THE ROLE OF MOTOR PROTEIN KIF4 DURING VIRAL INFECTION AND ITS CLINICAL POTENTIAL

1. Institute of Pathological Sciences, Department of Medical Microbiology, Laboratory of Molecular Virology, Semmelweis University Nagyváradter 4, 1086, Budapest, Hungary. 2. Immunology Department, EötvösLoránd University PázmányPéterstny. 1, 1117, Budapest, Hungary. 3. Clinical Experimental Research Institute, Department of Translational Medicine, Semmelweis University Tuzoltó u. 37-47, 1094, Budapest, Hungary. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 05 January 2020 Final Accepted: 07 February 2020 Published: March 2020

110 known to play a role in many types of cancer development although this role might differ in different types of cancer, and they have also been described as having a role in viral infection, notably during viral egress (Nelson and Guyer, 2012).
Kinesin consists of two heavy chains and several light chains. The kinesin heavy chain (KHC) consists of an NH 2terminal globular motor domain that has an alleged ATP-binding site and a microtubule-binding site, a central αhelical coiled coil stalk domain, and a COOH-terminal fan-like domain that interacts with light chains and vesicles (Sekine et al., 1994). An ~360-residue globular domain is their main feature. This well-conserved domain contains both a catalytic pocket for the hydrolysis of ATP and the binding sites for microtubules, being often referred to as the 'catalytic core'. Most KIFs form a long filamentous structure, with the globular domain at one end and a fanshaped structure that associates with light chains at the other end in the case of the kinesin heavy chain, as revealed by electron microscopy (Miki, Okada and Hirokawa, 2005). The catalytic core domain at one end is also called the 'head', followed by the stalk region and finally the 'tail' domain at the opposite end of the molecule. The 'head' domain is responsible for the movement driven by the hydrolysis of ATP whereas the 'stalk/tail' domain is important for the interaction with other subunits of the holoenzyme or with cargo molecules. A short region between the 'head' and 'stalk', namely the 'neck', often contains family-specific features.
KIF4 is plus-end-directed and contains a characteristic N-terminal motor domain that binds to microtubules and ATP, being responsible for force generation along microtubules. It also contains a central stalk region involved in dimerization and a C-terminal tail believed to mediate binding of cargo (Martinez et al., 2008). Its motor domain (Figure 1a) has been described by (Chang et al., 2013) as having a layer of central β-sheets between two layers of αhelices (Figure 1b), and the N-terminal of the catalytic core forming both the top α-helix and the central β-sheet layers, containing the ATPase reaction center. ATP is trapped in a shallow groove on the top surface of the catalytic core formed by the phosphate-binding loop (L4 or P-loop). The C-terminal half of the helix (α3) and the following loop (L9 or switch I) are also located near the catalytic center and contribute to the effective hydrolysis of ATP. The C-terminal half of the catalytic core forms the bottom α-helix layer that contains five structural elements: loop L11, helix α4, loop L12, helix α5, and loop L13. This region is known as switch II because of its analogy to the switch II structural element in G proteins, which serves as the binding surface for the microtubule (Figure 1b). 111

Kif4's Interactions with Gag Polyprotein During Viral Infection:
Just like our cells, viruses also need to maintain stability during their life cycle, and for that they use and manipulate the host cell's machinery for membrane trafficking, transcription, splicing, nuclear pore transport and protein synthesis (Sodeik, 2004). Almost all viruses are capable of taking advantage of their hosts' cytoskeleton to facilitate their replication and spread, with several viruses using the microtubules to transport their genetic material from the plasma membrane to the replication center of the infected cell, and, in some cases, also during viral egress of newly synthesized viral proteins from the nucleus to the plasma membrane where assembly of new virions takes place, after which they can exit the cell either through exocytosis or by budding to the plasma membrane ( Stabilization of microtubules increases the efficiency of retroviral infection (Naghavi et al., 2007), and this is achieved by microtubule plus-end tracking proteins (+TIPs) that are recruited to dynamic microtubule ends by the end-binding protein EB1. (Sabo et al., 2013) has demonstrated that during HIV infection the HIV-1 matrix protein targets KIF4 that subsequently binds to the EB1 protein, therefore inducing microtubule stabilization through posttranslational modifications such as detyrosination and acetylation. The EB1 protein also recruits plus-end tracking proteins (+TIPs) to the dynamic microtubule ends for regulation of the microtubule stabilization process.
The motor protein KIF4 has been shown to help maintain stability of retroviruses during infection, and most studies until the present time have been focused on human immunodeficiency virus type 1 and murine leukemia virus and their interactions with the retroviral Gag polyprotein, a very important protein that directs the assembly and release of virus-like particles from the cell (Tang et al., 1999), thus being essential for viral survival and spread. Gag polyproteins must multimerize, bind to membranes, and then assemble into a virion that is released from the cell for a viral particle to form. KIF4 binds to Gag polyproteins in vivo and KIF4-Gag association can be detected in normal cells infected by a retrovirus (Kim et al., 1998). Disrupting its function slowed temporal progression of Gag through its trafficking intermediates and inhibited virus-like particle production. Knockdown of KIF4 also led to increased Gag degradation, resulting in reduced intracellular Gag polyprotein levels (Martinez et al., 2008). When KIF4 is reintroduced, normal levels of virus-like particles production are restored. These studies identify a novel transit station through which Gag traffics en-route to particle assembly and highlight the importance of KIF4 in regulating HIV-1 Gag trafficking and stability, flagging Gag degradation as a unique antiviral strategy that could be exploited as a therapeutic target for intervention during HIV infection (Martinez et al., 2008).

Kif4's Clinical Potential:
The ability to genetically tag viral proteins with fluorescent proteins (FP) has advanced the study of viral entry and egress by allowing the real-time visualization of these chimeras in live cells (Nelson and Guyer, 2012). It is now possible to visualize and modify the dynamics of single motor proteins with unprecedented spatial and temporal resolutions, while structural studies have provided detailed information on the molecular conformations during the biochemical processes associated with the molecular motor motion (Kolomeisky, 2013).
Viruses can use alternative strategies for intracellular transport. One of them is to invade cytoplasmic membrane traffic. What happens is that viruses pass through the endocytic pathway to the cell center during viral entry, and after budding, virions can travel inside vesicles derived from the endoplasmic reticulum and the Golgi apparatus to the plasma membrane for viral egress. Viral components can also interact directly with the cytoskeletal transport system through direct interactions between cytosolic viral components and the cytoskeleton (Sodeik, 2004).
As pathogenic cargos, viruses require microtubules to transport to and from their intracellular sites of replication. Changes in the spatial organization and dynamics of the microtubule array mediated by virus or host-induced changes to microtubule regulatory proteins, not only play a central role in the intracellular transport of virus particles but also regulate a wider range of processes critical to the outcome of infection (Naghavi and Walsh, 2017).
Analyzing and understanding the underlying principles of KIF4's retroviral cytosolic transport can aid the design of viral vectors to be used in research as well as human gene therapy, and in the identification of new antiviral target molecules. This may lead to the identification of new targets for the development of antiviral therapy with drugs that do not inhibit viral enzymes, but specific host or virus-host interactions and are thus less likely to promote the evolution of drug-resistant viral strains.
Virus-based expression vectors should include all factors that modulate the host cytoskeleton during entry and transcription, but not those involved in virus replication and egress (Radtke, Döhner and Sodeik, 2006). According to (Elsner and Bohne, 2017), the main principle of viral vector design is the identification of viral genes or elements needed for transgene delivery and deletion of the remaining sequences to generate coding capacity for the gene of interest. In a second step, the essential genes for vector production are provided on separate plasmids yielding single-round infectious viral particles.
All retroviruses share the overall genome structure including the gag, pol, and env genes ( Figure 2). Gag and pol encode the structural proteins and replication enzymes, whereas env gives rise to the envelope protein anchored in the membrane enclosing the capsid. However, complex retroviruses like lenti-and spuma viruses encode additional proteins that impact on virulence and pathogenesis (Coffin, Hughes and Varmus, 1997). Although the intracellular transport of Human Immunodeficiency Virus type 1 (HIV-1) Gag to the plasma membrane remains poorly understood, and cellular motor proteins responsible for Gag movement are not known, the interactions between retroviral Gag polyproteins and the kinesin motor protein KIF-4 suggest that large multiprotein complexes are translocated on the microtubule-based cytoskeleton in virus-expressing cells (Tang et al., 1999). Martinez et. al (2008) showed that disruption of endogenous KIF4 slows progression of newly synthesized Gag through its trafficking intermediates and that KIF4 might control Gag polyprotein stability. As previously mentioned, when KIF4 is disrupted, degradation of intracellular Gag is greatly increased. This example provides evidence that viral ribonucleoprotein translocation is dictated by the activities of a variety of viral and cellular proteins (Cochrane, Mcnally and Mouland, 2006) KIF4's interactions with retroviral Gag polyproteins could mediate a plus-end-directed transport of the reverse transcription complex during entry, as well as the transport of RNA granules to the plasma membrane for assembly (Sodeik, 2004). This could lead to the identification of new targets for the development of antiviral therapy with drugs that do not inhibit viral enzymes but instead inhibit specific host or virus-host interactions, being less likely to promote the evolution of drug-resistant viral strains (Radtke, Döhner and Sodeik, 2006). All this could be potentially exploited for the development of therapeutic agents that block HIV-1 or other retroviruses viral particles production through KIF4 targeting.