**5. Influenza Virus Propagation and Sphingolipids**

Seasonal influenza epidemics are responsible for over 200,000 hospitalizations in the United States and up to 500,000 deaths worldwide each year. Influenza A (IAV) is an enveloped virus possessing a negative sense, single-stranded, segmented RNA genome. Eight RNA segments encode 11 different viral proteins (Figure 5). The envelope spike glycoproteins HA and NA mediate viral entry and release, respectively, and serve as antigenic determinants of the virus. M2 is proton-selective ion channel involved in uncoating once the virus has entered the cell. Matrix protein (M1) forms a matrix under the viral envelope which is critical for maintaining the integrity and shape of the intact viral particle. Each segment of viral RNA is encapsulated with nucleocapsid protein (NP) and associated with the trimeric polymerase complex (PB1, PB2, and PA), forming what are referred to as a viral ribonucleoprotein complex (vRNP). Non-structural protein 1 (NS1) plays a critical role in evasion of host immunity. Non-structural protein 2 (NS2) is involved in the export of newly synthesized vRNPs from the nucleus to the cytoplasm for packaging.

The two classes of flu antiviral drugs include M2 ion channel blockers (e.g., amantadine and rimantadine) and neuraminidase inhibitors (e.g., zanamivir, oseltamivir). Early administration of these antivirals reduces disease symptoms, shortens the duration of illness, reduces hospitalization rates, and reduces viral transmission [67,68]. However, replication of the IAV genome involves a high error rate (10−<sup>3</sup> to 10−<sup>4</sup> substititution per genome) [69,70], resulting in the frequent accumulation of amino acid changes in IAV proteins. These changes enable IAV to evade host immunity acquired by prior exposure or vaccination and is the reason why IAV vaccines must be reformulated annually. Additionally, these amino acid changes may allow the virus to develop resistance against currently available antiviral agents that target the activity of the flu NA protein. As such, novel antivirals against influenza are critically needed.

**Figure 5.** Organization of the Influenza A virus's genome. The influenza A virus's (IAV's) genome consists of eight segments of negative-sense, single-stranded RNA. Encoded by the genome are three polymerase proteins (PA, PB1, and PB2), nucleoprotein (NP), and two envelope proteins (HA and NA). The M and NS mRNAs can be alternatively spliced to yield M1 and M2, and NS1 and NS2 proteins, respectively. Boxes indicate coding regions, sized relative to the other gene segments. Black lines at each end represent the 3' and 5' untranslated regions. The total length of each segment (conding and non-coding regions) in nucleotides (nt) is indicated. Figure adapted from Bouvier and Palese [71].

#### *5.1. Sphingolipids and IAV Entry*

The influenza virus's envelope, derived from the host cell's plasma membrane, consists of a lipid bilayer decorated with the viral hemagglutinin (HA), neuraminidase (NA), and M2 proteins. The lipidome of purfied IAV particles consists of glycerophospholipids and sterols (primarily cholesterol) [72,73]. Further, almost all sphingolipid classes were detected in the viral envelope [72]. Attachment and entry into host cells requires interactions between the viral HA, concentrated in microdomains on the viral envelope, and sialic acid residues present on the cell surface. These microdomains, paralleling the lipids rafts present on the cell surface, are enriched with cholesterol and various sphingolipids, including sphingomyelin and glycosphingolipids [74–76]. Residues in the transmembrane domain and cytoplasmic tail of IAV HA are important for its association with lipid microdomains [75,76]. Disruption of these microdomains has an adverse effect on viral attachment. Sun and Whittaker showed that pretreatment of IAV virions with methly-β-cyclodextrin to deplete envelope cholesterol resulted in reduced viral fusion and infectivity [77]. Though not examined in their study, the authors suggested that cholesterol depletion perturbed the organization of HA in envelope lipid microdomains. Viral infectivity has been found to be reduced by approximately a thousandfold when HA fails to associate with lipid microdomains [75]. Similarly, it is expected that depletion of the IAV envelope sphingolipds would adversely impact IAV binding and infectivity.

#### *5.2. Sphingolipids and IAV Replication*

Following binding to host target cells, influenza virus enters the cytoplasm via receptor-mediated endocytosis. The viral M2 ion channel protein allows the influx of protons into the virion, triggering the release of viral ribonucleoproteins (vRNPs) into the cytoplasm. The vRNPs, made up of viral negative-stranded RNA, viral NP, and the viral RNA polymerase complex, then travel to the nucleus for transcription and replication of the influenza virus genome. Research suggests that products derived from sphingolipids are involved in influenza virus's genome replication. Seo et al. [78] found that cells infected with influenza virus possessed increased levels of the enzyme sphingosine kinase (SK1), which converts sphingosine into sphingosine 1-phosphate (S1P). The authors further showed that inhibition of SK1 impaired viral RNA synthesis and the subsequent nuclear export of newly generated vRNPs. Similarly, it was demonstrated that SK1 is critical for the nuclear export of viral proteins (NP, NS2, and M1) involved in transporting vRNPs from the nucleus to the cytoplasm [79].

#### *5.3. Sphingolipids and IAV Egress*

Like several other enveloped viruses, influenza uses "raft-like" microdomains on the cell surface as platforms for viral assembly. Newly synthesized HA and NA concentrate in microdomains enriched for sphingomyelin and cholesterol [76,80–82]. Tafesse et al. [12] showed that perturbation of host sphingomyelin biosynthesis adversely impacted the trafficking of influenza virus HA and NA to the cell surface, which in turn impaired viral maturation, budding, and release. Additionally, though abbreviated treatment with methyl-β-cyclodextrin at the late stages of infection was found to increase the release of viral particles from infected MDCK cells, the infectivity of the released particles was significantly reduced, as their envelope possessed lower contents of cholesterol and disrupted raft microdomains [83].

#### **6. Conclusions**

The role of sphingolipids and glycosphingolipids has been overlooked for many years in viral infection due to the difficulty of detecting or measuring these lipids. However, with the advent of lipid mass spectrometry, it is now possible to accurately determine the level of sphingolipids and glycosphingolipids in virus-infected cells and virus particles. Recent findings clearly imply that many enveloped RNA viruses have evolved to leverage sphingolipids and/or glycosphingolipids to enter target cells, replicate their genome, or form new virus particles enriched with these lipids (Figure 6).

Despite differences in the viral proteins and corresponding host cell receptor(s), the presence of sphingolipids in the envelopes of HCV, flaviviruses, HIV, and IAV appears critical for the proper organization of viral envelope proteins within microdomains to facilitate viral entry. Correspondingly, cell surface receptors required for viral adsorption and subsequent entry are concentrated in sphingolipid-rich microdomains present on host cell membranes, with sphingolipid itself serving as an alternative receptor for some viruses (e.g., HIV). Several of the viruses (e.g., HCV and DENV) discussed utilize specialized sites for genome replication. The trafficking of viral genomes and required replication platforms appear dependent on vesicular networks which involve lipid moieties, including sphingolipids. Lastly, the morphogeneses and egressions of the viruses discussed require the trafficking and assembly of components to budding sites on host membranes enriched for sphingolipids. Continued research is needed to ascertain the role of sphingolipids in the pathogenesis of these viruses and other enveloped RNA viruses.

Sphingolipids and glycosphingolipids are critical for membrane integrity and depleting them can have deleterious impact on distinct tissues or organs. However, minor changes in host glycosphingolipids can have a much greater impact on viruses that need them for successful infection. Hence, there is a need to develop more pharmacological inhibitors targeting sphingolipid and glycosphingolipid metabolic pathways. These inhibitors have the potential for a broad-spectrum antiviral activity. In addition, they can be combined with existing antivirals to increase their effectiveness and reduce the cost of these drugs.

**Figure 6.** Sphingolipid involvement in the infection cycle of clinically important viruses. The three lines indicate the steps involved in the replication of HCV and flaviviruses (red), HIV (green), and influenza virus (yellow). Reported roles for lipids (sphingomyelin [SM], glycosphingolipids [GSL], and/or cholesterol (Chol)) in the lifecycle of each virus are indicated.

**Author Contributions:** K.V.K. and E.J.Y. contributed equally to the final manuscript.

**Funding:** K.V.K. is supported by the Albany Medical College Bridge Grant. E.J.Y. is supported by an Albany College of Pharmacy and Health Sciences Scholarship of Discovery Grant.

**Acknowledgments:** The authors thank Tammy Garren for her input on the design of figures and Carlos de Noronha for his assistance with the editing of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
