Peder Digre BIOL 325 The Tree of Life Scott Freeman 1 June 2012 Introduction Viruses impact the

e entire spectrum of life from bacteria and archaea to protists, plants, fungi, and animals. Although viruses consist of simple machinery and genetic information, they outnumber host cells by at least one order of magnitude. A virus success in propagating its genetic information depends on its ability to transfer its genes to the host cell, replicate those genes, and then produce new virions to infect new cells. With such a wide range of cell morphology across the tree of lifebacterial, archaeal, eukaryotichow viruses parasitize cells, and specifically how viruses use cell receptors to attach to cells, must be significantly different. This review will address the entry of viruses into host cells, with an emphasis on cell receptors if applicable. In many sections, a case study approach is taken based on current research and the mechanisms presented may not necessarily apply to the entire kingdom. Bacteria and Archaea To enter a bacterial cell, viral genetic material needs to migrate through a cell wall containing peptidoglycan (PG) the major morphologic trait that distinguishes bacteria from archaea. Entering the cell from the cell wall, bacteria either have an outer membrane (if gramnegative) or no outer membrane (if gram-positive) followed by a PG layer and the cytoplasmic membrane (CM) (Jakutyt et al. 2011). Normal transport across the cell wall and membrane occurs through the exploitation of a proton gradient with a pH above 7 in the extracellular space. Viruses use this proton gradient to transfer their genetic material into the cell. The same set of steps occurs in almost every bacteriophage (bacterial virus) infection: a virion binds to a general receptor on either the host cell wall or membrane reversibly, meaning

that the virion can potentially detach; the virion binds tightly to a more-specific cell envelope receptor irreversibly; and the genetic information transcends the cell wall and membrane to enter the cytoplasm (Jakutyt et al. 2011). Although the binding sequence determines the specific infectivity of a bacteriophage, the primary interest in this sequence lies in how the genetic information enters the cell. Typically, the phage will have a spiked tail with lysin enzymes to degrade the PG to access the CM, where a pore needs to form to allow further movement of the genetic information into the cytoplasm. The initial infection uses the normal proton gradient to depolarize the cell by the movement of Ca2+ ions to both bacterial envelope structures and the viral structure to support the transfer of nucleic acid. The depolarization of the cell is the main driving force of the viral machinery. Consider viral entry in the infection of Bacillus subtilis by bacteriophage SPP1 as studied by Jakutyt et al. (Figure 1). SPP1 first binds reversibly and then binds irreversibly to the YueB receptor. This interaction causes a conformational change in the phage tail that transfers the viral capsid into the cytoplasm of the host cell. The genetic information enters the cytoplasm. The initial interaction of SPP1 with YueB receptors likely causes the depolarization of the CM. The degree of depolarization is dependent on both the number of YueB receptors and the number of virions attacking the cell. The depolarization causes the movement of Ca2+ and K+ ions to induce conformational changes of the YueB receptor. This allows the DNA of SPP1 to enter through the phage tail. Although the cell exterior in archaea is more similar to eukarya than bacteria, except for the isoprene units in their lipid bilayers, the mechanism for viral gene entry is similar to phage invasion. Although Archaea may be the largest lineage on earth, in terms of number of individuals, as of 2009 about 55 viruses were known to attack archaeans and very few of those

had been studied (Pietil et al. 2009). The phages with tails (similar to SPP1) have been proposed to have a similar mechanism to that which was previously described. While viral infections in archaea are most likely similar to those in bacteria, the methods bacteriophages use to inject their genetic information into the host cell are far from universal, as will be seen in Big6 Eukaryotes, Plants, Fungi, and Animals.

Figure 1: This figure from Jakutyt et al. 2011 shows the 1) reversible attachment of SPP1 2) irreversible attachment to YueB 3) structural modification 4) cell wall degradation 5) and DNA passage.

Big6 Eukaryotes As viruses are essentially fragments of nucleic acid that parasitize host cells to simply replicate their genome, many mechanisms that viruses employ are highly conserved across the tree of life. Bacteriophages and viruses that infect archaea often only inject genetic material into the cell and the host cell infrequently internalizes the capsid. Internalization of the entire virion is much more common in eukarya. This is especially evident with endocytosis of enveloped virusesviruses that are coated in a phospholipid bilayer acquired from the host cells membrane. The process of endocytosis generally begins with the virion attaching to receptors and coreceptors on the host cell surface and then an invagination of the cell membrane forms with the assistance of coat-proteins such clathrin or caveolin. This process is very general and the specific mechanisms are as diverse as the viruses themselves and will be further examined with the examples of Giardiavius infecting Giardia lamblia and Mimivirus infecting amoeba. Giardia lamblia virus (GLV) is a non-enveloped double-stranded RNA virus that infects the eukaryotic protozoa G. lamblia. In a study by Sepp et al., GLV was shown to bind to a specific unknown receptor on the cell surface that mediates the endocytosis of GLV into the host cell. In a related study by Tai et al., GLV was shown to be absorbed into the cell membrane and then peripheral vacuoles migrated to the membrane in response to binding of GLV to a receptor where the absorbed virions were engulfed by the vacuoles and brought into the cell. The GLV virions are further released into the cytoplasm to mediate genome replication. The process described here is typical of non-enveloped RNA viruses, which are replicated in the cytosol and do not need to be transported to the nucleus to undergo replication using the cells polymerases. Unlike GLV, which likely uses clathrin-mediated endocytosis, mimivirus is a doublestranded DNA virus that infects several species of amoeba (but also causes pneumonia in

humans). Mimivirus uses macropinocytosis or phagocytosis for viral entry to the host cell. Unlike clathrin-mediated endocytosis, macropinocytosis occurs independent of receptors and typically only engulfs macromolecules and fluid to form large vesicles. Phagocytosis is mediated by receptors on the cell surfaces and uptakes large particles into the cell. Mimivirus has been shown to enter Acanthamoeba polyphaga, an amoeba, through phagocytosis. However, macropinocytosis and phagocytosis are routes for viral entry typically seen in bacteria, but in a study by Ghigo et al., it was found that human macrophage cells, as well as the amoeba cells, used these methods to uptake viral cells. This provides an alternative to viral entry through clathrin-mediated pit formation endocytosis. This also illustrates that the viral entry method affects the type of cells that can be infected. In this case, only human macrophage cells can be infected. This can provide further opportunity to develop treatment for pneumonia. Although most viral infections begin with a virion attaching to receptor on the host cell surface, how that virion enters the cell and ultimately causes infection can take many forms. From bacterial and archaeal viruses which inject genetic information into the cell to be replicated to eukaryotic viruses which are fully taken up by the cell through endocytosis, many different entry methods can be observed. Giardiavirus and mimivirus are only two viruses that affect eukaryotic cells, but how they infect those cells are vastly different. Land Plants Although the discovery the tobacco mosaic virus in 1886 contributed to the founding of virology, relatively little is known about plant viruses. They are unlike viruses that infect other kingdoms of life because plants do not have receptors on the cell surface that viruses can attach to and enter cells. In this way, healthy plants are essentially impervious to viruses. The only known way for a virus to enter a plant cell is through a wound, most likely caused by an insect,

which are known to be a vectors of plant virus transmission. Therefore the entry of the viruses into plant cells is through straightforward mechanical movement caused by a break in the physical barrier of a cell wall. As such, viruses do not exit plant cells through a membrane or by lysing a cell. The real intrigue of viral transmission in plants lies through the intercellular propagation of viruses. Plants are highly compartmentalized and cell-to-cell communication occurs through plasmodesmata (PD), which are endoplasmic reticulum derived membrane-lined holes that transcend cell walls and link the cytoplasm of two cells. Once a cell is infected with a virus, it spreads throughout the plant via PD. Plant viruses have evolved to be quite small only three to fifteen proteins are encoded as it is advantageous for travel through the small passageway provided by PD (Schoelz et al. 2011). However, PD are still too narrow for a virus to pass through freely so much of their genome is dedicated to proteins that aid their transport. Some plant viruses produce movement proteins (MP) that guide viruses through tubule or non-tubule movement. In non-tubule guided movement, these MPs modify the structure of the PD to expand it slightly to allow the virus to travel through the PD into an adjacent cell. Some MPs form tubules, which result in tunnels completely formed from viral-encoded proteins through which the virus can freely travel. (Figure 2) It is likely that the MP in place also play an active role in transporting the virus through the PD by mechanical movement as opposed to just making a large enough channel (Schoelz et al. 2011) In addition, it is has been suggested that viruses may be able to switch between tubule-guided or non-tubule guided movement depending on the host species or environment (Niehl & Heinlein 2011). In either situation, MPs create a channel large enough for viral material to propagate throughout the plant.

How the MPs physically facilitate the modification of PD to allow viruses to flow between cells was largely unknown until recently. In a study by Amari et al., it was found that there are proteins located in plasmodesmata (PDLPs) that interact with tubule-forming MPs in almost a receptor-like fashion that allow for the assembly of tubules and the transport of virions between cells. When the researchers knocked out genes for PDLPs, tubule formation was significantly reduced. PDLPs are conserved among higher-order plants. The conservation of PDLPs is likely unaffected by evolution as plant viruses do not typically decrease the fitness of the plant significantly. The interaction between PDLPs and viral MPs was an evolutionary development that allowed plant viruses to spread throughout the organism. Since plant viruses typically do not destroy their host cells in their own replication, the propagation of plant viruses often do not elicit a response from the plant. Plant viruses are obligate parasites and depend on the continued life of the plant for their own spread throughout the system (Lee & Lu 2011). As previously mentioned, plants are highly sectorial and do not grow in one symplast, all cells connected by PD. The combination of cell walls and being sectorial has allowed plants to be highly resistant to the effects of viruses and as such viruses have evolved mechanisms such as the production of MPs to continue their life cycle and reproduce.

Figure 2: This figure from Niehl and Heinlein 2011 shows a) non-modified PD b) tubule formed by MPs and c) PD modified by MPs to allow viral transport between cells.

Fungi Perhaps the least amount of information is known about fungi of any kingdom of life. Although viruses were discovered in the late 19th century, mycoviruses viruses that infect fungi were not discovered until 1962. From what is known about fungi, it is apparent that fungi reproduce through hyphal fusion, which produces large continuously compounding structures that can grow to be what are believed to be the largest organisms in existence, as measured by area covered. Because of this unique growth pattern, how mycoviruses reproduce and spread through these large aggregates of fungal cells is remarkably different than any other kingdom of life. Mycoviruses have evolved to use the continued mitotic division taking place through hyphal fusion to propagate throughout an entire organism, with very little extracellular cell entry. Although it is unknown how viruses came to be in fungi, most of the transmission observed today is a result of hyphal fusion. Viruses can spread through mating of two fungi or through hyphal anastomosis (Buck 1988). Because hyphal fusion causes a high frequency of mitosis and cell fusion, it is very efficient for mycoviruses to propogate throughout the organism during mitosis. As such, mycoviruses would be present in hypal compartments from their very formation (Wickner 1992). Since the continued growth of the fungus is also advantageous for the virus, mycoviruses do not lyse their host. Because of how mycoviruses spread and replicate, it is quite common for two or more mycoviruses to infect a fungus at a time. Going further, it is not unreasonable to assume that the majority of fungal organisms are infected by at least one mycovirus (Ghabrial 2009). As mycoviruses are little more than genetic information within the cytoplasm of fungi that replicate, they are also unique among viruses because they do not typically cause symptoms within the host A few exceptions are noted during symbiotic relationship that benefits the fungus

 usually also in a symbotic relationship with a plant by causing fungal hypovirulence, or the ability to cause more infection within a plant (Herrero 2009). This suggests that unlike most viruses, which parasitize host cells, mycocytes have coevolved with fungi to benefit the virus through the reproduction pattern of fungi and the fungi through symbiotic hypovirulence in plant symbiotic relationships. Furthermore, a recent study by Gker et al. in 2011 has shown that phylogenies of two viral familes which infect endophytic fungi, Partitiviridae and Totiviridae, have a significant positive correlation. This finding suggests that codivergence of fungi and viruses is the main cause of mycovirus diversification. The evidence shows that co-speciation has occurred in viral strains and fungal species because mycoviruses are not transmitted outside the host. The specific pattern of viral transmission through hyphal fusion of fungal cells has been the driving force in the evolution of mycoviruses. This example illustrates the importance of the mode of viral transmission even if it is not per the virion/receptor route that we commonly associate with viral transmission. Animals Embarking upon a discussion of viruses that affect animals is familiar territory because it is important to remember that we, as humans, are animals and we spend the vast majority of research time and funding studying viruses that affect us. Like other eukaryotic cells, animal viruses use viral entry proteins, which are typically glycosylated oligomers, to bind to receptors on the cell surface and then deliver the viral genome either through endocytosis or nonendocytosis (Dimitrov 2004). This is the basic mechanism for viral entry into animal cells, which will be examined further with the examples of human immunodeficiency virus 1 (HIV-1) after a discussion of the basics of the modes of entry. (Figure 3)

As discussed with Big6 Eukaryotes, endocytosis involves a virion binding to a receptor, which remains bound until normal cellular processes transport the bound virion into the cell using clathrin or caveolae-coated vesicles where it is released into the cytoplasm. It has also been found that viruses can induce endocytosis (Dimitrov 2004). Non-endocytotic routes are similar to bacteriophage entry as it is often just viral genetic information that enters the cell. Viruses are not restricted to one mode of entry and may use both routes. (Dimitrov 2004). Perhaps the most important attribute of a virus in regards to cell entry is whether it is enveloped or non-enveloped. After a virus binds to a receptor, a conformational change of viral entry proteins or host-cell receptors takes place either by penetration (for non-enveloped viruses) or fusion (for enveloped viruses). A conformational change is effective for entry into the cell as these changes expose hydrophobic sequences in the membrane, which destabilizes it (Dimitrov 2004). Perhaps the most studied of all viruses is HIV-1. From its origins in Africa in the 20th century, it has spread globally and has become one of the leading causes of mortality around the world. HIV-1 is a single-stranded RNA enveloped virus, which has glycoprotein 120 (gp120) on its envelope surface (Bour 1995). Gp120 binds with high affinity to the CD4 receptor on the surface of T-cells. This binding event induces a conformational change that exposes the CCR5 co-receptor for binding to gp120 (Dimitrov 2004). As Dimitrov states, CD4 serves as an attachment receptor that ensures specific binding to CD4-expressing cells and the co-receptor serves as a fusion receptor (114) After these events take place, conformational changes induce fusion. Retroviral genetic material then enters the cell through a non-endocytotic route as seen in Figure 3. Although this is a simplification of the true entry mechanism, it can be seen that entry proteins on the virus itself and multiple receptors on the cell surface play key roles in the entry of HIV-1.

Figure 3: This figure from Dimitrov 2004 shows a)clathrin-mediated endocytosis and b)fusion at the cell membrane (which occurs in HIV)

Conclusion As examined throughout this review, only bacteriophages and animal viruses have a large wealth of knowledge available from years of research. Relatively, not much is known about Big6 eukaryotic viruses, plant viruses, and mycoviruses. Although only a few examples have been presented along with some general mechanisms for viral entrysome involving cell surface receptors, some notit is evident that viral entry mechanisms are as diverse as viruses themselves. Although there are patterns that can be drawn and are helpful to understanding when confronted with a newly discovered virus, new mutations occur frequently and modes of viral entry change. The success of a virus lies with its ability to change and continue to propagate genetic information using a host cell. By understanding how viruses enter a host cell, more is

learned about the host cells themselves and therefore a better understanding about the entire tree of life is attained.

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