ABSTRACT
Background and Objectives: Herpes simplex keratitis (HSK) is the leading infectious cause of corneal damage and associated loss of visual acuity. Because of its frequent recurrence, it represents a major health problem; thus, timely and accurate diagnosis is the key to successful treatment. To enable this, we aimed to determine HSK patients' demographic and clinical features. Materials and Methods: This prospective study included 55 patients diagnosed with HSK between March 2019 and August 2022 at the Department of Ophthalmology, Clinical Hospital Rijeka. Results: We found that HSK is most prevalent in the elderly, with 72.73% of patients older than 60. The most common HSK types were dendritic (HSK-D; 43.64%) and stromal with epithelial ulceration (HSK-SEU 23.64%). HSK recurrences occurred in 65.45% of patients, with most having two to five recurrences (55.56%). Visual acuity at presentation (65.5%) and after treatment (50.9%) was mostly in the 20/50 range. The longest period until the disease symptoms were resolved was in the group with stromal HSK without epithelial ulceration (HSK-SnEU), for which symptoms lasted more than 11 weeks in 87.5% of patients. The overall incidence of HSK-related complications was high (85.45%), with 76.4% of patients having corneal scarring. The average time from symptom to treatment was 15.78 days. Interestingly, we observed a strong seasonality in the incidence of HSK, which was most prevalent in the colder months, with 63.6% of cases occurring between October and March. Conclusions: To the best of our knowledge, this is the first prospective study in Croatia, and one of the few in Europe, to describe the demographic and clinical features of HSK patients. We found that HSK is most common in the elderly population, with its dendritic form as a clinical presentation. We have shown that HSK is prone to recurrence and secondary complications, with a worryingly long time between symptom and treatment, indicating the need for diagnostic testing in routine practice.
Subject(s)
Keratitis, Herpetic , Humans , Keratitis, Herpetic/epidemiology , Keratitis, Herpetic/diagnosis , Female , Male , Middle Aged , Aged , Prospective Studies , Adult , Visual Acuity , Aged, 80 and over , Recurrence , Tertiary Care Centers/statistics & numerical data , DemographyABSTRACT
Many potential immune therapeutic targets are similarly affected in adult-onset neurodegenerative diseases, such as Alzheimer's (AD) disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD), as well as in a seemingly distinct Niemann-Pick type C disease with primarily juvenile onset. This strongly argues for an overlap in pathogenic mechanisms. The commonly researched immune targets include various immune cell subsets, such as microglia, peripheral macrophages, and regulatory T cells (Tregs); the complement system; and other soluble factors. In this review, we compare these neurodegenerative diseases from a clinical point of view and highlight common pathways and mechanisms of protein aggregation, neurodegeneration, and/or neuroinflammation that could potentially lead to shared treatment strategies for overlapping immune dysfunctions in these diseases. These approaches include but are not limited to immunisation, complement cascade blockade, microbiome regulation, inhibition of signal transduction, Treg boosting, and stem cell transplantation.
ABSTRACT
The Adenosine Deaminases Acting on RNA (ADAR) catalyze the posttranscriptional deamination of adenosine residues to inosine in double-stranded RNAs (dsRNAs, A-to-I editing), preventing the overactivation of dsRNA sensor molecules and interferons. RNA editing is the cornerstone of innate immunity that distinguishes between self and non-self (virus), and it is essential for normal regulation of cellular homeostasis. Although much is already known about the role of ADAR proteins in RNA virus infection, the role of ADAR proteins in herpesvirus infection remains largely unexplored. In this review, we provide several lines of evidence from studies of different herpesviruses for another level of complexity in regulating the already intricate biphasic life cycle of herpesviruses.
Subject(s)
Adenosine Deaminase , Herpesviridae Infections , Humans , Adenosine Deaminase/metabolism , RNA Editing , RNA, Double-Stranded , Adenosine/metabolismABSTRACT
IMPORTANCE: Herpes simplex virus 1 is an important human pathogen that has been intensively studied for many decades. Nevertheless, the molecular mechanisms regulating its establishment, maintenance, and reactivation from latency are poorly understood. Here, we show that HSV-1-encoded miR-H2 is post-transcriptionally edited in latently infected human tissues. Hyperediting of viral miRNAs increases the targeting potential of these miRNAs and may play an important role in regulating latency. We show that the edited miR-H2 can target ICP4, an essential viral protein. Interestingly, we found no evidence of hyperediting of its homolog, miR-H2, which is expressed by the closely related virus HSV-2. The discovery of post-translational modifications of viral miRNA in the latency phase suggests that these processes may also be important for other non-coding viral RNA in the latency phase, including the intron LAT, which in turn may be crucial for understanding the biology of this virus.
Subject(s)
Herpes Simplex , Herpesvirus 1, Human , MicroRNAs , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Herpesvirus 1, Human/physiology , Virus Latency/genetics , Viral Proteins/metabolism , Ganglia/metabolism , Trigeminal Ganglion , Virus Activation/geneticsABSTRACT
Photodynamic therapy (PDT) is broadly used to treat different tumors, and it is a rapidly developing approach to inactivating or inhibiting the replication of fungi, bacteria, and viruses. Herpes simplex virus 1 (HSV-1) is an important human pathogen and a frequently used model to study the effects of PDT on enveloped viruses. Although many photosensitizers (PSs) have been tested for their antiviral properties, analyses are usually limited to assessing the reduction in viral yield, and thus the molecular mechanisms of photodynamic inactivation (PDI) remain poorly understood. In this study, we investigated the antiviral properties of TMPyP3-C17H35, a tricationic amphiphilic porphyrin-based PS with a long alkyl chain. We show that light-activated TMPyP3-C17H35 can efficiently block virus replication at certain nM concentrations without exerting obvious cytotoxicity. Moreover, we show that the levels of viral proteins (immediate-early, early, and late genes) were greatly reduced in cells treated with subtoxic concentrations of TMPyP3-C17H35, resulting in markedly decreased viral replication. Interestingly, we observed a strong inhibitory effect of TMPyP3-C17H35 on the virus yield only when cells were treated before or shortly after infection. In addition to the antiviral activity of the internalized compound, we show that the compound dramatically reduces the infectivity of free virus in the supernatant. Overall, our results demonstrate that activated TMPyP3-C17H35 effectively inhibits HSV-1 replication and that it can be further developed as a potential novel treatment and used as a model to study photodynamic antimicrobial chemotherapy.
ABSTRACT
Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus.
Subject(s)
Herpes Simplex , Herpesvirus 1, Human , MicroRNAs , Herpes Simplex/genetics , Herpesvirus 1, Human/physiology , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Up-Regulation , Virus ReplicationABSTRACT
We developed a next-generation SARS-CoV-2 sequencing platform and obtained the first SARS-CoV-2 sequences from patients in Croatia at the beginning of the COVID-19 outbreak in the spring of 2020. Integrating the sequencing and the epidemiological data, we show that patients were infected with different SARS-CoV-2 variants belonging to different clades (mostly G and GH). This result confirms that there was widespread virus transmission early in 2020. Interestingly, we identified a unique mutation resulting in a V13I substitution in Nsp5A, the main viral protease, in a patient who had not received antiviral therapy.
Subject(s)
COVID-19/epidemiology , COVID-19/virology , Genetic Variation , SARS-CoV-2/genetics , Coronavirus 3C Proteases/chemistry , Croatia/epidemiology , Genome, Viral , Humans , Models, Molecular , Phylogeny , Protein Conformation , Whole Genome SequencingABSTRACT
Since the original publication of sRNAtoolbox in 2015, small RNA research experienced notable advances in different directions. New protocols for small RNA sequencing have become available to address important issues such as adapter ligation bias, PCR amplification artefacts or to include internal controls such as spike-in sequences. New microRNA reference databases were developed with different foci, either prioritizing accuracy (low number of false positives) or completeness (low number of false negatives). Additionally, other small RNA molecules as well as microRNA sequence and length variants (isomiRs) have continued to gain importance. Finally, the number of microRNA sequencing studies deposited in GEO nearly triplicated from 2014 (280) to 2018 (764). These developments imply that fast and easy-to-use tools for expression profiling and subsequent downstream analysis of miRNA-seq data are essential to many researchers. Key features in this sRNAtoolbox release include addition of all major RNA library preparation protocols to sRNAbench and improvements in sRNAde, a tool that summarizes several aspects of small RNA sequencing studies including the detection of consensus differential expression. A special emphasis was put on the user-friendliness of the tools, for instance sRNAbench now supports parallel launching of several jobs to improve reproducibility and user time efficiency.
Subject(s)
MicroRNAs/chemistry , MicroRNAs/metabolism , Software , Gene Expression Profiling , Genetic Variation , Sequence Analysis, RNAABSTRACT
Herpes simplex virus 1 (HSV-1) switches between two infection programs, productive ("lytic") and latent infection. Some HSV-1 microRNAs (miRNAs) have been hypothesized to help control this switch, and yet little is known about regulation of their expression. Using Northern blot analyses, we found that, despite inherent differences in biogenesis efficiency among six HSV-1 miRNAs, all six exhibited high pre-miRNA/miRNA ratios during lytic infection of different cell lines and, when detectable, in acutely infected mouse trigeminal ganglia. In contrast, considerably lower ratios were observed in latently infected ganglia and in cells transduced with lentiviral vectors expressing the miRNAs, suggesting that HSV-1 lytic infection blocks miRNA biogenesis. This phenomenon is not specific to viral miRNAs, as a host miRNA expressed from recombinant HSV-1 also exhibited high pre-miRNA/miRNA ratios late during lytic infection. The levels of most of the mature miRNAs remained stable during infection in the presence of actinomycin D, indicating that the high ratios are due to inefficient pre-miRNA conversion to miRNA. Cellular fractionation experiments showed that late (but not early) during infection, pre-miRNAs were enriched in the nucleus and depleted in the cytoplasm, indicating that nuclear export was blocked. A mutation eliminating ICP27 expression or addition of acyclovir reduced pre-miRNA/miRNA ratios, but mutations drastically reducing Us11 expression did not. Thus, HSV-1 lytic infection inhibits miRNA biogenesis at the step of nuclear export and does so in an ICP27- and viral DNA synthesis-dependent manner. This mechanism may benefit the virus by reducing expression of repressive miRNAs during lytic infection while permitting elevated expression during latency.IMPORTANCE Various mechanisms have been identified by which viruses target host small RNA biogenesis pathways to achieve optimal infection outcomes. Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen whose successful persistence in the host entails both productive ("lytic") and latent infection. Although many HSV-1 miRNAs have been discovered and some are thought to help control the lytic/latent switch, little is known about regulation of their biogenesis. By characterizing expression of both pre-miRNAs and mature miRNAs under various conditions, this study revealed striking differences in miRNA biogenesis between lytic and latent infection and uncovered a regulatory mechanism that blocks pre-miRNA nuclear export and is dependent on viral protein ICP27 and viral DNA synthesis. This mechanism represents a new virus-host interaction that could limit the repressive effects of HSV-1 miRNAs hypothesized to promote latency and may shed light on the regulation of miRNA nuclear export, which has been relatively unexplored.
Subject(s)
Active Transport, Cell Nucleus , Herpes Simplex/virology , Herpesvirus 1, Human/growth & development , Host-Pathogen Interactions , MicroRNAs/metabolism , RNA Precursors/metabolism , Animals , Blotting, Northern , Cell Line , Disease Models, Animal , Gene Expression Regulation , Herpes Simplex/pathology , Humans , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism , Mice , Mutation , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Trigeminal Ganglion/pathology , Trigeminal Ganglion/virology , Viral Proteins/genetics , Viral Proteins/metabolism , Virus LatencyABSTRACT
Viruses utilize microRNAs (miRNAs) in a vast variety of possible interactions and mechanisms, apparently far beyond the classical understanding of gene repression in humans. Likewise, herpes simplex virus 1 (HSV-1) expresses numerous miRNAs and deregulates the expression of host miRNAs. Several HSV-1 miRNAs are abundantly expressed in latency, some of which are encoded antisense to transcripts of important productive infection genes, indicating their roles in repressing the productive cycle and/or in maintenance/reactivation from latency. In addition, HSV-1 also exploits host miRNAs to advance its replication or repress its genes to facilitate latency. Here, we discuss what is known about the functional interplay between HSV-1 and the host miRNA machinery, potential targets, and the molecular mechanisms leading to an efficient virus replication and spread.
ABSTRACT
Human herpes simplex virus 1 (HSV-1) expresses numerous miRNAs, the function of which is not well understood. Several qualitative and quantitative analyses of HSV-1 miRNAs have been performed on infected cells in culture and animal models, however, there is very limited knowledge of their expression in human samples. We sequenced small-RNA libraries of RNA derived from human trigeminal ganglia latently infected with HSV-1 and Varicella zoster virus (VZV) and detected only a small subset of HSV-1 miRNA. The most abundantly expressed miRNAs are miR-H2, miRNA that regulates the expression of immediate early gene ICP0, and miR-H3 and -H4, both miRNAs expressed antisense to the transcript encoding the major neurovirulence factor ICP34.5. The sequence of many HSV-1 miRNAs detected in human samples was different from the sequences deposited in miRBase, which might significantly affect targeted functional analyses.
Subject(s)
Genetic Variation , Herpes Simplex/virology , Herpesvirus 1, Human/genetics , MicroRNAs/genetics , RNA, Viral/genetics , Trigeminal Ganglion/virology , Virus Latency , Herpesvirus 3, Human/genetics , Humans , Sequence Analysis, DNAABSTRACT
Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors.
Subject(s)
Herpesvirus 1, Human/enzymology , Host-Pathogen Interactions , MicroRNAs/genetics , Ubiquitin-Protein Ligases/metabolism , Zinc Finger E-box-Binding Homeobox 1/metabolism , Cell Nucleus , Cells, Cultured , Fibroblasts/virology , Gene Expression Regulation , Herpesvirus 1, Human/genetics , Humans , Immediate-Early Proteins/deficiency , Immediate-Early Proteins/genetics , Nerve Tissue Proteins/genetics , Neurons/virology , Protein Binding , Proteolysis , RNA-Binding Proteins/genetics , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , Virus Replication , Zinc Finger E-box-Binding Homeobox 1/geneticsABSTRACT
Photodynamic therapy (PDT) combines a photosensitiser, light and molecular oxygen to induce oxidative stress that can be used to kill pathogens, cancer cells and other highly proliferative cells. There is a growing number of clinically approved photosensitisers and applications of PDT, whose main advantages include the possibility of selective targeting, localised action and stimulation of the immune responses. Further improvements and broader use of PDT could be accomplished by designing new photosensitisers with increased selectivity and bioavailability. Porphyrin-based photosensitisers with amphiphilic properties, bearing one or more positive charges, are an effective tool in PDT against cancers, microbial infections and, most recently, autoimmune skin disorders. The aim of the review is to present some of the recent examples of the applications and research that employ this specific group of photosensitisers. Furthermore, we will highlight the link between their structural characteristics and PDT efficiency, which will be helpful as guidelines for rational design and evaluation of new PSs.
ABSTRACT
To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.
Subject(s)
Herpes Simplex/metabolism , Herpesvirus 1, Human/genetics , MicroRNAs/metabolism , Virus Latency/genetics , Animals , Cell Culture Techniques , Fibroblasts/metabolism , Herpes Simplex/genetics , Herpesvirus 1, Human/metabolism , High-Throughput Nucleotide Sequencing , Humans , Mice , Neurons/metabolism , RatsABSTRACT
Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3' untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection.
Subject(s)
DNA Helicases/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Simplexvirus/immunology , Simplexvirus/pathogenicity , 3' Untranslated Regions , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , DNA Helicases/genetics , DNA Helicases/immunology , Down Syndrome , HEK293 Cells , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/immunology , Herpesvirus 1, Human/pathogenicity , Herpesvirus 1, Human/physiology , Herpesvirus 2, Human/genetics , Herpesvirus 2, Human/immunology , Herpesvirus 2, Human/pathogenicity , Herpesvirus 2, Human/physiology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Host-Pathogen Interactions/physiology , Humans , Immediate-Early Proteins/metabolism , MicroRNAs/genetics , MicroRNAs/immunology , MicroRNAs/physiology , Nuclear Proteins/genetics , Nuclear Proteins/immunology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/immunology , RNA, Viral/physiology , Repressor Proteins/genetics , Repressor Proteins/immunology , Repressor Proteins/physiology , Simplexvirus/genetics , Simplexvirus/physiology , Transfection , Ubiquitin-Protein Ligases/metabolism , Vero Cells , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/physiology , X-linked Nuclear ProteinABSTRACT
Mammalian alphaherpesviruses are major causes of human and veterinary disease. During productive infection, these viruses exhibit complex and robust patterns of gene expression. These viruses also form latent infections in neurons of sensory ganglia in which productive cycle gene expression is highly repressed. Both modes of infection provide advantageous opportunities for regulation by microRNAs. Thus far, published data regarding microRNAs are available for six mammalian alphaherpesviruses. No microRNAs have yet been detected from varicella zoster virus. The five other viruses-herpes simplex viruses-1 and -2, herpes B virus, bovine herpesvirus-1, and pseudorabies virus-representing both genera of mammalian alphaherpesviruses have been shown to express microRNAs. In this article, we discuss these microRNAs in terms of where they are encoded in the viral genome relative to other viral transcripts; whether they are expressed during productive or latent infection; their potential targets; what little is known about their actual targets and functions during viral infection; and what little is known about the interactions of these viruses with the host microRNA machinery. This article is part of a Special Issue entitled: "MicroRNAs in viral gene regulation".
Subject(s)
Alphaherpesvirinae/genetics , MicroRNAs/metabolism , Animals , Gene Expression Regulation, Viral , Genome, Viral , Herpesvirus 1, Human/genetics , Herpesvirus 2, Human/genetics , Herpesvirus 3, Human/genetics , Humans , MicroRNAs/genetics , Virus Latency/geneticsABSTRACT
Several herpes simplex virus 1 microRNAs are encoded within or near the latency associated transcript (LAT) locus, and are expressed abundantly during latency. Some of these microRNAs can repress the expression of important viral proteins and are hypothesized to play important roles in establishing and/or maintaining latent infections. We found that in lytically infected cells and in acutely infected mouse ganglia, expression of LAT-encoded microRNAs was weak and unaffected by a deletion that includes the LAT promoter. In mouse ganglia latently infected with wild type virus, the microRNAs accumulated to high levels, but deletions of the LAT promoter markedly reduced expression of LAT-encoded microRNAs and also miR-H6, which is encoded upstream of LAT and can repress expression of ICP4. Because these LAT deletion mutants establish and maintain latent infections, these microRNAs are not essential for latency, at least in mouse trigeminal ganglia, but may help promote it.
Subject(s)
Gene Expression Regulation, Viral , Herpesvirus 1, Human/pathogenicity , MicroRNAs/biosynthesis , Trigeminal Ganglion/virology , Virus Latency , Animals , Carrier State/virology , Disease Models, Animal , Herpes Simplex/virology , Herpesvirus 1, Human/genetics , Male , Mice , MicroRNAs/genetics , Sequence DeletionABSTRACT
Cytomegaloviruses, representatives of the Betaherpesvirinae, cause opportunistic infections in immunocompromised hosts. They infect various cells and tissues in their natural host but are highly species specific. For instance, human cytomegalovirus (HCMV) does not replicate in mouse cells, and human cells are not permissive for murine cytomegalovirus (MCMV) infection. However, the underlying molecular mechanisms are so far poorly understood. In the present study we isolated and characterized a spontaneously occurring MCMV mutant that has gained the capacity to replicate rapidly and to high titers in human cells. Compared to the parental wild-type (wt) virus, this mutant formed larger nuclear replication compartments and replicated viral DNA more efficiently. It also disrupted promyelocytic leukemia (PML) protein nuclear domains with greater efficiency but caused less apoptosis than did wt MCMV. Sequence analysis of the mutant virus genome revealed mutations in the M112/M113-coding region. This region is homologous to the HCMV UL112-113 region and encodes the viral early 1 (E1) proteins, which are known to play an important role in viral DNA replication. By introducing the M112/M113 mutations into wt MCMV, we demonstrated that they are sufficient to facilitate MCMV replication in human cells and are, at least in part, responsible for the efficient replication capability of the spontaneously adapted virus. However, additional mutations probably contribute as well. These results reveal a previously unrecognized role of the viral E1 proteins in regulating viral replication in different cells and provide new insights into the mechanisms of the species specificity of cytomegaloviruses.
Subject(s)
Antigens, Viral/genetics , Immediate-Early Proteins/genetics , Muromegalovirus/growth & development , Muromegalovirus/genetics , Mutation, Missense , Virus Replication , Animals , Cell Survival , Cells, Cultured , DNA Mutational Analysis , DNA, Viral/chemistry , DNA, Viral/genetics , Humans , Mice , Sequence Analysis, DNA , Viral LoadABSTRACT
Certain viruses use microRNAs (miRNAs) to regulate the expression of their own genes, host genes, or both. Previous studies have identified a limited number of miRNAs expressed by herpes simplex viruses 1 and 2 (HSV-1 and -2), some of which are conserved between these two viruses. To more comprehensively analyze the miRNAs expressed by HSV-1 or HSV-2 during productive and latent infection, we applied a massively parallel sequencing approach. We were able to identify 16 and 17 miRNAs expressed by HSV-1 and HSV-2, respectively, including all previously known species, and a number of previously unidentified virus-encoded miRNAs. The genomic positions of most miRNAs encoded by these two viruses are within or proximal to the latency-associated transcript region. Nine miRNAs are conserved in position and/or sequence, particularly in the seed region, between these two viruses. Interestingly, we did not detect an HSV-2 miRNA homolog of HSV-1 miR-H1, which is highly expressed during productive infection, but we did detect abundant expression of miR-H6, whose seed region is conserved with HSV-1 miR-H1 and might represent a functional analog. We also identified a highly conserved miRNA family arising from the viral origins of replication. In addition, we detected several pairs of complementary miRNAs and we found miRNA-offset RNAs (moRs) arising from the precursors of HSV-1 and HSV-2 miR-H6 and HSV-2 miR-H4. Our results reveal elements of miRNA conservation and divergence that should aid in identifying miRNA functions.