Large and small viral RNAs and their role in viral gene expression and evading host antiviral defense

Question 1

miRNA Details

Answer 1

• Originate mostly from capped & polyadenylated full length precursors (pri-miRNA)
• Generally transcribed by RNA polymerase II
• Hairpin precursor ~70 nt (pre-miRNA) long
• Mature miRNA ~22 nt long
• First miRNA discovered in 1993 by Victor Ambros (lin-4)
• Let-7 discovered in 2000 by Frank Slack and Gary Ruvkun

Question 2

Small RNAs may regulate the function and stability of target mRNAs by different mechanisms

Answer 2

A: imperfect matches -> translational repression
B: perfect complementarity -> miRNA- mediated RISC cleavage and mRNA degradation

Question 3

Biological significance of miRNA-mediated regulation

Answer 3

1 miRNAs emerge as key post transcriptional regulators of gene expression
2 Computational predictions suggest that a single miRNA may regulate more than hundred mRNAs
3 30-70% of animal genes may be subject to miRNA-mediated regulation
4 specific miRNAs have been implicated in diverse biological processes:
- Development
- Cellular differentiation
- Proliferation
- Apoptosis
- Oncogenesis

Question 4

Model for microRNA-mediated regulation

Answer 4

• several different miRNAs can regulate a single mRNA (mRNA 5, mRNA 2 and mRNA 6)
• several different mRNAs can be regulated by one miRNA (mRNA 2, mRNA 5 and mRNA 6)
• the same mRNA target can contain multiple miRNA docking sites for the same miRNA (mRNA 7)

Question 5

Production of miRNAs

Answer 5

Production of functional miRNAs is a multi-step process:
* Transcription of pri-miRNA precursors
* Imperfect hairpin release in the nucleus
* Export to the cytoplasm
* Dicer processing in the cytoplasm
* Strand selection by RISC complex

Question 6

Biogenesis of miRNA

Answer 6

canonical pathway:
pri-miRNA processing occurs in two steps, catalysed by two members of the RNase III family of enzymes, Drosha and Dicer, operating in complexes with dsRNA-binding proteins (dsRBPs), for example DGCR8 and transactivation-responsive (TAR) RNA- binding protein (TRBP) in mammals.

non-canonical pathway:
pre-miRNAs are produced from very short introns (mirtrons) as a result of splicing and debranching, thereby bypassing the Drosha– DGCR8 step

Question 7

Viruses interfere with host miRNA biogenesis

Answer 7

• Expression of viral proteins inhibiting miRNA loading into the host RISC complex (tomato bushy stunt virus p19 protein) or Ago2 activity (cucumber virus 2b protein).
• Flaviviruses sfRNA and Vaccinia virus inhibit or reduce the expression of Dicer activity in the infected cell (also Adenoviruses by expression of VA1 RNA)
• Herpesviruses encode viral sequences complementary to mature miRNAs miR-17 and miR-27 resulting in degradation and inhibition of the miRNA-induced regulation of mRNA targets.

Question 8

miRNAs and Viruses

Answer 8

• miRNAs of viral and cellular origin can positively or negatively influence viral replication
• viral miRNAs can directly alter host cell physiology, including components of the immune system
• host miRNAs can alter virus life cycle

Why would viruses exploit cellular and virus-encoded miRNAs?
* miRNAs are non-immunogenic
* take up a small amount of genomic space
* can be expressed in large amounts
* are powerful regulators of gene expression
* remarkable functional flexibility
* function without the need of translation into a protein product- fast response

Question 9

Potential effects of cellular miRNAs on virus replication

Answer 9

1) Cellular miRNAs may bind directly viral RNAs and promote viral replication: proviral
2) Cellular miRNAs may mediate the recognition of viral RNAs by RISC thereby directly limiting viral replication: antiviral
3) Cellular miRNAs might indirectly promote or limit virus replication through regulation of their endogenous cellular mRNA targets: proviral or antiviral

Effect of cellular miRNAs on viruses:
1. impact on viral genome evolution
2. Regulation of tissue tropism of viruses in vivo

Question 10

Host miRNAs Can Directly Block Viral Replication

Answer 10

• Enterovirus 71 (EV71): Elevated levels of miR-296-5p were detected in Enterovirus 71 (EVtq)- infected cells. miR-296-5p targets both capsid protein VP1 and VP3 coding regions in the viral genome as a response to viral infection.
• Coxsackievirus B3 (CVB3): miR-342-5p targets the 2C-coding region of the viral RNA, which results in its degradation
• Herpes simplex virus type 1 (HSV-1): Lytic replication and reactivation form latency depend on the expression of viral infected Cell Protein 0 (ICP0), which is controlled by the cell-specific miR-138 in neurons

Question 11

Host miRNAs can directly improve RNA virus replication

Answer 11

-> The unusual interaction between host miRNA and increasing amounts of viral RNA during replication results in a reduction of the interaction of the host miRNA with its cellular targets (“sponge effect”).

Question 12

RNA viruses can be dependent on cellular miRNAs and connect virus-induced miRNA sequestration to host transcriptome regulation

Answer 12

*Pestiviruses critically depend on cellular miR-17 and let-7
*Pestiviral RNA functionally reduces miR-17 binding on endogenous mRNA targets

Question 13

Hepatitis C Virus RNA Functionally Sequesters cellular miR-122

Answer 13

• HCV RNA functionally reduces miR-122 binding on endogenous mRNA targets
• HCV miRNA sponging can be redirected by swapping viral miRNA tropism

Question 14

Viruses exploit direct interactions with host miRNAs: Modulation of hepatitis C virus RNA abundance by a liver-specific MIcroRNA

Answer 14

• miRNA122 is preferentially expressed in the liver
• miRNA122 regulates fatty acid and cholesterol synthesis central to liver function
  *HCV RNA synthesis is stimulated by cellular miRNA-122
  *Binding of miR-122 to the 5‘UTR increases the abundance of HCV RNA
  *Stimulation is conferred by direct interaction of miR-122 with two target sites in the 5‘UTR of the HCV genome.

Liver-specific expression of miRNA122 may contribute to the tissue tropism of HCV

Question 15

miR-122 Stimulates Hepatitis C Virus RNA synthesis by Altering the Balance of Viral RNAs Engaged in Replication versus Translation

Answer 15

• The microRNA miR-122 and PCBP2 jointly regulate HCV genome circularization and thereby influence engagement of the RNAn in the mutually incompatible processes of viral RNA synthesis and translation.
• Binding of PCBP2 to sequences near the 5’ and 3’ ends of the viral positive-strand RNA ((*)RNA) provides a protein bridge that promotes genome circularization and facilitates IRES-initiated translation.
• Competition of miR-122 with PCBP2 for binding to the 5’ UTR: promoting open, noncircular genome conformation
• miR-122 may also induce changes in the conformation of the IRES.
• Consequence: reduction in translation and an increase in the initiation of negative-strand RNA ((_)RNA) synthesis (possibly owing to a greater availability of the 3’ UTR for interactions with the HCV replicase)

Question 16

A model for interaction of miR-122 with the 5’ UTR of the HCV RNA and miravirsen’s mechanism of action

Answer 16

Miravirsen (SPC3649) is a high-affinity 15-mer LNA modified antimiR oligonucleotide that acts by sequestering mature miR-122, leading to inhibition of miR-122 function and thereby suppression of HCV

Question 17

Hepatitis B virus-human chimeric transcript HBx-LINE1 promotes hepatic injury via sequestering cellular microRNA-122

Answer 17

• hybrid HBV-human transcript common in HBV-associated HCC cases serves to functionally sequester miR-122
• by depleting cellular miR-122, HBx-LINE1 promotes the expression of WNT1 protein
• Enhanced WNT1-expression causes up regulation of beta-catenin signaling, epithelial-mesenchymal transition (EMT), enhanced cell migration and invasion
• promoting HBV-linked HCC progression

Question 18

The differentially expressed miRNAs induced by EV71 infection: up-regulated levels of cellular miR-141 suppress eIF4E expression

Answer 18

eIF4E mRNA is a target of miR-141!

Question 19

Model for the regulatory role of miR-141 in enterovirus infection

Answer 19

The upregulation of EGR1 induced by the Enterovirus infection increases cellular miR-141 expression.
miR-141 represses the expression of eIF4E via imperfect base pairing between miR-141 and the 3‘UTR of eIF4E mRNA.
The reduction in eIF4E causes a protein synthesis switch from cap- dependent to cap-independent translation
Contribution to viral pathogenesis and virus propagation.

Question 20

Host miRNAs Can Act As Proviral Factors by Inhibiting Antiviral Host Factors

Answer 20

miR-146a expression is increased upon virus infection (VSV, JEV or DenV)
The proviral function of miR-146a is explained by the reduction of target mRNA expression for IRAK1, IRAK2, or TRAF6 which are essential partners of the type I interferon response

Question 21

Viruses can up regulate Host miRNAs to promote their replication via the miRNA-mediated inhibition of the Jak/STAT signaling pathway

Answer 21

• Cellular miRNAs (miR-9041, miR-9850, miR29a and miR-373) are upregulated during DNA or RNA virus infections.
• The proviral function of cellular miRNAs is explained by the reduction of target mRNAs coding for key elements of the JAK/STAT signaling pathway resulting in a reduced expression of interferon-induced genes.

Question 22

Virus encoded miRNAs

Answer 22

Virus-encoded miRNAs can be grouped into two classes
- Viral miRNAs that are analogs of host miRNAs
- Viral miRNAs that are viral specific

most functions ascribed to virus-encoded miRnAs can be grouped in the following categories:
1) Prolonging longevity of infected cells
2) evading the immune response
3) regulating host or viral genes to limit the lytic cycle

Question 23

Most of the virus-encoded miRNAs share their biogenesis pathway with cellular miRNAs

Answer 23

• Virus-encoded miRNAs can affect both viral and cellular transcripts

Question 24

The minority of viruses utilize non canonical mechanisms in the biogenesis of pre-miRNA molecules

Answer 24

• Herpesvirus saimiri (HVS) encodes Sm class U RNAs (HSURs) that are transcribed by RNA Pol II and subsequently processed by the host Integrator complex to generate pre-miRNAs
• The miRNAs encoded by mouse gammaherpesvirus 68 (MHV68) and BLV are both transcribed by host RNA Pol III
• MHV68 miRNAs are processed from larger tRNA-like RNAs by host tRNase Z and possibly additional factors to generate pre-miRNAs

Question 25

Virus families that encode miRNAs or miRNA-like molecules

Answer 25

*Tom Tuschl‘s group reported in 2004 the existence of viral miRNAs for the first time in Epstein- Barr Virus (EBV) *(>225) miRNAs have been identified in 15 viruses from 3 viral families (mostly from the large DNA genome herpesvirus family)

Question 26

Why have viral-encoded miRNAs not been discovered in more virus families?

Answer 26

1. lack of access to nuclear miRNA processing machinery (e.g. Poxviruses) 2. destabilizing effects of miRNA processing on RNA genomes (e.g. Flaviviruses, Picornaviruses) (exception: Flaviviruses produce sfRNA)

Question 27

Potenital mechanisms of viral miRNA function

Answer 27

1. Viral miRNAs may inhibit translation of viral transcripts carrying imperfect matches to the miRNA 2. dsDNA virus-derived miRNAs may target viral transcripts that are transcribed antisense to the miRNA precursor for degradation by RISC e.g. viral mRNA (LT-Ag mRNA) degradation SV40 miR-S1 -> antisense v-miRNAs are generally involved in the lytic or latency phase transition e.g. EBV promotes the latency phase by targeting immediate-early genes (BZLF1 and BRLF1 ) 3. Viral miRNAs may engage in novel interactions with host mRNAs or function as orthologs of cellular miRNAs and therefore inhibit the normal mRNA targets of the orthologous host cell miRNA

Question 28

hCMV infection targets host innate immune system by expressing a viral miRNA

Answer 28

hCMV -> Expression of hCMV-miR-UL122 and miR-UL148D-1 - miR-UL112 and miR-UL148D-1 engage in interactions with host cellular mRNA -> hCMV exploits miRNA-based immunoevasion mechanism for ist own survival

Question 29

Herpes Viral miRNAs in Immune Evasion

Answer 29

- HCMV miR-UL122-1 target the MICB (major histocompatibility complex class I-related chain B) decreased NK cell killing - EBV miR-BART2-5p and KSHV miR-K12-7 target the 3' UTR of MICB at different locations - miR-UL-148D-1 directly targets RANTES, a chemokine that recruits immune cells to the site of infection

Question 30

hCMV uses a viral miRNA to establish latency in infected cells by targeting a viral transcription factor

Answer 30

hCMV infection -> Expression of hCMV-miR-UL112-1 (expressed early and accumulates during viral infection) -> viral strategy to maintain latency or to promote the transition from productive replication to latent infection

Question 31

hCMV infection targets viral proteins to establish latency in infected cells by expressing a viral miRNA

Answer 31

- viral immediate-early protein 1 (IE 1, also known as IE72)- transcription factor required for the expression of many viral genes -> hCMV miR-UL 112-1 expression downregulates viral IE1 protein expression and viral DNA glycosylase UL114 : viral strategy to maintain latency or to promote the transition from productive replication to latent infection (viral IE gene also targeted by the cellular miR200 in CD34+ cells)

Question 32

SV40-encoded microRNAs regulate viral gene expression by miRNAs-mediated degradation of viral transcripts

Answer 32

Simian virus 40 (SV40): polyomavirus, small DNA viruses in immunocompromised hosts SV40 infection is associated with tumorigenesis and various diseases (nephropathy, neural demyelination) Block of translation of protein products encoded by the early mRNAs (highly immunogenic, recognized by CTLs) -> Reduction of large and small T antigen protein levels -> Decreased visibility to SV40-specific cytotoxic T Lymphocytes (CTLs) Role for SV40 miRNA in evading the adaptive immune response by reducing the expression of highly immunogenic early viral proteins

Question 33

Map of the polyomavirus genome

Answer 33

The miRNAs are expressed from the same strand as the late transcripts and are therefore perfectly complementary to the early mRNAs. As such, they target the early mRNAs for degradation.

Question 34

Genome organization and location of miRNAs encoded by the Epstein-Barr Virus

Answer 34

* The EBV genome is maintained as a circular episome in latently infected cells. * EBV genome contain three miRNA cluster: BHRF cluster and BART clusters 1 and 2. * Each miRNA is processed from pre-miRNA precursors and transported from nucleus to cytoplasm.

Question 35

Viral miRNAs regulate gene expression-EBV infection targets viral and cellular transcripts by expressing viral miRNAs

Answer 35

EBV infection -> Expression of clustered viral miRNAs from polycistronic transcripts: co-regulation

Question 36

Virus-encoded mircoRNAs as orthologs of cellular microRNAs

Answer 36

Virus-encoded miRNAs usually don‘t share seed homology with cellular miRNAs they usually target novel sites on cellular mRNAs - some virus-encoded miRNAs share seed- sequences with existing cellular microRNAs -regulate a common set of host mRNA targets -strategy of viruses to block host antiviral defence *Use of pre-existing host miRNA network of mRNA targets and binding sites *not unique to KSHV

Question 37

KSHV uses pre-existing host miRNA network - a virus-encoded miRNAs as an orthodox of a cellular miRNA

Answer 37

-KSHV-encoded miR-K12-11 shares the seed-sequence with the cellular microRNA miR- 155 -exogenous expression of miR-K12-11 was shown to downregulate numerous host mRNAs with functions in innate immunity and apoptosis -KSHV express miR-K12-11 to block host antiviral defense - KSHV miR-K12-11 target mRNAs which are part of existing host regulatory network

Question 38

Model of a potential role for miR-K12-11 in lymphogenesis during KSHV infection

Answer 38

* miR-155 is transiently expressed in macrophages, T and B lymphocytes upon treatment with inflammatory stimuli * Germinal center-dependent B-cell maturation is dependent on precise miRNA155 expression * Constitutive expression of miR-155 in B cells is associated with B cell lymphomas in human, mice and chicken * miR-155 is therefore classified as oncomir -> possible role for miR-12-11 in the development of KSHV-associated lymphomas

Question 39

Model for some miRNAs during viral latency

Answer 39

During viral latency: * lytic mRNAs are not expressed or expressed at typically undetectable levels. * viral miRNAs are expressed at relatively high levels during latency and may suppress ‘‘leaky’’ lytic transcripts. During lytic replication: * large changes in the transcriptional activity of lytic genes ‘‘overrun’’ the imposed inhibition by miRNAs and initiate lytic gene expression programs

Question 40

The role of virus-encoded miRNAs in promoting KSHV latency

Answer 40

KSHV miR-K 12-5 and miR-K 12-9 target the viral- encoded RTA, the master lytic switch transactivating protein that is both necessary and sufficient to trigger lytic replication in KSHV-infected cells. KSHV miR-K 12-1 targets the host- encoded IκBα, an inhibitor of NFκB signaling pathway * KSHV miRNA-mediated inhibition of RTA reduces the expression of lytic transcripts * KSHV miRNA-mediated inhibition of IκBα (an inhibitor of NFκB) is activating NFκB signaling and promotes latency A function of the KSHV miRNAs as a whole, is to negatively regulate the initiation of lytic replication.

Question 41

Nanocanonical miRNA Functions

Answer 41

* In addition to canonical miRNA repressive activities on trans targets, the biogenesis of some miRNAs can convey cis regulation. Several viruses encode pre-miRNAs embedded in cis within viral mRNA transcripts. During lytic replication the balance is shifted toward protein expression In latency miRNA biogenesis by the microprocessor complex results in reduced expression of Kaposin B * KapB transcripts may function as either an mRNA or pri-miRNA, but not both since pre- miRNA biogenesis occurs in the nucleus and destroys the mRNA. * The levels of the host Microprocessor component Drosha is decreased during lytic replication and thus allows a shift to the pathway favoring KapB protein production Subgenomic flaviviral RNA is produced as a result of incomplete degradation of genomic RNA by host enzyme XRN-1.

Question 42

Functions of sfRNA in arthropod and vertebrate hosts

Answer 42

- sfRNA inhibits XRN-1 and Dicer in both hosts, causing disruption of mRNA decay and siRNA7miRNA production - In vertebrates sfRNA inhibits IFN-alpha/beta response and induces apoptosis - Inhibitory effect of sfRNA on IFN-alpha/beta response is in part mediated by sfRNA binding to TRIM25 and to CAPRIN1/G3BP1/2 - In mosquitos, sfRNA inhibits expression of Toll-pathway components Cecil and Rel1a and supresses Toll-signalling in addition to inhibiting RNAi response