Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Techniques for Characterizing Cytomegalovirus-Encoded miRNAs.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to sites within the 3' untranslated regions of messenger RNA (mRNA) transcripts. The discovery of this completely new mechanism of gene regulation necessitated the development of a variety of techniques to further characterize miRNAs, their expression, and function. In this chapter, we will discuss techniques currently used in the miRNA field to detect, express and inhibit miRNAs, as well as methods used to identify and validate their targets, specifically with respect to the miRNAs encoded by human cytomegalovirus.

Human Cytomegalovirus Immediate Early 86-kDa Protein Blocks Transcription and Induces Degradation of the Immature Interleukin-1ß Protein during Virion-Mediated Activation of the AIM2 Inflammasome.

Secretion of interleukin-1ß (IL-1ß) represents a fundamental innate immune response to microbial infection that, at the molecular level, occurs following activation of proteolytic caspases that cleave the immature protein into a secretable form. Human cytomegalovirus (HCMV) is the archetypal betaherpesvirus that is invariably capable of lifelong infection through the activity of numerous virally encoded immune evasion phenotypes. Innate immune pathways responsive to cytoplasmic double-stranded DNA (dsDNA) are known to be activated in response to contact between HCMV and host cells. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) genome editing to demonstrate that the dsDNA receptor absent in melanoma 2 (AIM2) is required for secretion of IL-1ß following HCMV infection. Furthermore, dsDNA-responsive innate signaling induced by HCMV infection that leads to activation of the type I interferon response is also shown, unexpectedly, to play a contributory role in IL-1ß secretion. Importantly, we also show that rendering virus particles inactive by UV exposure leads to substantially increased IL-1ß processing and secretion and that live HCMV can inhibit this, suggesting the virus encodes factors that confer an inhibitory effect on this response. Further examination revealed that ectopic expression of the immediate early (IE) 86-kDa protein (IE86) is actually associated with a block in transcription of the pro-IL-1ß gene and, independently, diminishment of the immature protein. Overall, these results reveal two new and distinct phenotypes conferred by the HCMV IE86 protein, as well as an unusual circumstance in which a single herpesviral protein exhibits inhibitory effects on multiple molecular processes within the same innate immune response.IMPORTANCE Persistent infection with HCMV is associated with the operation of diverse evasion phenotypes directed at antiviral immunity. Obstruction of intrinsic and innate immune responses is typically conferred by viral proteins either associated with the viral particle or expressed immediately after entry. In line with this, numerous phenotypes are attributed to the HCMV IE86 protein that involve interference with innate immune processes via transcriptional and protein-directed mechanisms. We describe novel IE86-mediated phenotypes aimed at virus-induced secretion of IL-1ß. Intriguingly, while many viruses target the function of the molecular scaffold required for IL-1ß maturation to prevent this response, we find that HCMV and IE86 target the IL-1ß protein specifically. Moreover, we show that IE86 impairs both the synthesis of the IL-1ß transcript and the stability of the immature protein. This indicates an unusual phenomenon in which a single viral protein exhibits two molecularly separate evasion phenotypes directed at a single innate cytokine.

Human Cytomegalovirus Encodes a Novel FLT3 Receptor Ligand Necessary for Hematopoietic Cell Differentiation and Viral Reactivation.

The ability of human cytomegalovirus (HCMV) to reactivate from latent infection of hematopoietic progenitor cells (HPCs) is intimately linked to cellular differentiation. HCMV encodes UL7 that our group has shown is secreted from infected cells and induces angiogenesis. In this study, we show that UL7 is a ligand for Fms-like tyrosine kinase 3 receptor (Flt-3R), a well-known critical factor in HPC differentiation. We observed that UL7 directly binds Flt-3R and induces downstream signaling cascades, including phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. Importantly, we show that UL7 protein induces differentiation of both CD34+ HPCs and CD14+ monocytes. Last, we show that an HCMV mutant lacking UL7 fails to reactivate in CD34+ HPCs in vitro as well as in humanized mice. These observations define the first virally encoded differentiation factor with significant implications not only for HCMV reactivation but also for alteration of the hematopoietic compartment in transplant patients.IMPORTANCE Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients. CD34+ hematopoietic progenitor cells (HPCs) represent a critical reservoir of latent HCMV in the transplant population, thereby providing a source of virus for dissemination to visceral organs. HCMV reactivation has been linked to HPC/myeloid cellular differentiation; however, the mechanisms involved in these events are poorly understood at the molecular level. In this study, we show that a viral protein is a ligand for Fms-like tyrosine kinase 3 receptor (Flt-3R) and that the binding of HCMV UL7 to the Flt-3R triggers HPC and monocyte differentiation. Moreover, the loss of UL7 prevents viral reactivation in HPCs in vitro as well as in humanized mice. These observations define the first virally encoded differentiation factor with significant implications not only for HCMV reactivation but also for alteration of the hematopoietic compartment in transplant patients.

Human Cytomegalovirus miR-UL112-3p Targets TLR2 and Modulates the TLR2/IRAK1/NFκB Signaling Pathway.

Human Cytomegalovirus (HCMV) encodes multiple microRNAs (miRNAs) whose functions are just beginning to be uncovered. Using in silico approaches, we identified the Toll-Like Receptor (TLR) innate immunity pathway as a possible target of HCMV miRNAs. Luciferase reporter assay screens further identified TLR2 as a target of HCMV miR-UL112-3p. TLR2 plays a major role in innate immune response by detecting both bacterial and viral ligands, including HCMV envelope proteins gB and gH. TLR2 activates a variety of signal transduction routes including the NFκB pathway. Furthermore, TLR2 plays an important role in controlling CMV infection both in humans and in mice. Immunoblot analysis of cells transfected with a miR-UL112-3p mimic revealed that endogenous TLR2 is down-regulated by miR-UL112-3p with similar efficiency as a TLR2-targeting siRNA (siTLR2). We next found that TLR2 protein level decreases at late times during HCMV infection and correlates with miR-UL112-3p accumulation in fibroblasts and monocytic THP1 cells. Confirming direct miR-UL112-3p targeting, down-regulation of endogenous TLR2 was not observed in cells infected with HCMV mutants deficient in miR-UL112-3p expression, but transfection of miR-UL112-3p in these cells restored TLR2 down-regulation. Using a NFκB reporter cell line, we found that miR-UL112-3p transfection significantly inhibited NFκB-dependent luciferase activity with similar efficiency as siTLR2. Consistent with this observation, miR-UL112-3p transfection significantly reduced the expression of multiple cytokines (IL-1ß, IL-6 and IL-8) upon stimulation with a TLR2 agonist. Finally, miR-UL112-3p transfection significantly inhibited the TLR2-induced post-translational activation of IRAK1, a kinase located in the upstream section of the TLR2/NFκB signaling axis. To our knowledge, this is the first identified mechanism of TLR2 modulation by HCMV and is the first report of functional targeting of TLR2 by a viral miRNA. These results provide a novel mechanism through which a HCMV miRNA regulates the innate immune response by down-regulating TLR-2 expression.

Cytomegalovirus microRNAs.

The discovery that animals, plants and DNA viruses encode microRNAs (miRNAs) has transformed our understanding of the regulation of gene expression. miRNAs are ubiquitous small non-coding RNAs that regulate gene expression post-transcriptionally, generally by binding to sites within the 3' untranslated regions (UTR) of messenger RNA (mRNA) transcripts. To date, over 250 viral miRNAs have been identified primarily in members of the herpesvirus family. These viral miRNAs target both viral and cellular genes in order to regulate viral replication, the establishment and maintenance of viral latency, cell survival, and innate and adaptive immunity. This review will focus on our current knowledge of the targets and functions of human cytomegalovirus (HCMV) miRNAs and their functional equivalents in other herpesviruses.

Cytomegalovirus miRNAs target secretory pathway genes to facilitate formation of the virion assembly compartment and reduce cytokine secretion.

Herpesviruses, including human cytomegalovirus (HCMV), encode multiple microRNAs (miRNA) whose targets are just being uncovered. Moreover, miRNA function during the virus life cycle is relatively unknown. We find that HCMV miRs UL112-1, US5-1, and US5-2 target multiple components of the host secretory pathway, including VAMP3, RAB5C, RAB11A, SNAP23, and CDC42. A HCMV miR UL112-1, US5-1, and US5-2 triple mutant displayed aberrant morphogenesis of the virion assembly compartment (VAC), increased secretion of noninfectious particles, and increased IL-6 release from infected cells. Ectopic expression of miRs UL112-1, US5-1, and US5-2 or siRNAs directed against RAB5C, RAB11A, SNAP23, and CDC42 caused the loss of Golgi stacks with reorganization into structures that resemble the VAC and a decrease in cytokine release. These observations indicate that multiple HCMV miRNAs coordinately regulate reorganization of the secretory pathway to control cytokine secretion and facilitate formation of the VAC for efficient infectious virus production.

Techniques for characterizing cytomegalovirus-encoded miRNAs.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level, by binding to sites within the 3' untranslated regions of messenger RNA (mRNA) transcripts. The discovery of this completely new mechanism of gene regulation necessitated the development of a variety of techniques to further characterize miRNAs, their expression, and function. In this chapter, we will discuss techniques currently used in the miRNA field to express, detect, and inhibit miRNAs as well as methods used to identify their targets.

Functional genomics approaches to understand cytomegalovirus replication, latency and pathogenesis.

Cytomegalovirus (CMV) is a species-specific herpesvirus that is ubiquitous in the population and has the potential to cause significant disease in immunocompromised individuals as well as in congenitally infected infants. CMV establishes latency in cells of the myeloid lineage following primary infection. High-throughput functional genomics approaches have provided insight into the mechanisms of CMV replication, but although CMV latency cell models have been useful in elucidating the mechanisms of viral latency and reactivation, omics approaches have proven challenging in these cell systems. This review will summarize the current state of knowledge concerning the use of functional genomics technologies to understand mechanisms of CMV replication, latency and pathogenesis.

Human cytomegalovirus US7 is regulated synergistically by two virally encoded microRNAs and by two distinct mechanisms.

Human cytomegalovirus (HCMV) encodes at least 14 microRNAs (miRNAs) that act posttranscriptionally to repress gene expression. Although several HCMV miRNA targets of both cellular and viral origin have been identified, our knowledge of their function remains limited. HCMV miRNA targets, as well as phenotypes associated with HCMV miRNA mutants, have been difficult to identify since the downregulation of targets by a single miRNA is often less than 2-fold. Several factors can contribute to the strength of repression, including the mechanism of translational inhibition, the degree of complementarity between the miRNA and target mRNA, the number of binding sites for one miRNA, and cooperativity or antagonism between miRNAs. To determine the effect of multiple miRNAs on one gene, we examined the repression of a viral gene, US7. Here we demonstrate that the HCMV-encoded miRNAs miR-US5-1 and miR-US5-2 function in a highly synergistic manner to regulate US7, even at very low miRNA concentrations. Regulation of US7 involves three functional miRNA binding sites: two that are completely complementary to the 3' untranslated region (3'UTR) and one that is imperfectly matched. Surprisingly, we observed equal contributions to inhibition from both complete and partially complementary sites, and repression was not completely abrogated until all three sites were mutated simultaneously. We also observed that the miRNA binding sites did not follow the spacing constraints for corepressive miRNAs observed in earlier reports. These results underscore the importance of evaluating the contribution of multiple miRNAs on gene regulation and shed new insight into miRNA:mRNA interactions.

The Fanconi anemia protein FANCM is controlled by FANCD2 and the ATR/ATM pathways.

Genomic stability requires a functional Fanconi anemia (FA) pathway composed of an upstream "core complex" (FA proteins A/B/C/E/F/G/L/M) that mediates monoubiquitination of the downstream targets FANCD2 and FANCI. Unique among FA core complex members, FANCM has processing activities toward replication-associated DNA structures, suggesting a vital role for FANCM during replication. Using Xenopus egg extracts, we analyzed the functions of FANCM in replication and the DNA damage response. xFANCM binds chromatin in a replication-dependent manner and is phosphorylated in response to DNA damage structures. Chromatin binding and DNA damage-induced phosphorylation of xFANCM are mediated in part by the downstream FA pathway protein FANCD2. Moreover, phosphorylation and chromatin recruitment of FANCM is regulated by two mayor players in the DNA damage response: the cell cycle checkpoint kinases ATR and ATM. Our results indicate that functions of FANCM are controlled by FA- and non-FA pathways in the DNA damage response.

Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors.

BACKGROUND: The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as ATM (Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity. RESULTS: Using a replication-free assay in Xenopus extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells. CONCLUSIONS: These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds.

A novel cell-free screen identifies a potent inhibitor of the Fanconi anemia pathway.

The Fanconi Anemia (FA) DNA damage response pathway is involved in the processing of DNA interstrand crosslinks (ICLs). As such, inhibition of the FA pathway could chemosensitize FA-competent tumor cells to commonly used ICL agents like cisplatin. Moreover, suppression of the FA pathway is synthetic lethal with deficiencies in several other DNA repair pathways, suggesting that FA pathway inhibitors could be used in targeted therapies against specific tumors. To identify such inhibitors, we designed a novel in vitro screening assay utilizing Xenopus egg extracts. Using the DNA-stimulated monoubiquitylation of Xenopus FANCD2 (xFANCD2-L) as readout, a chemical library screen identified DDN (2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone) as a novel and potent FA pathway inhibitor. DDN inhibited xFANCD2-L formation in a dose-dependent manner in both extracts and human cells without disruption of the upstream FA core complex. DDN also inhibited the characteristic subnuclear FANCD2 foci formation following DNA damage. Moreover, DDN displayed a greater synergistic effect with cisplatin in a FA-proficient cancer cell line compared to its FA-deficient isogenic counterpart, suggesting that DDN might be a good lead candidate as cisplatin chemosensitizer in both FA-deficient and FA-competent tumors. This system constitutes the first cell-free screening assay for identifying compounds that inhibit the FA pathway and provides a new biochemical platform for mapping the functions of its various components with specific chemical inhibitors.

Human SSRP1 has Spt16-dependent and -independent roles in gene transcription.

The facilitating chromatin transcription (FACT) complex, a heterodimer of SSRP1 and Spt16, has been shown to regulate transcription elongation through a chromatin template in vitro and on specific genes in cells. However, its global role in transcription regulation in human cells remains largely elusive. We conducted spotted microarray analyses using arrays harboring 8308 human genes to assess the gene expression profile after knocking down SSRP1 or Spt16 levels in human non-small cell lung carcinoma (H1299) cells. Although the changes of these transcripts were surprisingly subtle, there were approximately 170 genes whose transcript levels were either reduced or induced >1.5-fold. Approximately 106 genes with >1.2-fold change at the level of transcripts were the common targets of both SSRP1 and Spt16 ( approximately 1.3%). A subset of genes was regulated by SSRP1 independent of Spt16. Further analyses of some of these genes not only verified this observation but also identified the serum-responsive gene, egr1, as a novel target for both SSRP1 and Spt16. We further showed that SSRP1 and Spt16 are important for the progression of elongation RNA pol II on the egr1 gene. These results suggest that SSRP1 has Spt16-dependent and -independent roles in regulating gene transcription in human cells.

Functional analysis of evolutionary conserved clustering of bZIP binding sites in the baculovirus homologous regions (hrs) suggests a cooperativity between host and viral transcription factors.

The genome of the Autographa californica Multinucleocapsid Polyhedrosis Virus (AcMNPV) contains nine interspersed homologous regions (hrs) that function as potent enhancer sequences when linked in cis to either viral or heterologous RNA polymerase II-dependent promoters. Their activity is strongly increased by the binding of the major immediate early viral transregulator IE1 on 28-mer palindromic sites present in hrs. We show that hrs of AcMNPV additionally carry, in the interpalindromic sequences, a large number of cAMP response elements (CRE) and TPA response elements (TRE), known to bind ubiquitous cellular transcription factors of the bZIP family. Moreover, these clusters of CRE and TRE motifs are concentrated in hrs. Analysis of the 25 baculovirus genomes sequenced so far reveals that these motifs are evolutionary conserved in Lepidoptera NPVs, suggesting a functional role in the hr enhancer function. Consistently, EMSA experiments indicate that CRE and on a lesser extent TRE sites specifically bind insect host factors. Moreover, reporter assays reveal that these CRE sites have an additive stimulatory effect on RNAPol II-dependent transcription in Sf9 cells and are potentially able to synergize with the IE1-binding palindrome.

Vitellogenin mRNA expression in Cherax quadricarinatus during secondary vitellogenic at first maturation females.

PCR products of 1.1 and 0.9 kb were generated using Cherax quadricarinatus genomic DNA in the first case, and hepatopancreas and ovary cDNAs in the second case. These PCR products were cloned and analyzed for nucleotide sequences. The 1.1 kb fragment was used as a probe for Northern hybridization, revealing a transcript of approximately 8 kb in both tissues. Results from both Northern blot and RT-PCR analyses showed that the mRNA enconding the 3' end of the vitellogenin cDNA was present simultaneously in both hepatopancreas and ovary tissues in secondary vitellogenic at first maturation females, but was not detected in male hepatopancreas. The deduced amino acid sequences of Vitellogenin (Vg) cDNAs from ovary and hepatopancreas confirmed the existence at least two different Vg genes, and two different sites of synthesis.

Characterization and transcriptional profiles of three Spodoptera frugiperda genes encoding cysteine-rich peptides. A new class of defensin-like genes from lepidopteran insects?

The present work describes sequence and transcription of three Spodoptera frugiperda genes encoding 6-cysteine-rich peptides. Sequence alignments indicate that the predicted peptides belong to the insect defensin family, although phylogenetic analyses suggest they form a cluster distinct from that of other neopteran insect defensins. The three genes were identified in a non-immune-challenged Sf9 cells cDNA (DNA complementary to RNA) library (Landais et al., Bioinformatics, in press) and were named spodoptericin, Sf-gallerimycin and Sf-cobatoxin. Spodoptericin is a novel defensin-like gene that appears to be weakly up-regulated following injection of bacteria and fungi. Interestingly, no sequence motif clearly homologous to cis regulatory element involved in the regulation of antimicrobial genes was found. An homologue of the spodoptericin gene was identified in the SilkBase Bombyx mori cDNA library. Sf-gallerimycin is related to the Galleria mellonella gallerimycin gene and is induced after immune challenge by injection of bacteria in the larval fat body as well as in hemocytes. In silico analysis of the sequence upstream from the cDNA reveals the presence of at least one motif homologous to a nuclear factor kappaB (NF-kappaB) binding site. Finally, Sf-cobatoxin is related to the G. mellonella cobatoxin-like gene. Despite high levels of constitutive expression compared to the two previous genes, transcription of Sf-cobatoxin is increased after immune, in particular, bacterial challenge. We therefore confirm that these three genes encode potential candidate molecules involved in S. frugiperda innate humoral response.