Influenza Virus Host Restriction Factors: The ISGs and Non-ISGs

Influenza virus has been one of the most prevalent and researched viruses globally. Consequently, there is ample information available about influenza virus lifecycle and pathogenesis. However, there is plenty yet to be known about the determinants of influenza virus pathogenesis and disease severity. Influenza virus exploits host factors to promote each step of its lifecycle. In turn, the host deploys antiviral or restriction factors that inhibit or restrict the influenza virus lifecycle at each of those steps. Two broad categories of host restriction factors can exist in virus-infected cells: (1) encoded by the interferon-stimulated genes (ISGs) and (2) encoded by the constitutively expressed genes that are not stimulated by interferons (non-ISGs). There are hundreds of ISGs known, and many, e.g., Mx, IFITMs, and TRIMs, have been characterized to restrict influenza virus infection at different stages of its lifecycle by (1) blocking viral entry or progeny release, (2) sequestering or degrading viral components and interfering with viral synthesis and assembly, or (3) bolstering host innate defenses. Also, many non-ISGs, e.g., cyclophilins, ncRNAs, and HDACs, have been identified and characterized to restrict influenza virus infection at different lifecycle stages by similar mechanisms. This review provides an overview of those ISGs and non-ISGs and how the influenza virus escapes the restriction imposed by them and aims to improve our understanding of the host restriction mechanisms of the influenza virus.


Introduction
The influenza virus is an obligate intracellular pathogen and infects various mammalian and avian species.In humans, the influenza virus causes an acute febrile respiratory disease, influenza, which is commonly known as the 'flu'.Influenza virus exists in four types: A, B, C, and D. Influenza A virus is the most significant and researched among four types because it infects both mammalian and avian species and causes recurring seasonal epidemics, occasional pandemics, and zoonotic outbreaks.An influenza A virus particle possesses a lipid bilayer envelope, a matrix protein 1 (M1) skeleton underlying the envelope, and a core of eight viral ribonucleoprotein (vRNPs) complexes.The envelope harbors surface antigens, hemagglutinin (HA) and neuraminidase (NA), and an ion channel, matrix protein 2 (M2).The antigenicity of HA and NA glycoproteins continues to evolve due to genetic evolution; hence, influenza A viruses are further subtyped as, e.g., H1N1 and H5N1, based on their HA and/or NA properties.Each vRNP complex is composed of nucleoprotein (NP), three RNA polymerase subunits: polymerase acidic (PA), polymerase basic 1 (PB1), and polymerase basic 2 (PB2), and one of the eight single-stranded, negative-sense RNA genome segments: HA, M, NA, NP, NS, PA, PB1, or PB2 [1].
Influenza virus targets the epithelial cells of the respiratory tract (in mammals) or gastrointestinal tract (in waterfowl) to initiate the infection.For this, influenza virus particle attaches to the host cell by binding the receptor, α-2.6-linked sialic acids (in humans), or α-2.3-linked sialic acids (in avian sp.) through HA.Subsequently, the virus particle is internalized to the host cell, mainly by endocytosis.Through the combined action of low endosomal pH, viral ion channel M2, host proteases, and other factors, the viral envelope fuses with the endosomal membrane, and eight vRNPs are released from the endosomes into the cytoplasm [2,3].The vRNPs are transported through the cytoplasm and imported into the nucleus, where the viral RNA segment in each vRNP is transcribed and replicated into viral mRNA and viral RNA, respectively.In addition to viral NPs and RNA polymerase subunits (PA, PB1, PB2), various host factors facilitate viral transcription and replication [3].Then, mature viral mRNAs are exported to the cytoplasm for translation.The viral proteins, HA, M2, and NA, are trafficked to the plasma membrane, whereas the NP, M1, PA, PA-X, PB1, PB2, NS1 (non-structural 1), and nuclear export protein (NEP, formerly known as NS2) are transported to the nucleus.The vRNPs are formed in the nucleus and then exported off the nucleus and trafficked through the cytoplasm to the plasma membrane.The virus assembly occurs at the plasma membrane, and viral progeny is released by budding.
A variety of host factors facilitate and restrict the influenza virus lifecycle at each stage [3].The host factors that restrict the infection are called host restriction factors or antiviral factors and, broadly, can be of two types: (1) encoded by the interferon-stimulated genes (ISGs) and (2) encoded by the genes that are constitutively expressed or are not stimulated by interferons (non-ISGs).Many host restriction factors in both categories have been identified, some through the latest genetic techniques, such as RNA interference and CRISPR-Cas9 (Supplementary Table S1), and characterized to restrict influenza virus infection.This review compiles those host restriction factors and summarizes their infection restriction mechanisms.Also, this review identifies any strategies the influenza virus employs to escape the restriction imposed by host restriction factors.

ISGs
The expression of ISGs, as the name suggests, is induced by interferons.Interferons are the first line of defense molecules produced by host cells after sensing the virus infection through pattern recognition receptors.The existence of ISGs was first detected in the later part of the 20th century [4,5].Since then, several hundreds of ISGs have been identified [6] and characterized to inhibit the infection of many viruses [7].Likewise, many ISGs, encoding both proteins and non-coding RNAs (ncRNAs), have been identified to express in response to the influenza virus infection and restrict its infection at different stages of the viral lifecycle.Table 1 summarizes the individual ISGs known to restrict influenza virus infection at different stages of the lifecycle with their antiviral targets.

IFIT Proteins
The IFIT (interferon-induced proteins with tetratricopeptide repeats) family has four proteins, IFITs 1, 2, 3, and 5 (or ISGs 56, 54, 60, and 58, respectively), which have been characterized in humans [139].IFIT1 is the prototypic member of the family and was the first to be identified as an ISG in the IFIT family [140,141], followed by the rest [139].The indication of an antiviral function of human IFITs 1, 2, and 3 during influenza virus infection was first discovered in a proteomic screen [142].Later, it was demonstrated that human IFITs 1, 2, and 3 and avian IFIT5 exhibit antiviral properties during influenza virus infection [143][144][145][146][147]. The human and chicken IFITs exert their antiviral function by sequestering the viral RNA by binding its 5 ′ -triphosphate group, called PPP-RNA [142,145,148], whereas the duck IFIT sequesters viral NPs [144].However, Pinto et al. found no antiviral activity of human IFIT1 during influenza virus function [149], while Tran et al. found influenza virus rather exploiting the RNA binding property of IFIT2 to promote viral mRNA translation [150].

Tetherin
Tetherin, also known as BST-2/CD317/HN1.24, is a GPI-anchored transmembrane protein [157] and restricts virus infection by tethering the viral progeny to the cell surface.The antiviral role of tetherin during influenza virus infection is inconclusive and has been controversial.However, tetherin expression is induced in influenza virus-infected cells in an interferon-dependent manner [158].Human tetherin was observed to effectively tether the budding influenza virus-like particles to the plasma membrane [159][160][161][162]; however, the same was not observed with live influenza virus particles [158,159,163] or tetherin from other host species [164,165].In other studies, tetherin was observed to inhibit the influenza virus release [161,162,166,167], but this restriction was either NA-dependent [161,167] or countered by M2 protein, which facilitated the downregulation of tetherin on the cell surface [162].
2.9.PKR PKR (protein kinase R) is a dsRNA-activated serine/threonine protein kinase and phosphorylates the eukaryotic translation initiation factor 2 (eIF-2α); this leads to the inhibition of the initiation of global protein synthesis [180].This leads to the inhibition of viral protein synthesis too, and consequently, the influenza virus infection [181,182].Influenza virus counteracts this restriction through NS1, which binds to dsRNA and blocks PKR activation [182][183][184].Influenza virus NP also can block PKR activation by activating the cellular PKR inhibitor, P58 [185].

Other Proteins
CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) expression was first shown to be induced by interferon-gamma [186].CEACAM1 inhibits influenza virus infection by suppressing the mTOR (mammalian target of rapamycin) activity, consequently inhibiting the global protein synthesis in infected cells [187].
IFI16 (interferon γ-inducible 16) is a ~80-kDa nucleic acid-binding protein [188,189].It is a PYHIN (pyrin and hematopoietic interferon-inducible nuclear (HIN) domain) family protein and was initially identified as an intracellular DNA sensor [190].Recently, IFI16 has been discovered to inhibit influenza virus infection by sensing the viral RNA and promoting the RIG-I-mediated innate antiviral response [191,192].ISG20 (interferon-stimulated gene 20), as the name suggests, is a 20-kDa protein with 3 ′ to 5 ′ exonuclease activity that is specific for single-stranded RNA [193,194].ISG20 inhibits influenza virus infection by interfering with viral RNA transcription and replication [195,196].MOV10 (Moloney leukemia virus 10) is a member of the RNA helicase superfamily [197], and its expression can be stimulated by interferons [7].MOV10 inhibits influenza virus infection by binding to NP and sequestering the incoming vRNPs in the cytoplasm, consequently inhibiting their nuclear import [198][199][200].However, the antiviral function of MOV10 is independent of its RNA helicase activity [199,200].
MUC1 (mucin 1) is a member of mucins, a family of highly glycosylated proteins that are expressed on the surface of respiratory epithelial cells, which are the target of influenza virus infection.MUC1 potentially acts as a receptor decoy and inhibits influenza virus infection by binding to virus particles and blocking their attachment to target cells [201][202][203].
NCOA7 (nuclear receptor coactivator 7) expression is induced by the interferonbeta [204].NCOA7 inhibits influenza virus infection by inhibiting the fusion of the viral envelope with the endosomal membrane during entry [205].
p21 is a cyclin-dependent kinase inhibitor and inhibits influenza virus infection by interfering with viral RNA polymerase activity [206].
Serpin 1 or plasminogen activator inhibitor 1 (PAI-1) inhibits influenza virus infection by neutralizing host proteases, like trypsin, and preventing the cleavage of HA, which is required for influenza virus entry [207].However, influenza virus may escape this restriction in humans carrying the naturally occurring SNP rs6092 in serpin 1 gene [207].
SERTAD3 (SERTA domain containing 3), also called RBT1 (replication protein A binding transactivator 1), is one of the SERTA family transcription factors, and its expression is induced by interferons [208].SERTAD3 inhibits influenza virus infection by disrupting the formation of the viral RNA polymerase complex [208].
SLFN11 and SLFN14 are Schlafen family proteins and possess an RNA helicase domain [209].SLFN11 and SLFN14 expression is induced by interferons, and both inhibit influenza virus infection by contributing to host innate defenses [210,211].
SPOCK2 (SPARC/osteonectin CWCV and Kazal-like domains 2) or testican 2 is a secreted proteoglycan, and it inhibits influenza virus infection by blocking the attachment of virus particles to the cell surface [212].
Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) protein [214], also called RSAD2, inhibits influenza virus infection by disrupting the lipid rafts on the plasma membrane and inhibiting the viral progeny release [215,216].
ZAP (zinc finger antiviral) or ZC3HAV1 (Zinc finger CCCH-type antiviral 1) protein exists in two forms, short (ZAPS) and long (ZAPL), and both forms exhibit anti-influenza virus properties [217][218][219].The ZAPS exerts its antiviral function by promoting the degradation of viral mRNA but is antagonized by the NS1, which competes with ZAPS for viral mRNA binding [218].Whereas ZAPL promotes the degradation of viral PA and PB2 and is antagonized by viral PB1, which binds ZAPL and displaces PA and PB2 [217].

ncRNAs
Much of the human genome is transcribed into non-coding RNAs (ncRNAs), which do not translate into a protein.Based on their length, these ncRNAs are called microRNAs or miRNAs (~22 nucleotides), small-interfering RNAs or siRNAs (21-25 nucleotides), piwirelated RNAs or piRNAs (24-33 nucleotides), vault RNAs or vtRNAs (80-150 nucleotides), or long non-coding RNAs or lncRNAs (>200 nucleotides).Further, some lncRNAs exist as covalently closed circular RNAs or circRNAs.Many ncRNAs are upregulated in response to influenza virus infection and inhibit infection by targeting the viral proteins and critical host proteins [220].
lncRNAs are the prominent form of ncRNAs that have been identified to be upregulated in response to influenza virus infection or interferon treatment [221][222][223][224][225][226][227][228].These lncRNAs inhibit influenza virus infection primarily by strengthening the antiviral state in infected cells through various mechanisms, e.g., stabilization of the RIG-I-TRIM25 complex for host sensing of the influenza virus [222], epigenetic modifications of the regulatory regions of innate response genes [225,227], and manipulation of the regulators (including miRNAs) of interferon signaling [221,223,226,228].
Also, circRNAs, circVAMP3, and AIVRs are upregulated in response to influenza virus infection and restrict the infection by different mechanisms [229,230].The circVAMP3 acts as a decoy to viral NP and NS1 and interferes with their function [230], while the AIVR sequesters a microRNA, which degrades an enhancer of the interferon production [229].

Non-ISGs
Host proteins encoded by the constitutively expressed genes (called non-ISGs) also restrict influenza virus infection.The majority of these non-ISGs have been identified either through co-immunoprecipitation followed by mass spectrometry analyses or yeast-two hybrid, RNA interference, and CRISPR-Cas genetic screens.The non-ISGs known to restrict influenza virus infection at different stages of the lifecycle and their antiviral targets are summarized in Table 2.

MARCH Proteins
MARCH (membrane-associated RING-CH-type) proteins are RING (really interesting novel gene) finger E3 ligases and are known to downregulate the expression of cellular proteins on the cell surface.MARCH1 and MARCH8 inhibit influenza virus infection though it is unclear if, like in case of other enveloped viruses, they downregulate the expression of influenza virus membrane proteins on the cell surface [250][251][252].However, MARCH8 has been identified to block the furin-mediated cleavage of HA of avian influenza A virus H5N1 subtype [250].

Translation Factors
The eukaryotic translation initiation factor 4B (eIF4B) inhibits influenza virus infection indirectly by promoting the translation of ISGs, like ISG15 and IFITM3 [253].But, influenza virus overcomes this restriction by promoting the lysosome-mediated degradation of eIF4B [253].The eukaryotic translation elongation factor 1 delta (eEF1D) inhibits influenza virus infection by a different mechanism; it impedes the nuclear import of vRNPs by impairing the interaction of the NP and PB1 with their nuclear receptors [254].

Other Proteins
Annexin 6, a calcium-dependent membrane-binding protein involved in membrane organization, inhibits influenza virus infection by interacting with M2 and interfering with the budding of viral progeny [272,273].APOE (apolipoprotein E) restricts influenza virus infection by interfering with membrane cholesterol homeostasis and inhibiting the virion attachment to the cell surface [274].
BTN3A3 (butyrophilin subfamily 3 member A3) inhibits the infection of, specifically, avian influenza viruses by interfering with the replication of viral RNA [279].Hence, BTN3A3 is a barrier to the transmission of avian influenza viruses to humans.However, like Mx proteins, zoonotic avian influenza viruses escape this barrier by acquiring escape mutations in their NPs [279].
Cyclin D3, a key cell cycle regulator, like annexin 6, interacts with M2 and inhibits its interaction with M1, consequently inhibiting the formation of influenza virus progeny [281].
Galectin-1 is an S-type lectin and is secreted extracellularly in the lungs.It binds influenza virus particles and inhibits their attachment to the cell surface [282], consequently inhibiting the infection and reducing disease severity [282][283][284].Further, galectin-1 gene variants, SNPs rs4820294 and rs13057866, express galectin-1 at a higher level and protect the humans carrying those SNPs from severe influenza virus infection [283].
HAX-1 (HCLS1-associated X1), an anti-apoptotic protein, inhibits influenza virus infection by binding to PA and blocking its nuclear import [285].However, mostly zoonotic avian influenza viruses are sensitive to the HAX-1-mediated restriction [286].Nevertheless, zoonotic avian influenza viruses can overcome this restriction via viral PB1-F2, which also binds to HAX-1 and competes with PA for this binding [286,287].
hnRNPAB (heterogeneous nuclear ribonucleoprotein A/B) restricts influenza virus infection by promoting the nuclear retention of viral mRNA [288,289].
Hsp70 (heat shock protein 70) has been described to inhibit influenza virus infection by interacting with PB1 and PB2 and interfering with vRNP integrity, which results in the interference of viral RNA replication [290].But, another study claimed that HsP70-PB1-PB2 interaction promotes viral RNA replication [291].
MMP3 (matrix metalloproteinase 3) restricts influenza virus infection by translocating to the nucleus and promoting the expression of antiviral cytokines and chemokines [293].
PGRMC1 (progesterone receptor membrane component 1) restricts influenza virus infection by antagonizing the ubiquitination-mediated activation of RIG-I.Potentially, it determines the neurotropism of influenza viruses too [297].
PKP2 (plakophilin 2) protein, mainly known for the formation of desmosomes and stabilization of cell junctions, binds to PB1.It restricts influenza virus infection by competing with PB2 for binding to PB1 and impeding RNA polymerase activity [298].
PSMB4 (proteasome subunit beta type 4) restricts influenza virus infection by targeting NS1 and facilitating its degradation in infected cells [301].
RTF2 (replication termination factor 2) is a nucleus-localized protein and restricts influenza virus infection by inhibiting the viral transcription and promoting the interferon response [302].
SERINC5, one of the five SERINC (serine incorporator) family membrane proteins, inhibits influenza virus infection by interfering with the fusion of the viral envelope and endosomal membrane during entry [304,305].
TET2 (ten-eleven translocation 2), a methylcytosine dioxygenase, inhibits influenza virus infection by enhancing the expression of STAT1 and consequently the expression of various ISGs via DNA demethylation [307].However, influenza virus counters the TET2-mediated restriction by downregulating the TET2 expression through its host shutoff protein PA-X [307].
TRA2A (transformer 2 alpha homolog), an mRNA splicing regulator, restricts the infection of avian influenza viruses but not the human influenza viruses in humans.Human TRA2A binds to the ISS (intronic splicing silencer) motif of avian influenza virus M mRNA and inhibits its splicing into M1 mRNA and M2 mRNA [308].Some avian influenza viruses may have escaped the TRA2A restriction and adapted to humans by mutating the ISS motif in their M genes [308].
TUFM (Tu elongation factor, mitochondrial) protein acts as a barrier to interspecies transmission of avian influenza viruses to humans.TUMF binds to avian influenza virus PB2 in mitochondria and induces autophagy, which, in turn, restricts avian influenza virus growth in human cells [309].However, the E627K mutation in PB2 of avian influenza viruses impedes the binding of TUMF to PB2 and allows avian influenza viruses to escape this restriction and multiply in human cells [309].
ZMPSTE24 (zinc metallopeptidase STE24) is an effector of IFITMs and inhibits influenza virus infection by facilitating the antiviral function of IFITMs in endosomes [310,311].However, the antiviral function of ZMPSTE24 is independent of its protease activity [310,311].

Summary
A plethora of host restriction factors, ISGs and non-ISGs, have been identified, which restrict influenza virus infection by inhibiting the viral attachment, entry, synthesis, assembly, and release, and strengthening the host innate antiviral response (Tables 1 and 2).However, the influenza virus seems to have the upper hand and effectively antagonizes the restrictions imposed by these factors.Basically, there are three strategies that influenza virus employs to perform this: (1) the acquisition of escape mutations in viral proteins, like NP, targeted by the restriction factors, (2) the downregulation of the expression of restriction factors at both mRNA and protein levels via viral endonucleases, PA and PA-X, or host factors, ncRNAs, proteasome, lysosome, and caspases, and (3) the sequestration or interference of the restriction factors by viral proteins, like NS1 (Table 3).Furthermore, the genetic diversity of some restriction factors (galectin-1, IFITM3, Mx, OAS-1, Serpin-1) in various hosts and human populations also helps the influenza virus to escape the host restriction.Nevertheless, an exhaustive list of the influenza virus host restriction factors with their restriction mechanisms is yet to be compiled.A comprehensive knowledge of host restriction factors and influenza virus interplay is critical for designing targeted antiviral interventions to overcome the existing and newly emerging influenza virus variants.

Table 1 .
ISGs restricting the influenza virus lifecycle at different stages.

Table 2 .
Non-ISGs restricting the influenza virus lifecycle at different stages.

Table 3 .
Influenza virus strategies to escape the restriction from ISGs and non-ISGs.The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/pathogens13020127/s1,TableS1.Influenza virus restriction factors identified by RNA interference or CRISPR-Cas9 screenings/techniques.The author's research in recent times has been supported by the J C and H S Anderson Charitable Trust, New Zealand (2020), Maurice & Paykel Charitable Trust, New Zealand (2020, 2022), Maurice Wilkins Centre, New Zealand (2023), and the School of Biomedical Sciences (2022).
Funding:Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: Not applicable.