G-quadruplexes formed by Varicella-Zoster virus reiteration sequences suppress expression of glycoprotein C and regulate viral cell-to-cell spread.

G-quadruplex (G4) formed by repetitive guanosine-rich sequences plays important roles in diverse cellular processes; however, its roles in viral infection are not fully understood. In this study, we investigated the genome-wide distribution of G4-forming sequences (G4 motifs) in Varicella-Zoster virus (VZV) and found that G4 motifs are enriched in the internal repeat short and the terminal repeat short regions flanking the unique short region and also in some reiteration (R) sequence regions. A high density of G4 motifs in the R2 region was found on the template strand of ORF14, which encodes glycoprotein C (gC), a virulent factor for viral growth in skin. Analyses such as circular dichroism spectroscopy, thermal difference spectra, and native polyacrylamide gel electrophoresis with oligodeoxynucleotides demonstrated that several G4 motifs in ORF14 form stable G4 structures. In transfection assays, gC expression from the G4-disrupted ORF14 gene was increased at the transcriptional level and became more resistant to suppression by G4-ligand treatment. The recombinant virus containing the G4-disrupted ORF14 gene expressed a higher level of gC mRNA, while it showed a slightly reduced growth. This G4-disrupted ORF14 virus produced smaller plaques than the wild-type virus. Our results demonstrate that G4 formation via reiteration sequences suppresses gC expression during VZV infection and regulates viral cell-to-cell spread.

Human cytomegalovirus microRNA miR-US4-1 inhibits CD8(+) T cell responses by targeting the aminopeptidase ERAP1.

Major histocompatibility complex (MHC) class I molecules present peptides on the cell surface to CD8(+) T cells, which is critical for the killing of virus-infected or transformed cells. Precursors of MHC class I-presented peptides are trimmed to mature epitopes by the aminopeptidase ERAP1. The US2-US11 genomic region of human cytomegalovirus (HCMV) is dispensable for viral replication and encodes three microRNAs (miRNAs). We show here that HCMV miR-US4-1 specifically downregulated ERAP1 expression during viral infection. Accordingly, the trimming of HCMV-derived peptides was inhibited, which led to less susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes (CTLs). Our findings identify a previously unknown viral miRNA-based CTL-evasion mechanism that targets a key step in the MHC class I antigen-processing pathway.

Human Cytomegalovirus UL48 Deubiquitinase Primarily Targets Innermost Tegument Proteins pp150 and Itself To Regulate Their Stability and Protects Virions from Inclusion of Ubiquitin Conjugates.

Viral deubiquitinases (DUBs) regulate cellular innate immunity to benefit viral replication. In human cytomegalovirus (HCMV), the UL48-encoded DUB regulates innate immune responses, including NF-κB signaling. Although UL48 DUB is known to regulate its stability via auto-deubiquitination, its impact on other viral proteins is not well understood. In this study, we investigated the role of UL48 DUB in regulating the ubiquitination of viral proteins by comparing the levels of ubiquitinated viral peptides in cells infected with wild-type virus and DUB active-site mutants using mass spectrometry. We found that ubiquitinated peptides were increased in DUB mutant virus infection for 90% of viral proteins, with the innermost tegument proteins pp150 (encoded by UL32) and pUL48 itself being most significantly affected. The highly deubiquitinated lysine residues of pUL48 were mapped within its N-terminal DUB domain and the nuclear localization signal. Among them, the arginine substitution of lysine 2 (K2R) increased pUL48 stability and enhanced viral growth at low multiplicity of infection, indicating that K2 auto-deubiquitination has a role in regulating pUL48 stability. pUL48 also interacted with pp150 and increased pp150 expression by downregulating its ubiquitination. Furthermore, we found that, unlike the wild-type virus, mutant viruses expressing the UL48 protein with the DUB domain deleted or DUB active site mutated contain higher levels of ubiquitin conjugates, including the ubiquitinated forms of pp150, in their virions. Collectively, our results demonstrate that UL48 DUB mainly acts on the innermost tegument proteins pp150 and pUL48 itself during HCMV infection and may play a role in protecting virions from the inclusion of ubiquitin conjugates. IMPORTANCE Herpesviruses encode highly conserved tegument proteins that contain deubiquitinase (DUB) activity. Although the role of viral DUBs in the regulation of host innate immune responses has been established, their roles in the stability and function of viral proteins are not well understood. In this study, we performed a comparative analysis of the levels of ubiquitinated viral peptides between wild-type and DUB-inactive HCMV infections and demonstrated that the innermost tegument proteins pp150 and pUL48 (DUB itself) are major targets of viral DUB. We also show that ubiquitinated viral proteins are effectively incorporated into the virions of DUB mutant viruses but not the wild-type virus. Our study demonstrates that viral DUBs may play important roles in promoting the stability of viral proteins and inhibiting the inclusion of ubiquitin conjugates into virions.

Genome-wide analysis of regulatory G-quadruplexes affecting gene expression in human cytomegalovirus.

G-quadruplex (G4), formed by repetitive guanosine-rich sequences, is known to play various key regulatory roles in cells. Herpesviruses containing a large double-stranded DNA genome show relatively higher density of G4-forming sequences in their genomes compared to human and mouse. However, it remains poorly understood whether all of these sequences form G4 and how they play a role in the virus life cycle. In this study, we performed genome-wide analyses of G4s present in the putative promoter or gene regulatory regions of a 235-kb human cytomegalovirus (HCMV) genome and investigated their roles in viral gene expression. We evaluated 36 putative G4-forming sequences associated with 20 genes for their ability to form G4 and for the stability of G4s in the presence or absence of G4-stabilizing ligands, by circular dichroism and melting temperature analyses. Most identified sequences formed a stable G4; 28 sequences formed parallel G4s, one formed an antiparallel G4, and four showed mixed conformations. However, when we assessed the effect of G4 on viral promoters by cloning the 20 putative viral promoter regions containing 36 G4-forming sequences into the luciferase reporter and monitoring the expression of luciferase reporter gene in the presence of G4-stabilizing chemicals, we found that only 9 genes were affected by G4 formation. These results revealed promoter context-dependent gene suppression by G4 formation. Mutational analysis of two potential regulatory G4s also demonstrated gene suppression by the sequence-specific G4 formation. Furthermore, the analysis of a mutant virus incapable of G4 formation in the UL35 promoter confirmed promoter regulation by G4 in the context of virus infection. Our analyses provide a platform for assessing G4 functions at the genomic level and demonstrate the properties of the HCMV G4s and their regulatory roles in viral gene expression.

Transmembrane Protein pUL50 of Human Cytomegalovirus Inhibits ISGylation by Downregulating UBE1L.

Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that can be conjugated to proteins via an enzymatic cascade involving the E1, E2, and E3 enzymes. ISG15 expression and protein ISGylation modulate viral infection; however, the viral mechanisms regulating the function of ISG15 and ISGylation are not well understood. We recently showed that ISGylation suppresses the growth of human cytomegalovirus (HCMV) at multiple steps of the virus life cycle and that the virus-encoded pUL26 protein inhibits protein ISGylation. In this study, we demonstrate that the HCMV UL50-encoded transmembrane protein, a component of the nuclear egress complex, also inhibits ISGylation. pUL50 interacted with UBE1L, an E1-activating enzyme for ISGylation, and (to a lesser extent) with ISG15, as did pUL26. However, unlike pUL26, pUL50 caused proteasomal degradation of UBE1L. The UBE1L level induced in human fibroblast cells by interferon beta treatment or virus infection was reduced by pUL50 expression. This activity of pUL50 involved the transmembrane (TM) domain within its C-terminal region, although pUL50 could interact with UBE1L in a manner independent of the TM domain. Consistently, colocalization of pUL50 with UBE1L was observed in cells treated with a proteasome inhibitor. Furthermore, we found that RNF170, an endoplasmic reticulum (ER)-associated ubiquitin E3 ligase, interacted with pUL50 and promoted pUL50-mediated UBE1L degradation via ubiquitination. Our results demonstrate a novel role for the pUL50 transmembrane protein of HCMV in the regulation of protein ISGylation.IMPORTANCE Proteins can be conjugated covalently by ubiquitin or ubiquitin-like proteins, such as SUMO and ISG15. ISG15 is highly induced in viral infection, and ISG15 conjugation, termed ISGylation, plays important regulatory roles in viral growth. Although ISGylation has been shown to negatively affect many viruses, including human cytomegalovirus (HCMV), viral countermeasures that might modulate ISGylation are not well understood. In the present study, we show that the transmembrane protein encoded by HCMV UL50 inhibits ISGylation by causing proteasomal degradation of UBE1L, an E1-activating enzyme for ISGylation. This pUL50 activity requires membrane targeting. In support of this finding, RNF170, an ER-associated ubiquitin E3 ligase, interacts with pUL50 and promotes UL50-mediated UBE1L ubiquitination and degradation. Our results provide the first evidence, to our knowledge, that viruses can regulate ISGylation by directly targeting the ISGylation E1 enzyme.

Cooperative inhibition of RIP1-mediated NF-κB signaling by cytomegalovirus-encoded deubiquitinase and inactive homolog of cellular ribonucleotide reductase large subunit.

Several viruses have been found to encode a deubiquitinating protease (DUB). These viral DUBs are proposed to play a role in regulating innate immune or inflammatory signaling. In human cytomegalovirus (HCMV), the largest tegument protein encoded by UL48 contains DUB activity, but its cellular targets are not known. Here, we show that UL48 and UL45, an HCMV-encoded inactive homolog of cellular ribonucleotide reductase (RNR) large subunit (R1), target receptor-interacting protein kinase 1 (RIP1) to inhibit NF-κB signaling. Transfection assays showed that UL48 and UL45, which binds to UL48, interact with RIP1 and that UL48 DUB activity and UL45 cooperatively suppress RIP1-mediated NF-κB activation. The growth of UL45-null mutant virus was slightly impaired with showing reduced accumulation of viral late proteins. Analysis of a recombinant virus expressing HA-UL45 showed that UL45 interacts with both UL48 and RIP1 during virus infection. Infection with the mutant viruses also revealed that UL48 DUB activity and UL45 inhibit TNFα-induced NF-κB activation at late times of infection. UL48 cleaved both K48- and K63-linked polyubiquitin chains of RIP1. Although UL45 alone did not affect RIP1 ubiquitination, it could enhance the UL48 activity to cleave RIP1 polyubiquitin chains. Consistently, UL45-null virus infection showed higher ubiquitination level of endogenous RIP1 than HA-UL45 virus infection at late times. Moreover, UL45 promoted the UL48-RIP1 interaction and re-localization of RIP1 to the UL48-containing virion assembly complex. The mouse cytomegalovirus (MCMV)-encoded DUB, M48, interacted with mouse RIP1 and M45, an MCMV homolog of UL45. Collectively, our data demonstrate that cytomegalovirus-encoded DUB and inactive R1 homolog target RIP1 and cooperatively inhibit RIP1-mediated NF-κB signaling at the late stages of HCMV infection.

Consecutive Inhibition of ISG15 Expression and ISGylation by Cytomegalovirus Regulators.

Interferon-stimulated gene 15 (ISG15) encodes an ubiquitin-like protein that covalently conjugates protein. Protein modification by ISG15 (ISGylation) is known to inhibit the replication of many viruses. However, studies on the viral targets and viral strategies to regulate ISGylation-mediated antiviral responses are limited. In this study, we show that human cytomegalovirus (HCMV) replication is inhibited by ISGylation, but the virus has evolved multiple countermeasures. HCMV-induced ISG15 expression was mitigated by IE1, a viral inhibitor of interferon signaling, however, ISGylation was still strongly upregulated during virus infection. RNA interference of UBE1L (E1), UbcH8 (E2), Herc5 (E3), and UBP43 (ISG15 protease) revealed that ISGylation inhibits HCMV growth by downregulating viral gene expression and virion release in a manner that is more prominent at low multiplicity of infection. A viral regulator pUL26 was found to interact with ISG15, UBE1L, and Herc5, and be ISGylated. ISGylation of pUL26 regulated its stability and inhibited its activities to suppress NF-κB signaling and complement the growth of UL26-null mutant virus. Moreover, pUL26 reciprocally suppressed virus-induced ISGylation independent of its own ISGylation. Consistently, ISGylation was more pronounced in infections with the UL26-deleted mutant virus, whose growth was more sensitive to IFNß treatment than that of the wild-type virus. Therefore, pUL26 is a viral ISG15 target that also counteracts ISGylation. Our results demonstrate that ISGylation inhibits HCMV growth at multiple steps and that HCMV has evolved countermeasures to suppress ISG15 transcription and protein ISGylation, highlighting the importance of the interplay between virus and ISGylation in productive viral infection.

Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry.

UNLABELLED: Human cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(ΔDUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(ΔDUB), which lacks only the DUB, and UL48(Δ360-1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ΔDUB/NLS and Δ360-1200 mutant viruses did not grow in fibroblasts, the ΔDUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation in trans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360-1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ΔDUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry. IMPORTANCE: HCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.

Positive role of promyelocytic leukemia protein in type I interferon response and its regulation by human cytomegalovirus.

Promyelocytic leukemia protein (PML), a major component of PML nuclear bodies (also known as nuclear domain 10), is involved in diverse cellular processes such as cell proliferation, apoptosis, gene regulation, and DNA damage response. PML also acts as a restriction factor that suppresses incoming viral genomes, therefore playing an important role in intrinsic defense. Here, we show that PML positively regulates type I interferon response by promoting transcription of interferon-stimulated genes (ISGs) and that this regulation by PML is counteracted by human cytomegalovirus (HCMV) IE1 protein. Small hairpin RNA-mediated PML knockdown in human fibroblasts reduced ISG induction by treatment of interferon-ß or infection with UV-inactivated HCMV. PML was required for accumulation of activated STAT1 and STAT2, interacted with them and HDAC1 and HDAC2, and was associated with ISG promoters after HCMV infection. During HCMV infection, viral IE1 protein interacted with PML, STAT1, STAT2, and HDACs. Analysis of IE1 mutant viruses revealed that, in addition to the STAT2-binding domain, the PML-binding domain of IE1 was necessary for suppression of interferon-ß-mediated ISG transcription, and that IE1 inhibited ISG transcription by sequestering interferon-stimulated gene factor 3 (ISGF3) in a manner requiring its binding of PML and STAT2, but not of HDACs. In conclusion, our results demonstrate that PML participates in type I interferon-induced ISG expression by regulating ISGF3, and that this regulation by PML is counteracted by HCMV IE1, highlighting a widely shared viral strategy targeting PML to evade intrinsic and innate defense mechanisms.

DNA sensing-independent inhibition of herpes simplex virus 1 replication by DAI/ZBP1.

DNA-dependent activator of interferon regulatory factor (DAI) acts as a cytosolic B-form DNA sensor that induces type I interferons. However, DAI is not required for DNA sensing in certain cell types due to redundancy of the DNA sensing system. Here, we investigated the effect of DAI on herpes simplex virus 1 (HSV-1) infection in HepG2 hepatocellular carcinoma cells. DAI transcription was induced after gamma interferon (IFN-γ) treatment or HSV-1 infection. HSV-1 replication was enhanced by DAI knockdown, and ectopic DAI expression repressed viral replication in a manner requiring the Zß and D3 domains, but not the Zα domain. This activity of DAI was more prominent at low multiplicity of infection (MOI) and correlated with the reduced expression of viral immediate-early genes. Consistently, DAI repressed the activation of ICP0 promoter in reporter gene assays. DAI knockdown did not affect the B-DNA-mediated IFN-ß transcription and IRF3 activation, and overexpression of DAI and RIP1 did not enhance NF-κB activation by B-DNA treatment, demonstrating that DAI is not essential for the B-DNA-mediated IFN production in HepG2 cells. DAI colocalized with ICP0 in a subset of nuclear and cytoplasmic foci in infected cells and interacted with ICP0 in coimmunoprecipitation assays. The anti-HSV-1 effect of DAI was not observed in ICP0-deleted mutant virus infection at a high MOI in HepG2 cells and mouse embryonic fibroblasts. Degradation of IFI16 and PML by ICP0 was enhanced in infection of DAI-knockdown cells. Collectively, these results demonstrate that DAI can suppress HSV-1 growth independent of DNA sensing through mechanisms involving suppression of viral genomes and regulation of ICP0.

Biphasic regulation of A20 gene expression during human cytomegalovirus infection.

BACKGROUND: The A20 ubiquitin-editing enzyme is a target of nuclear factor kappa B (NF-κB) and also plays a key role in regulating the NF-κB signaling pathway. NF-κB activity is increased during human cytomegalovirus (HCMV) infection and HCMV appears to be adapted to this change. To better understand the regulation of NF-κB signaling during HCMV infection, we investigated how A20 expression is controlled during HCMV infection. METHODS: The expression level of A20 in human fibroblast cells infected with HCMV or UV-inactivated virus (UV-HCMV) was measured by immunoblot analysis, cell staining, and quantitative real-time PCR. Changes of histone modifications on the A20 promoter were determined by chromatin immunoprecipitation assays. Lentiviral vectors were used to knockdown A20 in fibroblast cells. RESULTS: A20 expression was increased at early times after HCMV infection. This increase of the A20 protein level was promoted by viral gene expression under low viral load conditions. The viral IE1 protein, which is known to activate NF-κB, increased the A20 promoter activity through the upstream NF-κB sites in reporter assays, suggesting that IE1 is at least partly involved in A20 induction. Analysis of A20 expression with a high viral load demonstrated that the A20 regulation by HCMV was biphasic; both A20 protein and mRNA levels were increased at the early stage of infection, but decreased at the late stage. Under high viral load conditions, A20 upregulation was more profound with UV-HCMV than with HCMV, indicating a role of the viral gene product(s) in limiting A20 induction. Consistently, more histone modifications for euchromatin were found on the A20 promoter during UV-HCMV infection than with HCMV infection. A20 knockdown by shRNA reduced HCMV growth. CONCLUSION: These results suggest that the biphasic regulation of A20 expression may be important for productive HCMV infection.

Upregulation of Nrf2 expression by human cytomegalovirus infection protects host cells from oxidative stress.

NF-E2 related factor 2 (Nrf2) is a transcription factor that plays a key role(s) in cellular defence against oxidative stress. In this study, we showed that the expression of Nrf2 was upregulated in primary human foreskin fibroblasts (HFFs), following human cytomegalovirus (HCMV/HHV-5) infection. The expression of haem oxygenase-1, a downstream target of Nrf2, was also increased by HCMV infection, and this induction was suppressed in HFFs expressing a small hairpin RNA (shRNA) against Nrf2. The HCMV-mediated increase in Nrf2 expression was abolished when UV-irradiated virus was used or when the activity of casein kinase 2 was inhibited. Host cells infected by HCMV had higher survival rates following oxidative stress induced by buthionine sulfoximine compared with uninfected control cells, but this cell-protective effect was abolished by the use of Nrf2 shRNA. Our results suggest that HCMV-mediated activation of Nrf2 might be beneficial to the virus by increasing the host cell's ability to cope with oxidative stress resulting from viral infection and/or inflammation.

Rapid discovery and classification of inhibitors of coronavirus infection by pseudovirus screen and amplified luminescence proximity homogeneous assay.

To identify potent antiviral compounds, we introduced a high-throughput screen platform that can rapidly classify hit compounds according to their target. In our platform, we performed a compound screen using a lentivirus-based pseudovirus presenting a spike protein of coronavirus, and we evaluated the hit compounds using an amplified luminescence proximity homogeneous assay (alpha) test with purified host receptor protein and the receptor binding domain of the viral spike. With our screen platform, we were able to identify both spike-specific compounds (class I) and broad-spectrum antiviral compounds (class II). Among the hit compounds, thiosemicarbazide was identified to be selective to the interaction between the viral spike and its host cell receptor, and we further optimized the binding potency of thiosemicarbazide through modification of the pyridine group. Among the class II compounds, we found raloxifene and amiodarone to be highly potent against human coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. In particular, using analogs of the benzothiophene moiety, which is also present in raloxifene, we have identified benzothiophene as a novel structural scaffold for broad-spectrum antivirals. This work highlights the strong utility of our screen platform using a pseudovirus assay and an alpha test for rapid identification of potential antiviral compounds and their mechanism of action, which can lead to the accelerated development of therapeutics against newly emerging viral infections.

The chromatin-tethering domain of human cytomegalovirus immediate-early (IE) 1 mediates associations of IE1, PML and STAT2 with mitotic chromosomes, but is not essential for viral replication.

Human cytomegalovirus (HCMV) immediate-early (IE) 1 protein associates with chromosomes in mitotic cells using its carboxyl-terminal 16 aa region. However, the role of this IE1 activity in viral growth has not been evaluated in the context of mutant virus infection. We produced a recombinant HCMV encoding mutant IE1 with the carboxyl-terminal chromosome-tethering domain (CTD) deleted. This IE1(ΔCTD) virus grew like the wild-type virus in fibroblasts, indicating that the CTD is not essential for viral replication in permissive cells. Unlike wild-type virus infections, PML and STAT2, which interact with IE1, did not accumulate at mitotic chromosomes in IE1(ΔCTD) virus-infected fibroblasts, demonstrating that their associations with chromosomes are IE1 CTD-dependent. IE1 SUMOylation did not affect IE1 association with chromosomes. Our results provide genetic evidence that the CTD is required for the associations of IE1, PML and STAT2 with mitotic chromosomes, but that these IE1-related activities are not essential for viral replication in fibroblasts.

Human cytomegalovirus infection causes degradation of Sp100 proteins that suppress viral gene expression.

The interferon-inducible Sp100 proteins are thought to play roles in the chromatin pathway and in transcriptional regulation. Sp100A, the smallest isoform, is one of the major components of PML nuclear bodies (NBs) that exhibit intrinsic antiviral activity against several viruses. Since PML NBs are disrupted by the immediate-early 1 (IE1) protein during human cytomegalovirus (HCMV) infection, the modulation of Sp100 protein expression or activity during infection has been suggested. Here, we show that Sp100 proteins are lost largely in the late stages of HCMV infection. This event required viral gene expression and involved posttranscriptional control. The mutant virus with deletion of the sequence for IE1 (CR208) did not have Sp100 loss. In CR208 infection, PML depletion by RNA interference abrogated the accumulation of SUMO-modified Sp100A and of certain high-molecular-weight Sp100 isoforms but did not significantly affect unmodified Sp100A, suggesting that the IE1-induced disruption of PML NBs is not sufficient for the complete loss of Sp100 proteins. Sp100A loss was found to require proteasome activity. Depletion of all Sp100 proteins by RNA silencing enhanced HCMV replication and major IE (MIE) gene expression. Sp100 knockdown enhanced the acetylation level of histones associated with the MIE promoter, demonstrating that the repressive effect of Sp100 proteins may involve, at least in part, the epigenetic control of the MIE promoter. Sp100A was found to interact directly with IE1 through the N-terminal dimerization domain. These findings indicate that the IE1-dependent loss of Sp100 proteins during HCMV infection may represent an important requirement for efficient viral growth.

Analysis of Novel Drug-Resistant Human Cytomegalovirus DNA Polymerase Mutations Reveals the Role of a DNA-Binding Loop in Phosphonoformic Acid Resistance.

The appearance of drug-resistant mutations in UL54 DNA polymerase and UL97 kinase genes is problematic for the treatment of human cytomegalovirus (HCMV) diseases. During treatment of HCMV infection in a pediatric hematopoietic cell transplant recipient, H600L and T700A mutations and E576G mutation were independently found in the UL54 gene. Foscarnet (FOS; phosphonoformic acid) resistance by T700A mutation is reported. Here, we investigated the role of novel mutations in drug resistance by producing recombinant viruses and a model polymerase structure. The H600L mutant virus showed an increase in resistance to ganciclovir (GCV) by 11-fold and to FOS and cidofovir (CDV) by 5-fold, compared to the wild type, while the E756G mutant virus showed an increase in resistance to FOS by 9-fold and modestly to CDV by 2-fold. With the FOS-resistant T700A mutation, only H600L produced increased FOS resistance up to 37-fold, indicating an additive effect of these mutations on FOS resistance. To gain insight into drug resistance mechanisms, a model structure for UL54 polymerase was constructed using the yeast DNA polymerase as a template. In this model, HCMV DNA polymerase contains a long palm loop domain of which H600 and T700 are located on each end and T700 interacts with the FOS binding pocket. Our results demonstrate that H600L and E756G mutations in UL54 polymerase are novel drug-resistant mutations and that the acquisition of both H600L and T700A mutations in the DNA-binding loop confers increased resistance to FOS treatment, providing novel insights for the mechanism acquiring foscarnet resistance.

Role of the specific interaction of UL112-113 p84 with UL44 DNA polymerase processivity factor in promoting DNA replication of human cytomegalovirus.

The human cytomegalovirus (HCMV) UL112-113 region encodes four phosphoproteins with common amino termini (p34, p43, p50, and p84) via alternative splicing and is thought to be required for efficient viral DNA replication. We have previously shown that interactions among the four UL112-113 proteins regulate their intranuclear targeting and enable the recruitment of the UL44 DNA polymerase processivity factor to viral prereplication foci. Here, we show that in virus-infected cells, the UL112-113 proteins form a complex with UL44 and other replication proteins, such as UL84 and IE2. In vitro assays showed that all four phosphoproteins interacted with UL44. Interestingly, p84 required both the shared amino-terminal region and the specific near-carboxy-terminal region for UL44 binding. UL44 required both the carboxy-terminal region and the central region, including the dimerization domain for p84 binding. The production of recombinant virus from mutant Towne bacterial artificial chromosome (BAC) DNA, which encodes intact p34, p43, and p50 and a carboxy-terminally truncated p84 defective in UL44 binding, was severely impaired compared to wild-type BAC DNA. A similar defect was observed when mutant BAC DNA encoded a carboxy-terminally truncated UL44 defective in p84 binding. In cotransfection replication assays using six replication core proteins, UL84, IE2, and UL112-113, the efficient replication of an HCMV oriLyt-containing plasmid required the regions of p84 and UL44 necessary for their interaction. Our data suggest that the UL112-113 proteins form a complex with other replication proteins such as UL44, UL84, and IE2 and that the specific interaction of UL112-113 p84 with UL44 is necessary for efficient viral DNA replication.

Role of noncovalent SUMO binding by the human cytomegalovirus IE2 transactivator in lytic growth.

The 86-kDa immediate-early 2 (IE2) protein of human cytomegalovirus (HCMV) is a promiscuous transactivator essential for viral gene expression. IE2 is covalently modified by SUMO at two lysine residues (K175 and K180) and also interacts noncovalently with SUMO. Although SUMOylation of IE2 has been shown to enhance its transactivation activity, the role of SUMO binding is not clear. Here we showed that SUMO binding by IE2 is necessary for its efficient transactivation function and for viral growth. IE2 bound physically to SUMO-1 through a SUMO-interacting motif (SIM). Mutations in SIM (mSIM) or in both SUMOylation sites and SIM (KR/mSIM), significantly reduced IE2 transactivation effects on viral early promoters. The replication of IE2 SIM mutant viruses (mSIM or KR/mSIM) was severely depressed in normal human fibroblasts. Analysis of viral growth curves revealed that the replication defect of the mSIM virus correlated with low-level accumulation of SUMO-modified IE2 and of viral early and late proteins. Importantly, both the formation of viral transcription domains and the association of IE2 with viral promoters in infected cells were significantly reduced in IE2 SIM mutant virus infection. Furthermore, IE2 was found to interact with the SUMO-modified form of TATA-binding protein (TBP)-associated factor 12 (TAF12), a component of the TFIID complex, in a SIM-dependent manner, and this interaction enhanced the transactivation activity of IE2. Our data demonstrate that the interaction of IE2 with SUMO-modified proteins plays an important role for the progression of the HCMV lytic cycle, and they suggest a novel viral mechanism utilizing the cellular SUMO system.

Development and Characterization of a Reverse Genetics System for a Human-Derived Severe Fever With Thrombocytopenia Syndrome Virus Isolate From South Korea.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, tick-borne Bandavirus that causes lethal disease in humans. As there are no licensed vaccines and therapeutics for SFTSV, there is an urgent need to develop countermeasures against it. In this respect, a reverse genetics (RG) system is a powerful tool to help achieve this goal. Herein, we established a T7 RNA polymerase-driven RG system to rescue infectious clones of a Korean SFTSV human isolate entirely from complementary DNA (cDNA). To establish this system, we cloned cDNAs encoding the three antigenomic segments into transcription vectors, with each segment transcribed under the control of the T7 promoter and the hepatitis delta virus ribozyme (HdvRz) sequences. We also constructed two helper plasmids expressing the nucleoprotein (NP) or viral RNA-dependent RNA polymerase (RdRp) under the control of the T7 promoter and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). After co-transfection into BHK/T7-9 cells with three transcription and two helper plasmids, then passaging in Vero E6 or Huh-7 cells, we confirmed efficient rescue of the recombinant SFTSV. By evaluating the in vitro and in vivo virological properties of the parental and rescued SFTSVs, we show that the rescued virus exhibited biological properties similar to those of the parental virus. This system will be useful for identifying molecular viral determinants of SFTSV infection and pathogenesis and for facilitating the development of vaccine and antiviral approaches.

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.