Late Promoter Research Articles

Differential induction of Shiga toxin in environmental Escherichia coli O145:H28 strains carrying the same genotype as the outbreak strains

Shiga toxin-producing Escherichia coli (STEC) O145 is a major serotype associated with severe human disease. Production of Shiga toxins (Stxs), especially Stx2a, is thought to be correlated with STEC virulence. Since stx genes are located in prophages genomes, induction of prophages is required for effective Stxs production. Here, we investigated the production of Stxs in 12 environmental STEC O145:H28 strains under stresses STEC encounter in natural habitats and performed comparative analysis with two O145:H28 clinical strains, one linked to a 2010 U.S. lettuce-associated outbreak (RM13514) and the other linked to a 2007 Belgium ice cream-associated outbreak (RM13516). Similar to the outbreak strains, all environmental strains belong to Sequence Type (ST)-78 using the EcMLST typing scheme. Although all Stx1a-prophages were grouped together, variations in Stx1a production were observed prior to or following the inductions. Among all stx2a positive environmental strains, only the Stx2a-prophage in cattle isolate RM9154-C1 was clustered with the Stx2a-prophages in RM13514, the Stx2a-phage induced from a STEC O104:H4 strain linked to the 2011 outbreak of enterohemorrhagic infection in Germany, and the Stx2a-prophage in STEC O157:H7 strain EDL933, a prototype of enterohemorrhagic E. coli. Furthermore, the Stx2a-prophage in RM9154-C1 shared the same chromosomal insertion site and carried the same antiterminator Q gene and the late promoter PR' as the Stx2a-prophage in RM13514. Following mitomycin C or enrofloxacin treatment, the production of Stx2a in RM9154-C1 was the highest among all environmental strains tested. In contrast, following acid challenge and recovery, the production of Stx2a in RM9154-C1 was the lowest among all the environmental strains tested, at a level comparable to the clinical strains. A significant increase in Stx2a production was detected in all strains when exposed to H2O2, although the induction fold was much lower than those by other inducers. This low-efficiency induction of Stx-prophages by H2O2, a natural inducer of Stx-prophages, supports the hypothesis of bacterial altruism in controlling Stxs production, a strategy that assures the survival of the STEC population as a whole by sacrificing a small fraction of cells for Stxs production and release. Differential induction of Stxs among strains carrying nearly identical Stx-prophages suggests a role of host bacteria in regulating Stxs production. Our study revealed diverse Stx-prophages in STEC O145:H28 strains that were genotypically indistinguishable. Identification of a cattle isolate harboring a Stx2a-prophage associated with high virulence supports the premise that cattle, a natural reservoir of STEC, serve as a source of hypervirulent STEC strains.

Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3’). We found that the PUF60–AdML3’ dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60’s two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3’ revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3’) stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3’ splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3’ splice sites.

Transcriptional Landscape of Staphylococcus aureus Kayvirus Bacteriophage vB_SauM-515A1.

The Twort-like myoviruses (Kayvirus genus) of S. aureus are promising agents for bacteriophage therapy due to a broad host range and high killing activity against clinical isolates. This work improves the current understanding of the phage infection physiology by transcriptome analysis. The expression profiles of a typical member of the Kayvirus genus (vB_SauM-515A1) were obtained at three time-points post-infection using RNA sequencing. A total of 35 transcription units comprising 238 ORFs were established. The sequences for 58 early and 12 late promoters were identified in the phage genome. The early promoters represent the strong sigma-70 promoters consensus sequence and control the host-dependent expression of 26 transcription units (81% of genes). The late promoters exclusively controlled the expression of four transcription units, while the transcription of the other five units was directed by both types of promoters. The characteristic features of late promoters were long -10 box of TGTTATATTA consensus sequence and the absence of -35 boxes. The data obtained are also of general interest, demonstrating a strategy of the phage genome expression with a broad overlap of the early and late transcription phases without any middle transcription, which is unusual for the large phage genomes (>100 kbp).

Early termination of the Shiga toxin transcript generates a regulatory small RNA

Enterohemorrhagic Escherichia coli is a significant human pathogen that causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome. The latter can lead to potentially fatal renal failure and is caused by the release of Shiga toxins that are encoded within lambdoid bacteriophages. The toxins are encoded within the late transcript of the phage and are regulated by antitermination of the PR' late promoter during lytic induction of the phage. During lysogeny, the late transcript is prematurely terminated at tR' immediately downstream of PR', generating a short RNA that is a byproduct of antitermination regulation. We demonstrate that this short transcript binds the small RNA chaperone Hfq, and is processed into a stable 74-nt regulatory small RNA that we have termed StxS. StxS represses expression of Shiga toxin 1 under lysogenic conditions through direct interactions with the stx1AB transcript. StxS acts in trans to activate expression of the general stress response sigma factor, RpoS, through direct interactions with an activating seed sequence within the 5' UTR. Activation of RpoS promotes high cell density growth under nutrient-limiting conditions. Many phages utilize antitermination to regulate the lytic/lysogenic switch and our results demonstrate that short RNAs generated as a byproduct of this regulation can acquire regulatory small RNA functions that modulate host fitness.

Alphaherpesvirus Latency and Reactivation with a Focus on Herpes Simplex Virus.

We are at an interesting time in the understanding of alpha herpesvirus latency and reactivation and their implications to human disease. Conceptual advances have come from both animal and neuronal culture models. This review focuses on the concept that the tegument protein and viral transactivator VP16 plays a major role in the transition from latency to the lytic cycle. During acute infection, regulation of VP16 transactivation balances spread in the nervous system, establishment of latent infections and virulence. Reactivation is dependent on this transactivator to drive entry into the lytic cycle. In vivo de novo expression of VP16 protein is mediated by sequences conferring pre-immediate early transcription embedded in the normally leaky late promoter. In vitro, alternate mechanisms regulating VP16 expression in the context of latency have come from the SCG neuron culture model and include the concepts that (i) generalized transcriptional derepression of the viral genome and sequestration of VP16 in the cytoplasm for ~48 hours (Phase I) precedes and is required for VP16-dependent reactivation (Phase II); and (ii) a histone methyl/phospho switch during Phase I is required for Phase II reactivation. The challenge to the field is reconciling these data into a unified model of virus reactivation. The task of compiling this review was uncomfortably humbling, as if cataloging the stars in the universe. While not completely dark, our night sky is missing a multitude of studies which are among the many points of light contributing to our field. This article is a focused review in which we discuss from the vantage point of our expertise, just a handful of concepts that have or are emerging. A lookback at some of the pioneering work that grounds our field is also included.

Degradation of SAMHD1 Restriction Factor Through Cullin-Ring E3 Ligase Complexes During Human Cytomegalovirus Infection.

Sterile alpha motif (SAM) and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) acts as a restriction factor for several RNA and DNA viruses by limiting the intracellular pool of deoxynucleoside triphosphates. Here, we investigated the regulation of SAMHD1 expression during human cytomegalovirus (HCMV) infection. SAMHD1 knockdown using shRNA increased the activity of the viral UL99 late gene promoter in human fibroblasts by 7- to 9-fold, confirming its anti-HCMV activity. We also found that the level of SAMHD1 was initially increased by HCMV infection but decreased partly at the protein level at late stages of infection. SAMHD1 loss was not observed with UV-inactivated virus and required viral DNA replication. This reduction of SAMHD1 was effectively blocked by MLN4924, an inhibitor of the Cullin-RING-E3 ligase (CRL) complexes, but not by bafilomycin A1, an inhibitor of vacuolar-type H+-ATPase. Indirect immunofluorescence assays further supported the CRL-mediated SAMHD1 loss at late stages of virus infection. Knockdown of CUL2 and to a lesser extent CUL1 using siRNA stabilized SAMHD1 in normal fibroblasts and inhibited SAMHD1 loss during virus infection. Altogether, our results demonstrate that SAMHD1 inhibits the growth of HCMV, but HCMV causes degradation of SAMHD1 at late stages of viral infection through the CRL complexes.

Rescue of a Vaccinia Virus Mutant Lacking IFN Resistance Genes K1L and C7L by the Parapoxvirus Orf Virus.

Type 1 interferons induce the upregulation of hundreds of interferon-stimulated genes (ISGs) that combat viral replication. The parapoxvirus orf virus (ORFV) induces acute pustular skin lesions in sheep and goats and can reinfect its host, however, little is known of its ability to resist IFN. Vaccinia virus (VACV) encodes a number of factors that modulate the IFN response including the host-range genes C7L and K1L. A recombinant VACV-Western Reserve (WR) strain in which the K1L and C7L genes have been deleted does not replicate in cells treated with IFN-β nor in HeLa cells in which the IFN response is constitutive and is inhibited at the level of intermediate gene expression. Furthermore C7L is conserved in almost all poxviruses. We provide evidence that shows that although ORFV is more sensitive to IFN-β compared with VACV, and lacks homologues of KIL and C7L, it nevertheless has the ability to rescue a VACV KIL- C7L- gfp+ mutant in which gfp is expressed from a late promoter. Co-infection of HeLa cells with the mutant and ORFV demonstrated that ORFV was able to overcome the block in translation of intermediate transcripts in the mutant virus, allowing it to progress to late gene expression and new viral particles. Our findings strongly suggest that ORFV encodes a factor(s) that, although different in structure to C7L or KIL, targets an anti-viral cellular mechanism that is a highly potent at killing poxviruses.

Arming Oncolytic Adenoviruses: Effect of Insertion Site and Splice Acceptor on Transgene Expression and Viral Fitness.

Oncolytic adenoviruses (OAds) present limited efficacy in clinics. The insertion of therapeutic transgenes into OAds genomes, known as “arming OAds”, has been the main strategy to improve their therapeutic potential. Different approaches were published in the decade of the 2000s, but with few comparisons. Most armed OAds have complete or partial E3 deletions, leading to a shorter half-life in vivo. We generated E3+ OAds using two insertion sites, After-fiber and After-E4, and two different splice acceptors linked to the major late promoter, either the Ad5 protein IIIa acceptor (IIIaSA) or the Ad40 long fiber acceptor (40SA). The highest transgene levels were obtained with the After-fiber location and 40SA. However, the set of codons of the transgene affected viral fitness, highlighting the relevance of transgene codon usage when arming OAds using the major late promoter.

Epstein–Barr virus co-opts TFIIH component XPB to specifically activate essential viral lytic promoters

Epstein-Barr virus (EBV) is associated with epithelial and lymphoid malignancies, establishes latent infection in memory B cells, and intermittently produces infectious virions through lytic replication. Released virions play a key role in latent reservoir maintenance and transmission. Lytic EBV transcription differs from cellular transcription in requiring a virus-encoded preinitiation complex that binds to TATT motifs unique to EBV late lytic promoters. Expression of 15 late lytic genes that are important for virion production and infectivity is particularly dependent on the EBV SM protein, a nuclear protein expressed early during lytic reactivation that binds to viral RNAs and enhances RNA stability. We recently discovered that spironolactone blocks EBV virion production by inhibiting EBV SM function. Since spironolactone causes degradation of xeroderma pigmentosum group B-complementing protein (XPB), a component of human transcription factor TFIIH, in both B lymphocytes and epithelial cells, we hypothesized that SM utilizes XPB to specifically activate transcription of SM target promoters. While EBV SM has been thought to act posttranscriptionally, we provide evidence that SM also facilitates EBV gene transcription. We demonstrate that SM binds and recruits XPB to EBV promoters during lytic replication. Depletion of XPB protein, by spironolactone treatment or by siRNA transfection, inhibits SM-dependent late lytic gene transcription but not transcription of other EBV genes or cellular genes. These data indicate that SM acts as a transcriptional activator that has co-opted XPB to specifically target 15 EBV promoters that have uniquely evolved to require XPB for activity, providing an additional mechanism to differentially regulate EBV gene expression.

Efficient production of HPV16 E2 protein from HPV16 late mRNAs spliced from SD880 to SA2709

Human papillomaviruses (HPVs) produce a large number of alternatively spliced mRNAs, including a number of differently spliced mRNAs with the potential to produce E2 protein. To identify the alternatively spliced HPV16 mRNA with the highest ability to produce E2 protein, we have generated E2 cDNA expression plasmids representing the most common, alternatively spliced E2 mRNAs, and assessed their translational potential. Our results revealed that an mRNA initiated at the HPV16 late promoter p670 and spliced from the HPV16 5′-splice site SD880 to the HPV16 3′-splice site SA2709, located immediately upstream of the E2 ATG, produced higher levels of E2 than any of the other alternatively spliced, E2-encoding mRNAs. Utilization of a known, alternative 3′-splice site located upstream of the E2 ATG named SA2582, generated mRNAs with lower ability to produce E2 than mRNAs spliced to SA2709. Finally, analysis of HPV16 mRNA splicing demonstrated that SA2709 was more efficiently spliced to the upstream 5′-splice site SD880 than to the upstream 5′-splice site SD226. In conclusion, the HPV16 mRNA with the greatest ability to produce E2 protein is generated from the HPV16 late promoter and is spliced between HPV16 5′-splice site SD880 and HPV16 3′-splice site SA2709.

Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling.

Short-chain fatty acids (SCFAs) are fermented dietary components that regulate immune responses, promote colonic health, and suppress mast cell-mediated diseases. However, the effects of SCFAs on human mast cell function, including the underlying mechanisms, remain unclear. Here, we investigated the effects of the SCFAs (acetate, propionate, and butyrate) on mast cell-mediated pathology and human mast cell activation, including the molecular mechanisms involved. Precision-cut lung slices (PCLS) of allergen-exposed guinea pigs were used to assess the effects of butyrate on allergic airway contraction. Human and mouse mast cells were co-cultured with SCFAs and assessed for degranulation after IgE- or non-IgE-mediated stimulation. The underlying mechanisms involved were investigated using knockout mice, small molecule inhibitors/agonists, and genomics assays. Butyrate treatment inhibited allergen-induced histamine release and airway contraction in guinea pig PCLS. Propionate and butyrate, but not acetate, inhibited IgE- and non-IgE-mediated human or mouse mast cell degranulation in a concentration-dependent manner. Notably, these effects were independent of the stimulation of SCFA receptors GPR41, GPR43, or PPAR, but instead were associated with inhibition of histone deacetylases. Transcriptome analyses revealed butyrate-induced downregulation of the tyrosine kinases BTK, SYK, and LAT, critical transducers of FcεRI-mediated signals that are essential for mast cell activation. Epigenome analyses indicated that butyrate redistributed global histone acetylation in human mast cells, including significantly decreased acetylation at the BTK, SYK, and LAT promoter regions. Known health benefits of SCFAs in allergic disease can, at least in part, be explained by epigenetic suppression of human mast cell activation.

Promoter activity of Merkel cell Polyomavirus variants in human dermal fibroblasts and a Merkel cell carcinoma cell line

BackgroundMerkel cell polyomavirus (MCPyV) is a human polyomavirus that establishes a life-long harmless infection in most individuals, with dermal fibroblasts believed to be the natural host cell. However, this virus is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. Several MCPyV variants with polymorphism in their promoter region have been isolated, but it is not known whether these differences affect the biological properties of the virus.MethodsUsing transient transfection studies in human dermal fibroblasts and the MCC cell line MCC13, we compared the transcription activity of the early and late promoters of the most commonly described non-coding control region MCPyV variant and six other isolates containing specific mutation patterns.ResultsBoth the early and late promoters were significantly stronger in human dermal fibroblasts compared with MCC13 cells, and a different promoter strength between the MCPyV variants was observed. The expression of full-length large T-antigen, a viral protein that regulates early and late promoter activity, inhibited early and late promoter activities in both cell lines. Nonetheless, a truncated large T-antigen, which is expressed in virus-positive MCCs, stimulated the activity of its cognate promoter.ConclusionThe promoter activities of all MCPyV variants tested was stronger in human dermal fibroblasts, a cell line that supports viral replication, than in MCC13 cells, which are not permissive for MCPyV. Truncated large T-antigen, but not full-length large T-antigen stimulated viral promoter activity. Whether, the difference in promoter strength and regulation by large T-antigen may affect the replication and tumorigenic properties of the virus remains to be determined.

Selective repression of the Drosophila cyclin B promoter by retinoblastoma and E2F proteins

The Cyclin B1 gene encodes a G2/M cyclin that is deregulated in various human cancers, however, the transcriptional regulation of this gene is incompletely understood. The E2F and retinoblastoma family of proteins are involved in this gene's regulation, but there is disagreement on which of the E2F and retinoblastoma proteins interact with the promoter to regulate this gene. Here, we dissect the promoter region of the Drosophila CycB gene, and study the role of Rbf and E2F factors in its regulation. This gene exhibits remarkable features that distinguish it from G1/S regulated promoters, such as PCNA. The promoter is comprised of modular elements with dedicated repressor and activator functions, including a segment spanning the first intron that interferes with a 5′ activator element. A highly active minimal promoter (−464, +100) is repressed by the Rbf1 retinoblastoma protein, but much more potently repressed by the Rbf2 protein, which has been linked in other studies to control of cell growth genes. Unlike many other cell-cycle genes, which are activated by E2F1 and repressed by E2F2, CycB is potently activated by E2F2, and repressed by E2F1. Although the bulk of Rbf binding is associated with a region 5′ of the core promoter, E2F and retinoblastoma proteins functionally interact with the basal promoter region, in part through a conserved E2F site at −80 bp. The specific regulatory requirements of this late cell cycle promoter appear to be linked to the unique activities of E2F and retinoblastoma family members acting on a complex cis-regulatory circuit.

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.

Herpes Simplex Virus 1-Specific CD8+ T Cell Priming and Latent Ganglionic Retention Are Shaped by Viral Epitope Promoter Kinetics.

Reactivation of herpes simplex virus 1 (HSV-1) from neurons in sensory ganglia such as the trigeminal ganglia (TG) is influenced by virus-specific CD8+ T cells that infiltrate the ganglia at the onset of latency and contract to a stable activated tissue-resident memory population. In C57BL/6 mice, half of HSV-specific CD8+ T cells (gB-CD8s) recognize one dominant epitope (residues 498 to 505) on glycoprotein B (gB498-505), while the remainder (non-gB-CD8s) recognize 19 subdominant epitopes from 12 viral proteins. To address how expression by HSV-1 influences the formation and ganglionic retention of CD8+ T cell populations, we developed recombinant HSV-1 with the native immunodominant gB epitope disrupted but then expressed ectopically from different viral promoters. In mice, the epitope expressed from the gB promoter restored full gB-CD8 immunodominance to 50%. Intriguingly, earlier expression from constitutive, immediate-early, and early promoters did not significantly increase immunodominance, indicating that these promoters cannot elicit more than half of the CD8 compartment. Epitope expressed from candidate viral promoters of "true late" HSV-1 genes either delayed or reduced the priming efficiency of gB-CD8s and their levels in the TG at early times. HSV expressing the epitope from the full latency-associated transcript promoter did not efficiently prime gB-CD8s; however, gB-CD8s primed by a concurrent wild-type flank infection infiltrated the TG and were retained long term, suggesting that latent epitope expression is sufficient to retain gB-CD8s. Taken together, the data indicate that viral promoters shape latent HSV-1-specific CD8+ T cell populations and should be an important consideration in future vaccine design.IMPORTANCE Latency of HSV-1 in host neurons enables long-term persistence from which reactivation may occur to cause recurrent diseases, such as blinding herpetic stromal keratitis. Latency is not antigenically silent, and viral proteins are sporadically expressed at low levels without full virion production. This protein expression is recognized by ganglion-resident HSV-1-specific CD8+ T cells that maintain a protective resident population. Since these T cells can influence lytic/latent decisions in reactivating neurons, we argue that improving their ganglionic retention and function may offer a strategy in vaccine design to reduce reactivation and recurrent disease. To understand factors driving the infiltration and retention of ganglionic CD8s, we examined several HSV recombinants that have different viral promoters driving expression of the immunodominant gB epitope. We show that the selection of epitope promoter influences CD8+ T cell population hierarchies and their function.

Conserved CxnC Motifs in Kaposi's Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34.

Late gene transcription in the beta- and gammaherpesviruses depends on a set of virally encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct it to a subset of viral genes that are required for completion of the viral replication cycle and capsid assembly. In Kaposi's sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. Assembly of the vTAs into a complex is critical for late gene transcription, and thus, deciphering the architecture of the complex is central to understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is incorporated into the vTA complex primarily through its interaction with ORF34, which is dependent upon a set of four conserved cysteine-rich motifs in the C-terminal domain of ORF66. While both ORF24 and ORF66 occupy the canonical K8.1 late gene promoter, their promoter occupancy requires the presence of the other vTAs, suggesting that sequence-specific, stable binding requires assembly of the entire complex on the promoter. Additionally, we found that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV viral preinitiation complex and suggests that it functions as a complex to recognize late gene promoters.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that is the causative agent of multiple human cancers. The release of infectious virions requires the production of capsid proteins and other late genes, whose production is transcriptionally controlled by a complex of six virally encoded proteins that hijack the host transcription machinery. It is poorly understood how this complex assembles or what function five of its six components play in transcription. Here, we demonstrate that ORF66 is an essential component of this complex in KSHV and that its inclusion in the complex depends upon its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examined the assembly of the viral preinitiation complex at late gene promoters and found that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral preinitiation complex.

Analysis of MHC Class I Processing Pathways That Generate a Response to Vaccinia Virus Late Proteins.

Use of recombinant viral vectors encoding nonnative Ags is an attractive mechanism for the generation of protective Ab, CD4+ T cell (TCD4+), and CD8+ T cell (TCD8+) responses in vivo following immunization. However, the life cycle and tropism of the viral vector, and its interactions with various components of the immune system, must be fully understood to maximize the efficacy of any vaccination strategies. Ab and TCD4+ responses typically target native Ags driven by late promoters in vaccinia virus (VACV)-based vectors. However, it has been demonstrated that model Ags driven by late promoters in recombinant VACV vectors do not stimulate TCD8+ responses, whereas identical Ags driven by early promoters stimulate strong responses. Conversely, TCD8+ can be generated against some natural late VACV Ags. We explored this dichotomy by investigating the Ag presentation pathways responsible for presentation of natural late VACV Ags in mice. We found that all of the late VACV Ags we examined could be cross-primed (i.e., presented by uninfected professional APC), as well as directly presented by infected dendritic cell populations. However, one Ag was only presented by professional APC populations and was not the target of a protective TCD8+ response. Therefore, there is no generalized blockade in Ag presentation of late VACV Ags, and expression of nonnative Ags driven by a late promoter allows production of large quantities of Ag that may allow simultaneous targeting of both TCD4+ and Ab responses, as well as TCD8+ responses, in the future.

Deletion of multiple genes induces virulence reduction of vaccinia virus Tiantan strain

The purpose of this study was to knock out two non-essential gene fragments (TC7L-TK2L and TJ2R) related to virulence, immunomodulation, and host range in the vaccinia virus Tian Tan strain (VTT), and combining with double-label screening and exogenous screening marker knockout techniques to construct attenuated strains with multiple gene deletions(rVTT-TC-TJ). The shuttle plasmids pSK-TC and pSK-TJ were constructed by designing 2 pairs of recombinant arms, combined with poxvirus early and late complex strong promoter pE/L and exogenous screening marker enhanced green fluorescent protein(EGFP). The results showed that knocking out the two gene fragments does not affect the replication ability of the virus and displays a good genetic stability. Furthermore, a series of in vivo and in vitro experiments demonstrate that although virulence of rVTT-TC-TJ is attenuated significantly, high immunogenicity was maintained. These results support the potential development of rVTT-TC-TJ as a safe viral vector or vaccine.

Major capsid protein of Autographa californica multiple nucleopolyhedrovirus contributes to the promoter activity of the very late viral genes

The baculovirus expression vector system (BEVS) is one of the most powerful eukaryotic expression systems. Recombinant protein expression is usually controlled by promoters of the baculovirus very late genes (i.e., polyhedrin and p10); therefore, identifying novel regulatory factors for these promoters is key to increasing BEVS productivity. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the viral vector most frequently used in BEVS. VP39 is the major nucleocapsid protein of AcMNPV and plays a pivotal role in nucleocapsid assembly in the nucleus. In this study, we found that knocking out vp39 from the AcMNPV genome resulted in decreased protein abundance of polyhedrin and P10. Further assays revealed that the mRNA transcripts and the promoter activities of polyhedrin and p10 were decreased in the absence of vp39, suggesting that VP39 contributes to the activity of the very late viral gene promoters and may represent a means of optimizing the current BEVS.

Development of a novel screening platform for the identification of small molecule inhibitors of human adenovirus

Human adenovirus (HAdV) can cause severe disease and death in both immunocompromised and immunocompetent patients. The current standards of treatment are often ineffective, and no approved antiviral therapy against HAdV exists. We report here the design and validation of a fluorescence-based high-content screening platform for the identification of novel anti-HAdV compounds. The screen was conducted using a wildtype-like virus containing the red fluorescent protein (RFP) gene under the regulation of the HAdV major late promoter. Thus, RFP expression allows monitoring of viral late gene expression (a surrogate marker for virus replication), and compounds affecting virus growth can be easily discovered by quantifying RFP intensity. We used our platform to screen ~1200 FDA-approved small molecules, and identified several cardiotonic steroids, corticosteroids and chemotherapeutic agents as anti-HAdV compounds. Our screening platform provides the stringency necessary to detect compounds with varying degrees of antiviral activity, and facilitates drug discovery/repurposing to combat HAdV infections.