MicrobiologyBytes

MicroRNAs (miRNAs) are ~22-nt regulatory RNAs expressed by all multicellular eukaryotes. Humans encode >700 miRNAs and similar numbers are likely to exist in other mammalian species. Almost all cellular miRNAs are initially transcribed by RNA polymerase II (Pol II) as part of a long, capped, polyadenylated primary miRNA (pri-miRNA) precursor. The miRNA forms part of one arm of an RNA stem-loop that consists of an ~32-bp imperfect stem flanked by unstructured RNA sequences. This stem-loop is recognized by the nuclear RNase III enzyme Drosha, which cleaves the stem to liberate an ~60-nt pre-miRNA hairpin. The pre-miRNA is then transported to the cytoplasm where it is cleaved by a second RNase III enzyme, called Dicer, which removes the terminal loop to generate the miRNA duplex intermediate. One strand of this duplex is incorporated into the RNA-induced silencing complex (RISC), where it acts as a guide RNA to direct RISC to complementary mRNA species. Depending on the level of complementarity, RISC can either cleave bound mRNAs and/or inhibit their translation. Inhibition of mRNA translation generally requires full complementarity of the mRNA to nucleotides 2 through 7 or 8 from the miRNA 5′ end – the miRNA seed region. The primary, and possibly sole, function of mammalian miRNAs is therefore to act as specific post-transcriptional inhibitors of mRNA function.

1. Do All Viruses Encode miRNAs? In general, we can divide mammalian viruses into three categories, i.e., the herpesviruses, which encode multiple viral miRNAs; other nuclear DNA viruses, which may encode one or two miRNAs; and the RNA viruses and cytoplasmic DNA viruses, which appear to lack any miRNAs.
2. What Do Viral miRNAs Do? Virus miRNAs may serve two major functions. First, to inhibit the expression of cellular factors that play a role in cellular innate or adaptive antiviral immune responses. Second, to downregulate the expression of virus regulatory proteins.
3. Are Virus miRNAs Conserved between Related Virus Species? No, but the miRNAs of individual viruses are conserved.
4. Do Viruses Regulate miRNA Transcription, Processing, or Function? At present, there is no evidence indicating that viruses modify the cellular environment to selectively favor the processing and expression of viral miRNAs, although viral miRNAs may be expressed at such high levels that they have the potential to competitively inhibit cellular miRNA function.
5. Can Viruses Use Cellular miRNAs to Promote Their Replication? At least one clear-cut example of a cellular miRNA that facilitates virus replication is known, activation of HCV replication by the liver-specific miRNA miR-122.

Five Questions about Viruses and MicroRNAs. PLoS Pathog 6(2): e1000787. doi:10.1371/journal.ppat.1000787

Related:

• Virus-encoded microRNAs in herpesvirus biology
• Human Cytomegalovirus-encoded microRNA regulates expression of multiple genes involved in replication
• Cellular Genes Targeted by KSHV MicroRNAs
• RNAi and Cold Sores

Human cytomegalovirus (HCMV) is an opportunistic human pathogen that establishes a lifelong latent infection that can periodically reactivate which, if unchecked by a robust immune response, can result in severe disease in immuno-compromised patients. Following primary infection of healthy individuals, human cytomegalovirus (HCMV) is met with a robust immune response resulting in an asymptomatic infection and subsequent establishment of latency. To date, HCMV remains one of the leading viral agents of disease in immuno-suppressed transplant patients and is a cause of severe morbidity in late stage AIDS sufferers and cancer patients.

The threat from HCMV is exacerbated by the dual threat of primary infection/re-infection, and virus reactivation within the host. Since many instances of virus disease result from HCMV reactivation, many studies have analysed the regulation of latency and reactivation using experimental models. An informed consensus supports the myeloid lineage as an important site of HCMV latency and carriage and that terminal myeloid differentiation is needed for HCMV reactivation. A recurrent theme in HCMV latency/reactivation, this differentiation-dependent permissiveness also applies to lytic infection. Latently infected cells are, by definition, unable to support the viral lytic transcription program – pivotal to this is failure of IE gene expression.

Reactivation of latent virus in myeloid progenitor cells is concomitant with cellular differentiation through regulation of the MIEP by chromatin remodelling. This study analyses the expression of the latent gene transcript UL81-82as (LUNA). LUNA is expressed in latently infected CD34+ cells and expression decreases as CD34+ cells differentiate to immature dendritic cells. Upon maturation (and HCMV reactivation) a second wave of transcription occurs consistent with expression during lytic infection also. Furthermore, it shows that the LUNA promoter is associated with acetylated histones during HCMV latency in experimentally and naturally infected CD34+ cells thus suggesting that latent gene promoters are, like the MIEP, regulated by post-translational modifications of their associated histone proteins.

Analysis of latent viral gene expression in natural and experimental latency models of human cytomegalovirus and its correlation with histone modifications at a latent promoter. J Gen Virol. Nov 11 2009

Related:

• Human Cytomegalovirus-encoded microRNA regulates expression of multiple genes involved in replication
• Human cytomegalovirus and the cell cycle

MicroRNAs regulate fundamental cellular processes in all metazoans The first discovered microRNA (miRNA), lin-4 of Caenorhabditis elegans, was found because of its role in a developmental timing defect. To date, more than 900 human miRNAs have been identified. MicroRNAs have been isolated from every metazoan and plant species examined thus far, and around 30% of all metazoan miRNAs are conserved between species. A hallmark of herpesvirus biology is the ability of the viruses to establish and maintain latent infections wherein the virus genome circularizes and persists as an episome, and where only very limited virus gene expression takes place. Herpesviruses establish infections that persist for the life of the host; an intricate balance therefore exists between host immune surveillance and virus immune evasion.

MicroRNAs (miRNAs) are short RNAs of about 22 nucleotides in length that post-transcriptionally regulate gene expression by binding to 3′ untranslated regions of mRNAs, thereby inducing translational silencing. Recently, more than 140 miRNAs have been identified in the genomes of herpesviruses. Deciphering their role in viral biology requires the identification of target genes, a challenging task because miRNAs require only limited complementarity. The subject of this review will be the herpesvirus miRNAs and their respective target genes that have been determined experimentally to date. These miRNAs regulate fundamental cellular processes including immunity, angiogenesis, apoptosis, and key steps in the herpesvirus life cycle, latency and the switch from latent to lytic replication.

Role of virus-encoded microRNAs in herpesvirus biology. Trends Microbiol. Oct 12 2009

Related:

• Human Cytomegalovirus-encoded microRNA regulates expression of multiple genes involved in replication
• Cellular Genes Targeted by KSHV MicroRNAs
• RNAi and Cold Sores

Players of contact sports like rugby and wrestling can end up with an infection caused by Herpes Gladiatorum (HG), a virus belonging to the herpes family. It gets into the body through cuts and abrasions and the disease is sometimes called scrumpox. Like all herpes viruses, once contracted, HG can remain dormant in its host and reactivate at any time. In this article in Microbiology Today (pdf) Julia Colston and Judy Breuer take a look at this unpleasant disease and show how it can even wreck an athletic career:

Herpes Gladiatorum (HG) is an active herpes simplex virus (HSV) infection associated with close-contact traumatic sports, such as wrestling, rugby and martial arts. Other names for the condition include scrumpox in association with rugby, and matpox in wrestling. It was first described in the literature in 1964, where five members of a small amateur wrestling group (and a further unfortunate gymnast who had volunteered themselves for a demonstration of the crossface manoeuvre!) developed lesions within a close time frame of individual fighting episodes. All five of these cases could be linked and they presented with similar symptoms of general malaise and an atypical vesicular rash affecting the exposed areas, namely the face and arms. Several further reports closely followed in 1965. It seems that outbreaks of HG were occurring well before this, with many unpublished epidemics taking place amongst wrestling groups. HG might have been described much earlier, had it not been for the ambiguity of the lesions produced, superimposed infections and a lack of appreciation for the relevance of the disease. Early reports list a vast array of alternative diagnoses, such as staphylococcal infections, herpes zoster, rickettsialpox and contact dermatitis, to name just a few. In fact, there are reports of unspecified disease in wrestling groups dating back as far as the 1920’s.

Read more

Related:

• How herpes simplex virus infects cells
• Herpesviruses – not all bad?
• A cream that could stop genital herpes?
• RNAi and Cold Sores

Regulation of gene expression at the level of mRNA translation is important for the control of numerous biological processes including cell growth, differentiation, development and the response to environmental stress. Unlike prokaryotes, the vast majority of eukaryotic mRNAs are unable to recognize ribosomes directly and rely instead on an intricate set of translation initiation factors that assemble a specialized multisubunit complex onto the mRNA 5′ terminus to recruit the 40S ribosome subunit. The responsiveness of individual constituents of this complex to a wide spectrum of cellular signals allows the translational machinery to respond rapidly to diverse physiological effectors. The 4E-BP translational repressor family, for example, sequesters eIF4E and prevents binding to eIF4G, limiting ribosome recruitment. Similarly, the ERK and p38-responsive eIF4G-associated kinase Mnk1 modulates eIF4E phosphorylation, which in specific instances has been associated with increased translation rates. Thus, regulated translation initiation factor complex assembly and modification is poised to potentiate important developmental decisions by controlling global and specific mRNA translation.

Viruses provide attractive models to study simple developmental decisions. In prokaryotes, much has been learned using bacteriophage λ to investigate how the lysis-lysogeny decision is made. In eukaryotes, latent herpesviruses exist in one of two developmental states within their hosts and must resolve an analogous question of whether to remain latent or initiate productive viral growth. Different herpesviruses permanently colonize distinct specialized host cell-types. Those that establish residency in dividing cell populations, exemplified by members of the γ-herpesvirus subfamily that includes Kaposi’s sarcoma associated herpesvirus (KSHV/HHV8), express a limited subset of viral genes that stimulate cell proliferation, allow for viral minichromosome replication and segregation, and evade antiviral defenses. In response to poorly understood environmental cues, these viruses can switch to a developmental program that results in productive replication. This alternate pathway involves activating a temporally coordinated cascade of viral lytic gene expression, which in turn results in massive viral DNA amplification, progeny virus production, and ultimately host cell destruction. To effectively switch its gene expression program, all herpesviruses produce a new population of viral mRNAs transcribed by the cellular RNA polymerase II, which are mostly capped and polyadenylated like their host counterparts, and these must successfully engage and reprogram the host cell translational apparatus. This is a critical component of the developmental switch because viruses are absolutely dependent upon the translational machinery resident in their hosts. Manipulating host translation initiation factors to ensure that nascent viral mRNAs successfully recruit ribosomes will therefore determine the overall level and efficacy with which the newly transcribed developmental instructions are executed. While we have a general understanding of how the viral transcriptome is altered for many viruses, a role for translational control in the developmental switch from a latent to a productively replicating state has not been described.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an important human pathogen and, like all herpesviruses, establishes a state of permanent residency in the infected host called latency. Major sites of KSHV latency are cells of the immune system and cells lining blood vessels. In individuals with weakened immunity, inappropriate growth of these cells driven by the resident virus can give rise to primary effusion lymphoma and Kaposi’s sarcoma, respectively. These life-threatening cancers are most common in patients with HIV/AIDS and have become a major source of mortality in parts of sub-Saharan Africa. Under appropriate stimuli, herpesviruses change their relationship with the host cell and begin to manufacture proteins required to assemble new infectious virus particles that can be released and spread. To achieve this, the virus hijacks key processes within the cell and conscripts them into producing viral proteins. A recent study describes for the first time how KSHV carefully manipulates the host protein synthesis machinery during the switch from latency to this specialized infectious virus production mode. The results show that although overall protein synthesis is diminished, key components of the host’s protein manufacturing machinery are actually stimulated, presumably to accelerate virus protein production.

Activation of Host Translational Control Pathways by a Viral Developmental Switch. PLoS Pathog 5(3): e1000334. doi:10.1371/journal.ppat.1000334
In response to numerous signals, latent herpesvirus genomes abruptly switch their developmental program, aborting stable host–cell colonization in favor of productive viral replication that ultimately destroys the cell. To achieve a rapid gene expression transition, newly minted capped, polyadenylated viral mRNAs must engage and reprogram the cellular translational apparatus. While transcriptional responses of viral genomes undergoing lytic reactivation have been amply documented, roles for cellular translational control pathways in enabling the latent-lytic switch have not been described. Using PEL-derived B-cells naturally infected with KSHV as a model, we define efficient reactivation conditions and demonstrate that reactivation substantially changes the protein synthesis profile. New polypeptide synthesis correlates with 4E-BP1 translational repressor inactivation, nuclear PABP accumulation, eIF4F assembly, and phosphorylation of the cap-binding protein eIF4E by Mnk1. Significantly, inhibiting Mnk1 reduces accumulation of the critical viral transactivator RTA through a post-transcriptional mechanism, limiting downstream lytic protein production, and impairs reactivation efficiency. Thus, herpesvirus reactivation from latency activates the host cap-dependent translation machinery, illustrating the importance of translational regulation in implementing new developmental instructions that drastically alter cell fate.

Related:

• Cellular Genes Targeted by KSHV MicroRNAs
• Modulation of the immune system by Kaposi’s sarcoma-associated herpesvirus

Adeno-Associated Viruses (AAV) are human parvoviruses widely used as a recombinant vector for gene transfer in animal studies and clinical trials designed to treat acquired or inherited genetic diseases. Wild type AAV is defined as a defective virus because it requires the presence of a helper virus to efficiently replicate. Although many viruses can provide helper functions to AAV, only those of Adenovirus were extensively studied, leading to the generation of molecular tools for recombinant AAV vector production. This study focuses on the helper activities of another virus, Herpes Simplex Virus type-1 (HSV-1), and demonstrates that nine HSV-1 proteins are able to fully and efficiently support the early steps of AAV replication. This study provides new information critical for the understanding of AAV replication and also opens the way for new biotechnological developments in the field of recombinant AAV vectors.

Definition of Herpes Simplex Virus Type 1 Helper Activities for Adeno-Associated Virus Early Replication Events. PLoS Pathog 5(3): e1000340. doi:10.1371/journal.ppat.1000340
The human parvovirus Adeno-Associated Virus (AAV) type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1); whereas, in the absence of a helper virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP) complex (UL5/8/52) and the single-stranded DNA-Binding Protein (ICP8) were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42) was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1–induced AAV replication.

Related:

• Adeno-associated virus vectors in gene therapy