Min-Hua Luo

Tau Protects Microtubules in the Axon from Severing by Katanin

Microtubules in the axon are more resistant to severing by katanin than microtubules elsewhere in the neuron. We have hypothesized that this is because of the presence of tau on axonal microtubules. When katanin is overexpressed in fibroblasts, the microtubules are severed into short pieces, but this phenomenon is suppressed by the coexpression of tau. Protection against severing is also afforded by microtubule-associated protein 2 (MAP2), which has a tau-like microtubule-binding domain, but not by MAP1b, which has a different microtubule-binding domain. The microtubule-binding domain of tau is required for the protection, but within itself, provides less protection than the entire molecule. When tau (but not MAP2 or MAP1b) is experimentally depleted from neurons, the microtubules in the axon lose their characteristic resistance to katanin. These results, which validate our hypothesis, also suggest a potential explanation for why axonal microtubules deteriorate in neuropathies involving the dissociation of tau from the microtubules.

Human Cytomegalovirus Disrupts both Ataxia Telangiectasia Mutated Protein (ATM)- and ATM-Rad3-Related Kinase-Mediated DNA Damage Responses during Lytic Infection

ABSTRACT Many viruses (herpes simplex virus type 1, polyomavirus, and human immunodeficiency virus type 1) require the activation of ataxia telangiectasia mutated protein (ATM) and/or Mre11 for a fully permissive infection. However, the longer life cycle of human cytomegalovirus (HCMV) may require more specific interactions with the DNA repair machinery to maximize viral replication. A prototypical damage response to the double-stranded ends of the incoming linear viral DNA was not observed in fibroblasts at early times postinfection (p.i.). Apparently, a constant low level of phosphorylated ATM was enough to phosphorylate its downstream targets, p53 and Nbs1. p53 was the only cellular protein observed to relocate at early times, forming foci in infected cell nuclei between 3.5 and 5.5 h p.i. Approximately half of these foci localized with input viral DNA, and all localized with viral UL112/113 prereplication site foci. No other DNA repair proteins localized with the virus or prereplication foci in the first 24 h p.i. When viral replication began in earnest, between 24 and 48 h p.i., there were large increases in steady-state levels and phosphorylation of many proteins involved in the damage response, presumably triggered by ATM-Rad3-related kinase activation. However, a sieving process occurred in which only certain proteins were specifically sequestered into viral replication centers and others were particularly excluded. In contrast to other viruses, activation of a damage response is neither necessary nor detrimental to infection, as neither ATM nor Mre11 was required for full virus replication and production. Thus, by preventing simultaneous relocalization of all the necessary repair components to the replication centers, HCMV subverts full activation and completion of both double-stranded break and S-phase checkpoints that should arrest all replication within the cell and likely lead to apoptosis.

Potential Role for p53 in the Permissive Life Cycle of Human Cytomegalovirus

ABSTRACT Infection of primary fibroblasts with human cytomegalovirus (HCMV) causes a rapid stabilization of the cellular protein p53. p53 is a major effector of the cellular damage response, and activation of this transcription factor can lead either to cell cycle arrest or to apoptosis. Viruses employ many tactics to avoid p53-mediated effects. One method HCMV uses to counteract p53 is sequestration into its viral replication centers. In order to determine whether or not HCMV benefits from this sequestration, we infected a p53−/− fibroblast line. We find that although these cells are permissive for viral infection, several parameters are substantially altered compared to wild-type (wt) fibroblasts. p53−/− cells show delayed and decreased accumulation of infectious viral particles compared to control fibroblasts, with the largest difference of 100-fold at 72 h post infection (p.i.) and peak titers decreased by approximately 10- to 20-fold at 144 h p.i. Viral DNA accumulation is also delayed and somewhat decreased in p53−/− cells; however, on average, levels of DNA are not more than fivefold lower than wt at any time p.i. and thus cannot account entirely for the observed differences in titers. In addition, there are delays in the expression of several key viral proteins, including the early replication protein UL44 and some of the late structural proteins, pp28 (UL99) and MCP (UL86). UL44 localization also indicates delayed formation and maturation of the replication centers throughout the course of infection. Localization of the major tegument protein pp65 (UL83) is also altered in these p53−/− cells. Partial reconstitution of the p53−/− cells with a wt copy of p53 returns all parameters toward wt, while reconstitution with mutant p53 does not. Taken together, our data suggest that wt p53 enhances the ability of HCMV to replicate and produce high concentrations of infectious virions in permissive cells.

Neonatal Neural Progenitor Cells and Their Neuronal and Glial Cell Derivatives Are Fully Permissive for Human Cytomegalovirus Infection

ABSTRACT Congenital human cytomegalovirus (HCMV) infection causes central nervous system structural abnormalities and functional disorders, affecting both astroglia and neurons with a pathogenesis that is only marginally understood. To better understand HCMVs interactions with such clinically important cell types, we utilized neural progenitor cells (NPCs) derived from neonatal autopsy tissue, which can be differentiated down either glial or neuronal pathways. Studies were performed using two viral isolates, Towne (laboratory adapted) and TR (a clinical strain), at a multiplicity of infection of 3. NPCs were fully permissive for both strains, expressing the full range of viral antigens (Ags) and producing relatively large numbers of infectious virions. NPCs infected with TR showed delayed development of cytopathic effects (CPE) and replication centers and shed less virus. This pattern of delay for TR infections held true for all cell types tested. Differentiation of NPCs was carried out for 21 days to obtain either astroglia (>95% GFAP+) or a 1:5 mixed neuron/astroglia population (β-tubulin III+/GFAP+). We found that both of these differentiated populations were fully permissive for HCMV infection and produced substantial numbers of infectious virions. Utilizing a difference in plating efficiencies, we were able to enrich the neuron population to ∼80% β-tubulin III+ cells. These β-tubulin III+-enriched populations remained fully permissive for infection but were very slow to develop CPE. These infected enriched neurons survived longer than either NPCs or astroglia, and a small proportion were alive until at least 14 days postinfection. These surviving cells were all β-tubulin III+ and showed viral Ag expression. Surprisingly, some cells still exhibited extended processes, similar to mock-infected neurons. Our findings strongly suggest neurons as reservoirs for HCMV within the developing brain.

Human Cytomegalovirus Infection Causes Premature and Abnormal Differentiation of Human Neural Progenitor Cells

ABSTRACT Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, largely manifested as central nervous system (CNS) disorders. The principal site of manifestations in the mouse model is the fetal brains neural progenitor cell (NPC)-rich subventricular zone. Our previous human NPC studies found these cells to be fully permissive for HCMV and a useful in vitro model system. In continuing work, we observed that under culture conditions favoring maintenance of multipotency, infection caused NPCs to quickly and abnormally differentiate. This phenotypic change required active viral transcription. Whole-genome expression analysis found rapid downregulation of genes that maintain multipotency and establish NPCs’ neural identity. Quantitative PCR, Western blot, and immunofluorescence assays confirmed that the mRNA and protein levels of four hallmark NPC proteins (nestin, doublecortin, sex-determining homeobox 2, and glial fibrillary acidic protein) were decreased by HCMV infection. The decreases required active viral replication and were due, at least in part, to proteasomal degradation. Our results suggest that HCMV infection causes in utero CNS defects by inducing both premature and abnormal differentiation of NPCs.

Long-Term Infection and Shedding of Human Cytomegalovirus in T98G Glioblastoma Cells

ABSTRACT Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, affecting primarily the central nervous system (CNS). To further understand this CNS pathology, cells from glioblastoma cell lines T98G and A172, the astrocytic glioblastoma cell line CCF-STTG1 (CCF), and the neuroblastoma cell line SH-SY5Y (SY5Y) were infected with HCMV. CCF and SY5Y cells were fully permissive for infection, while A172 cells were nonpermissive. In T98G cells, the majority of cells showed viral deposition into the nucleus by 6 h postinfection (hpi); however, viral immediate-early gene expression was observed in only ∼30% of cells in the first 72 h. In viral antigen (Ag)-positive cells, although the development of complete viral replication centers was delayed, fully developed centers formed by 96 hpi. Interestingly, even at very late times postinfection, a mixture of multiple small, bipolar, and large foci was always present. The initial trafficking of input pp65 into the nucleus was also delayed. Titer and infectious-center assays showed a small number of T98G cells shedding virus at very low levels. Surprisingly, both Ag-positive and Ag-negative cells continued to divide; because of this continuous division, we adopted a protocol for passaging the T98G cells every third day to prevent overcrowding. Under this protocol, detectable infectious-virus shedding continued until passage 5 and viral gene expression continued through eight passages. This evidence points to T98G cells as a promising model for long-term infections.

Novel isoforms of tau that lack the microtubule‐binding domain

Tau is a microtubule‐associated protein (MAP) whose transcript undergoes complex regulated splicing in the mammalian nervous system. Our previous work with exon 6 established that tau shows a unique expression pattern and splicing regulation profile, and that it utilizes alternative splice sites in several human tissues. The mRNAs from these splicing events, if translated, would result in truncated tau variants that lack the microtubule‐binding domain. In this study, we demonstrate that at least one of these tau variants is present as a stable protein in several tissues. The novel isoform shows a localization distinct from that of canonical tau in SH‐SY5Y neuroblastoma cells which stably overexpress it. In both normal and Alzheimers hippocampus, the novel isoform is found in dentate gyrus granular cells and CA1/CA3 pyramidal cells. However, it does not co‐localize with canonical tau but, rather, partly co‐localizes with MAP2.

MicroRNA miR-21 Attenuates Human Cytomegalovirus Replication in Neural Cells by Targeting Cdc25a

ABSTRACT Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs) and induces cell cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing the levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products—IE1, pp71, and UL26—were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS. IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.

Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection

Background Human cytomegalovirus (HCMV) encodes microRNAs (miRNAs) that function as post-transcriptional regulators of gene expression during lytic infection in permissive cells. Some miRNAs have been shown to suppress virus replication, which could help HCMV to establish or maintain latent infection. However, HCMV miRNA expression has not been comprehensively examined and compared using cell culture systems representing permissive (lytic) and semi-permissive vs. non-permissive (latent-like) infection. Methods Viral miRNAs levels and expression kinetics during HCMV infection were determined by miRNA-specific stem-loop RT-PCR. HCMV infected THP-1 (non-permissive), differentiated THP-1 (d-THP-1, semi-permissive) and human embryo lung fibroblasts (HELs, fully-permissive) were examined. The impact of selected miRNAs on HCMV infection (gene expression, genome replication and virus release) was determined by Western blotting, RT-PCR, qPCR, and plaque assay. Results Abundant expression of 15 HCMV miRNAs was observed during lytic infection in HELs; highest peak inductions (11- to 1502-fold) occurred at 48 hpi. In d-THP-1s, fourteen mRNAs were detected with moderate induction (3- to 288-fold), but kinetics of expression was generally delayed for 24 h relative to HELs. In contrast, only three miRNAs were induced to low levels (3- to 4-fold) during quiescent infection in THP-1s. Interestingly, miR-UL70-3p was poorly induced in HEL (1.5-fold), moderately in THP-1s (4-fold), and strongly (58-fold) in d-THP-1s, suggesting a potentially specific role for miR-UL70-3p in THP-1s and d-THP-1s. MiR-US33, -UL22A and -UL70 were further evaluated for their impact on HCMV replication in HELs. Ectopic expression of miR-UL22A and miR-UL70 did not affect HCMV replication in HELs, whereas miR-US33 inhibited HCMV replication and reduced levels of HCMV US29 mRNA, confirming that US29 is a target of miR-US33. Conclusions Viral miRNA expression kinetics differs between permissive, semi-permissive and quiescent infections, and miR-US33 down-regulates HCMV replication. These results suggest that miR-US33 may function to impair entry into lytic replication and hence promote establishment of latency.

A Novel Bat Herpesvirus Encodes Homologues of Major Histocompatibility Complex Classes I and II, C-Type Lectin, and a Unique Family of Immune-Related Genes

ABSTRACT Herpesviruses or herpesviral sequences have been identified in various bat species. Here, we report the isolation, cell tropism, and complete genome sequence of a novel betaherpesvirus from the bat Miniopterus schreibersii (MsHV). In primary cell culture, MsHV causes cytopathic effects (CPE) and reaches peak virus production 2 weeks after infection. MsHV was found to infect and replicate less efficiently in a feline kidney cell, CRFK, and failed to replicate in 13 other cell lines tested. Sequencing of the MsHV genome using the 454 system, with a 224-fold coverage, revealed a genome size of 222,870 bp. The genome was extensively analyzed in comparison to those of related viruses. Of the 190 predicted open reading frames (ORFs), 40 were identified as herpesvirus core genes. Among 93 proteins with identifiable homologues in tree shrew herpesvirus (THV), human cytomegalovirus (HCMV), or rat cytomegalovirus (RCMV), most had highest sequence identities with THV counterparts. However, the MsHV genome organization is colinear with that of RCMV rather than that of THV. The following unique features were discovered in the MsHV genome. One predicted protein, B125, is similar to human herpesvirus 6 (HHV-6) U94, a homologue of the parvovirus Rep protein. For the unique ORFs, 7 are predicted to encode major histocompatibility complex (MHC)-related proteins, 2 to encode MHC class I homologues, and 3 to encode MHC class II homologues; 4 encode the homologues of C-type lectin- or natural killer cell lectin-like receptors;, and the products of a unique gene family, the b149 family, of 16 members, have no significant sequence identity with known proteins but exhibit immunoglobulin-like beta-sandwich domains revealed by three-dimensional (3D) structural prediction. To our knowledge, MsHV is the first virus genome known to encode MHC class II homologues.