolving due to the detection of some lytic gene expression during latency. Here, we add to this literature by finding that the activity can be found for native lytic gene promoters as well as for constructs placed ectopically in the HSV genome. However, this activity was only detectable when these promoters were located close by a region known to be transcriptionally active during latency. These data have implications for our understanding of HSV gene regulation during latency and the extent to which transcriptionally active regions are insulated from adjacent parts of the viral genome.