Scott S. Terhune

Human Cytomegalovirus Protein UL38 Inhibits Host Cell Stress Responses by Antagonizing the Tuberous Sclerosis Protein Complex

Human cytomegalovirus proteins alter host cells to favor virus replication. These viral proteins include pUL38, which prevents apoptosis. To characterize the mode of action of pUL38, we modified the viral genome to encode an epitope-tagged pUL38 and used rapid immunoaffinity purification to isolate pUL38-interacting host proteins, which were then identified by mass spectrometry. One of the cellular proteins identified was TSC2, a constituent of the tuberous sclerosis tumor suppressor protein complex (TSC1/2). TSC1/2 integrates stress signals and regulates the mammalian target of rapamycin complex 1 (mTORC1), a protein complex that responds to stress by limiting protein synthesis and cell growth. We showed that pUL38 interacts with TSC1 and TSC2 in cells infected with wild-type cytomegalovirus. Furthermore, TSC1/2 failed to regulate mTORC1 in cells expressing pUL38, and these cells exhibited the enlarged size characteristic of cytomegalovirus infection. Thus, pUL38 supports virus replication at least in part by blocking cellular responses to stress.

Human Cytomegalovirus UL38 Protein Blocks Apoptosis

ABSTRACT Apoptosis is an innate cellular defense response to viral infection. The slow-replicating human cytomegalovirus (HCMV) blocks premature death of host cells prior to completion of the infection cycle. In this study, we report that the HCMV UL38 gene encodes a cell death inhibitory protein. A mutant virus lacking the pUL38 coding sequence, ADdlUL38, grew poorly in human fibroblasts, failed to accumulate viral DNA to wild-type levels, and induced excessive death of infected cells. Cells expressing pUL38 were resistant to cell death upon infection and effectively supported the growth of ADdlUL38. Cells infected with the pUL38-deficient virus showed morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, vesicle release, and chromatin condensation and fragmentation. The proteolytic cleavage of two key enzymes involved in apoptosis, namely, caspase 3 and poly(ADP-ribose) polymerase, was activated upon ADdlUL38 infection, and the cleavage was blocked in cells expressing pUL38. The pan-caspase inhibitor Z-VAD-FMK largely restored the growth of ADdlUL38 in normal fibroblasts, indicating that the defective growth of the mutant virus mainly resulted from premature death of host cells. Furthermore, cells expressing pUL38 were resistant to cell death induced by a mutant adenovirus lacking the antiapoptotic E1B-19K protein or by thapsigargin, which disrupts calcium homeostasis in the endoplasmic reticulum. Taken together, these results indicate that the HCMV protein pUL38 suppresses apoptosis, blocking premature death of host cells to facilitate efficient virus replication.

Human Cytomegalovirus pUL83 Stimulates Activity of the Viral Immediate-Early Promoter through Its Interaction with the Cellular IFI16 Protein

ABSTRACT The human cytomegalovirus (HCMV) virion protein pUL83 (also termed pp65) inhibits the expression of interferon-inducible cellular genes. In this work we demonstrate that pUL83 is also important for efficient induction of transcription from the viral major immediate-early promoter. Infection with a mutant virus containing a premature translation termination codon in the UL83 open reading frame (ORF) (UL83Stop) resulted in decreased transcription from the major immediate-early promoter in a time- and multiplicity-dependent manner. Expression of pUL83 alone is capable of transactivating the promoter in a reporter assay, and pUL83 associates with the promoter in infected cells. To investigate the mechanism by which the protein regulates the major immediate-early promoter, we utilized a mutant virus expressing an epitope-tagged pUL83 from its own promoter to identify protein binding partners for pUL83 during infection. We identified and confirmed the interaction of pUL83 with cellular IFI16 family members throughout the course of HCMV infection. pUL83 recruits IFI16 to the major immediate-early promoter, and IFI16 binding at the promoter is dependent upon the presence of pUL83. Consistent with the results obtained with the UL83Stop virus, infection of IFI16 knockdown cells with wild-type virus resulted in decreased levels of immediate-early transcripts compared to those of control cells. These data identify a previously unknown role for pUL83 in the initiation of the human cytomegalovirus gene expression cascade.

RNAs Are Packaged into Human Cytomegalovirus Virions in Proportion to Their Intracellular Concentration

ABSTRACT The assembly of human cytomegalovirus (HCMV) virions is a complex process and involves the incorporation of viral transcripts. These RNAs are delivered to the newly infected cells and have the potential to be translated in the absence of HCMV gene expression. We have quantified the relative amount of RNAs in HCMV virions and infected cells with real-time reverse transcription-PCR and observed that viral and cellular RNAs are packaged in proportion to the amount of RNA within the cell at the time of assembly. To determine whether cis elements influenced RNA packaging, we constructed a recombinant HCMV mutant virus that expressed the yellow fluorescence protein (YFP) gene fused to the virion RNA UL21.5. We also constructed a mutant virus in which the UL21.5 transcription unit was replaced with the YFP gene. YFP RNA was incorporated into both viruses, indicating that RNA is incorporated in the absence of a virus-specific signal motif. Furthermore, with in situ hybridization, packaged transcripts were observed throughout the cytoplasm of the infected cells, including the site of virus assembly. Several proteins that nonspecifically interact with RNA, including the tegument protein pp28, were found within HCMV virions. These studies demonstrate that both viral and cellular RNAs are nonspecifically incorporated into HCMV, potentially through interactions with several virion proteins.

Cellular Changes Induced by Low-Risk Human Papillomavirus Type 11 in Keratinocytes That Stably Maintain Viral Episomes

ABSTRACT Infections by low-risk papillomavirus types, such as human papillomavirus (HPV) type 6 (HPV-6) and HPV-11, induce benign genital warts that rarely progress to malignancy. In contrast, lesions induced by high-risk HPV types have the potential to progress to cancer. Considerable information is available concerning the pathogenesis of high-risk HPV types, but little is known about the life cycle of low-risk HPV types. Although functionally distinct, both high- and low-risk virus types infect keratinocytes and induce virion production upon differentiation. This information suggests that they may share common mechanisms for regulating their productive life cycles. Using tissue culture methods developed to study high-risk HPV types, we examined the ability of HPV-11 to be stably maintained as episomes following transfection of normal human keratinocytes with cloned viral DNA. HPV-11 genomes were found to be maintained in keratinocytes for extended passages in cultures in 14 independent experiments involving transfection of cloned HPV-11 DNA. Interestingly, the HPV-11-positive cells exhibited an extended life span that averaged approximately twofold longer than that of control neomycin-transfected cells. In organotypic cultures, HPV-11-positive cells exhibited altered differentiation patterns, but the extent of disruption was less severe than that seen with high-risk HPV types. In addition, the amplification of HPV-11 DNA, as well as the induction of several viral messages, was observed following differentiation of transfected cells in semisolid media. To determine whether global changes in cellular gene expression induced by HPV-11 were similar to those observed with high-risk HPV-31 (Y. E. Chang and L. A. Laimins, J. Virol. 74:4174–4182, 2000), microarray analysis of 7,075 expressed sequences was performed. A spectrum of cellular genes different from that previously reported for HPV-31 was found to be activated or repressed by HPV-11. The expression of only a small set of genes was similarly altered by both high- and low-risk HPV types. This result suggests that different classes of HPVs have distinct effects on global cellular transcription patterns during infection. The methods described allow for a genetic analysis of HPV-11 in the context of its differentiation-dependent life cycle.

Ubiquitin and Ubiquitin-Modified Proteins Activate the Pseudomonas aeruginosa T3SS Cytotoxin, ExoU

Pseudomonas aeruginosa is an opportunistic Gram‐negative pathogen that possesses a type III secretion system (T3SS) critical for evading innate immunity and establishing acute infections in compromised patients. Our research has focused on the structure–activity relationships of ExoU, the most toxic and destructive type III effector produced by P. aeruginosa. ExoU possesses phospholipase activity, which is detectable in vitro only when a eukaryotic cofactor is provided with membrane substrates. We report here that a subpopulation of ubiquitylated yeast SOD1 and other ubiquitylated mammalian proteins activate ExoU. Phospholipase activity was detected using purified ubiquitin of various chain lengths and linkage types; however, free monoubiquitin is sufficient in a genetically engineered dual expression system. The use of ubiquitin by a bacterial enzyme as an activator is unprecedented and represents a new aspect in the manipulation of the eukaryotic ubiquitin system to facilitate bacterial replication and dissemination.

Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs.

Antiviral Inhibition Targeting the HCMV Kinase pUL97 Requires pUL27-Dependent Degradation of Tip60 Acetyltransferase and Cell-Cycle Arrest

Infection with the β-herpesvirus human cytomegalovirus (HCMV) is lifelong, causing limited disease in healthy adults, but life threatening in immunocompromised individuals. The viral kinase pUL97, a functional ortholog of cellular cyclin-dependent kinases (CDKs), is critical for HCMV replication and a target for antiviral drug development. Upon kinase inhibition, drug-resistant strains emerge with mutations in UL27, an HCMV gene of unknown function. Using a proteomics approach, we discovered that pUL27 is necessary and sufficient to degrade Tip60, a host acetyltransferase and interacting partner of HIV Tat. Consistent with this, the expression of Tat restored antiviral inhibition of an otherwise resistant HCMV strain. The functional consequence of Tip60 degradation was the induction of the CDK inhibitor p21(Waf1/Cip1) and cell-cycle arrest, representing changes necessary for the antiviral effects of pUL97 inhibition. Consequently, either increasing p21(Waf1/Cip1) expression or decreasing Tip60 levels improved the antiviral activity of the HCMV kinase inhibitor maribavir.

Limited Variability of Glycoprotein Gene Sequences and Neutralizing Targets in Herpes Simplex Virus Type 2 Isolates and Stability on Passage in Cell Culture

Nucleotide sequence analyses of polymerase chain reaction-amplified genes were performed to determine whether adaptation of herpes simplex virus type 2 to replication in cultured cells or in internal organs during neonatal disseminated disease results in selection of variants with altered forms of three glycoproteins (gB, gC, or gD) that influence virus entry into cells. No variations in sequence were noted as a consequence of in vitro passage or replication in different organs. Five viruses from different subjects differed with respect to gB, gC, and gD gene sequences, expressing four distinct forms of gB, three of gC, and two of gD. These differences did not confer resistance to neutralization by guinea pig or human antisera from subjects immunized with recombinant gB or gD vaccines and may not be consequential for vaccine development.

Early Polyadenylation Signals of Human Papillomavirus Type 31 Negatively Regulate Capsid Gene Expression

ABSTRACT The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed upon keratinocyte differentiation from a promoter located in the E7 open reading frame (ORF) of the early region. Late transcripts must therefore pass through and ignore the early polyadenylation sequences to use the downstream late AAUAAA element located at the end of the L1 ORF. To identify sequences which modulate downstream capsid gene expression, a variety of substitution mutations were introduced into the early polyadenylation signal and studied first in the context of polycistronic luciferase reporter constructs. Removal of the G/U-rich cleavage stimulation factor (CstF) binding sites and the degenerate cleavage and polyadenylation specificity factor binding sites, UAUAUA, had minimal effect on downstream expression as defined by luciferase activities. This is in contrast to the deletion of the HPV-31 early AAUAAA element, which resulted in a dramatic increase in downstream expression. Additional sequences within the first 800 bp of the L2 ORF were also found to negatively regulate capsid expression in luciferase assays. To determine how these mutations influence gene expression in the context of the complete HPV-31 genome, recombinant genomes were constructed that contained a substitution in the AAUAAA sequence, an inserted strong CstF binding site, an inserted simian virus 40 (SV40) late poly(A) signal, or a substitution of the 5′-most 800 nucleotides of the L2 ORF. Reductions in both transient and stable replication were observed with the recombinant genomes containing the strong CstF site or the late SV40 signal, suggesting that alterations in the strength of the upstream poly(A) signal influence expression of viral replication factors. Similarly, disruption of the L2 ORF resulted in a significant reduction in genome replication and an inability to be maintained stably. In contrast, genomes containing a substitution of the AAUAAA sequence had increased levels of transient and stable replication. Quantitation of late transcripts following keratinocyte differentiation in methylcellulose also showed a reduction in downstream capsid gene expression in lines containing genomes with the strong CstF site or the late SV40 signal mutations, while a significant increase in expression was detected in the lines with genomes lacking the AAUAAA sequence. These studies demonstrate that capsid gene expression in HPV-31 requires an inefficient early poly(A) signal which is defined primarily by the AAUAAA element as well as a major negative regulatory element located within the L2 ORF.