IS GENOME PACKAGING IN SMALL PLANT VIRUSES ENERGY INDEPENDENT?

A. Mohanty¹, J. Kumar¹, T. Ranjan^1,*, R. R. Kumar¹ and K. Rajani²

¹Department of Molecular Biology and Genetic Engineering, Bihar Agricultural University, Sabour, Bhagalpur 813 210, Bihar, India

²Department of Seed Science and Technology, Bihar Agricultural University, Sabour, Bhagalpur 813 210, Bihar, India

*Corresponding author’s E mail: mail2tusharranjan@gmail.com

ABSTRACT

Genome packaging is a critical step during the viral maturation process. Viruses employ a distinct approach to package their genetic materials inside capsid: ranging from very simple strategy of nucleation of capsid proteins onto genome to complex segro-packasome machinery. The majority of small plant viruses, which falls under type I passive system, package their genome into stable virions in the cytoplasmic compartment, where chances of co-packaging of host RNA is very high, indicates viruses evolved the mechanism of selective and stringent packaging of their genomes. Recent discoveries of the unique ATPase fold in the capsid proteins of smaller plant viruses and their direct or indirect role during genome packaging have changed the perception about genome packaging in type I system. We feel that viruses of type I system have acquired unique and independent innovations for genome packaging over the course of evolution. The molecular interactions, intriguingly, cross-talk between capsid proteins and conserved signal sequence situated at the end of genome, plays an important role while viral genome packaging and translocation. Strategy utilized by smaller plant viruses for enhancement of selective genome packaging does not depend simply on nucleation of capsid proteins over genome but interestingly, configuration of viral genome, replicase, tRNA, viral encoded movement proteins are the other important key players that regulate genome packaging. The main aim of this review is to discuss and revisit the mechanism of genome packaging among viruses of agronomic importance. This study will be also useful for understanding the origin and evolution of viral genome packaging apparatuses and their roles in eukaryogenesis.

https://doi.org/10.36899/JAPS.2023.1.0608
Published first online September 20, 2022

INTRODUCTION

Packaging of the genome into the capsid is the crucial step during the assembly of virion particles. Almost all viruses basically employ three unique mechanisms to translocate their genome inside the capsid coat. In the type I system, capsid proteins (CP) recognize and condense with the genome and form a shell (Chelikani et al. 2014a), employ in small plant and animal viruses with genome size lesser than 20kb (most of the plant viruses like TMV) (Burroughs et al., 2007; Kumar et al., 2022; Ranjan et al., 2021). The genome condensation and capsid assembly in these viruses are coupled by coating the genetic material with viral capsid proteins without utilizing ATP leading to virus assembly (Kutluay et al., 2010; Ranjan et al., 2021). On the basis of configuration, the genomes of viruses infecting plant can be either non-segmented or segmented and are encapsidated into more than one or a single virion, respectively. In some of the plant pathogenic viruses like Bomovirus, multipartite genomes are encapsidated in three different virions of identical size and morphology. This stringency towards selection and packaging of viral RNA is achieved by specific interaction between capsid protein and nucleic acids viz., (1) protein-protein interactions for capsid assembly, (2) DNA/RNA-protein interaction for nucleation of capsid proteins on the genome, (3) sequence independent and dependent DNA/RNA-protein interactions for stabilization of the encapsidated genome and to avoid encapsidation of non-viral genome respectively within the mature virion particles (Vriend et al.,1986; Catalano, 2005). The unique packaging signal or OAS (origin of assembly sequence) such as stem-loop like structures, 3’-tRNA-like sequence (TLS) and 5’-untranslated region (UTR), at the genome termini are recognized by capsid protein (CP) distinguishes the viral genome from cellular genetic material (Kumar et al., 2022; Ranjan et al., 2021). For example, in case of HIV, 5’UTR act as a nucleation target for capsid assembly (Catalano, 2005; Choi and Rao, 2003). Apart from these unique packaging sites, some other factors viz. host encoded tRNA, viral replicase and movement proteins, etc. increase the efficiency of genome packaging (Choi and Rao, 2003; Rixon, 1993). In majority of plant viruses, RNA is extraordinarily compact with high degree of folded structure after their packaging inside capsid (Larson et al., 2005; Tang et al., 2001). Initially, the partly folded RNA might serve as a platform for CP assembly and later lead to the energetically favorable double stranded helices, stem-loops and folded tertiary structure of RNA (Schneemann, 2006). The CP basically serves as chaperone for genome folding and provides optimal interactions required to retain the folded RNA/DNA inside the capsid (Chen et al., 1989; Fisher et al., 1993; Ranjan et al., 2021).

The discovery of CP with a unique ATP binding and hydrolysis motifs such as repeated Walker A, Walker B, sensor and arginine motif in several plant viruses has considerably changed the opinion about energy independent type I packaging system (Kumar et al., 2022; Rakitinaa et al., 2005; Ranjan et al., 2021). Interestingly, mutation and knockdown of the ATPase domain resulted in production of noninfectious with nucleic acid-deficit virions indicating its direct role in the genome packaging (Kumar et al., 2022). Surprisingly, apart from CP, recruitment of other accessory ATPases (for example P4 ATPase, VP3 ATPase, Hsp-70, Rep andP10 ATPase etc.) during process of virus assembly made type I packaging system very interesting (Alzhanova et al., 2001; Ranjan et al., 2021; Wang et al., 2005). However, the molecular machinery that carries out the complex operation of genome encapsidation in plant viruses with remarkable fidelity remains to be discovered and understood.

Both type II and III system are ATP dependent active packaging apparatus (Chelikani et al., 2014a; Ranjan et al., 2021) and operate in viruses with large genome size (>20kb) (Burroughs et al., 2007). The well-known type II packaging system also known as the phage terminal-portal system, makes use of terminase protein to dock the genome onto the portal situated at a vertex of preformed empty prohead and translocate inside (for ex T4, T7, N4 and lambda phages) (Kondabagil et al., 2006; Yang et al., 2003). The recently discovered type III system includes giant viruses (Mimivirus, Pandoravirus etc) with genome size up to 2.5Mbp employs a unique prokaryotic segro-packasome like machinery to encapsidate their genome inside pre-assembled prohead (Chelikani et al., 2014b; Iyer et al., 2004).

The present review aims to upgrade type I genome packaging system with more emphasize on viruses infecting plant. Until now, reported plant viruses are classified in to 80 genera; out of which 53 possess icosahedral and 25 genera exhibits helical symmetry. The remaining two have unknown capsid symmetry (Catalano, 2005). In spite of differences in terms of architecture and host, the packaging of infectious virions is basically coordinated by precision interaction and nucleation of CP onto the nucleic acid (Catalano, 2005; Ni et al., 2013; Ranjan et al., 2021; Sarah et al., 2011; Zhu et al., 2015). The final destination for matures plant virion particles is their long distance spreading from cell-to-cell and their procurement and dissemination to new hosts by vectors such as insect, nematode, mite and fungi (Vriend et al., 1986; Benitez-Alfonso et al., 2010). Thus the detailed knowledge of mechanism of assembly and genome packaging in plant viruses is essential for understanding their biology. Plant viruses are responsible for huge losses in crop production and cause 50-60% of total damage alone (Kreuze et al., 2020). Targeting viral maturation steps may prevent huge economical loss (Kumari et al., 2021; Kumar et al., 2022). This kind of study will not be only helpful for agricultural biologists but also would be useful for understanding the origin and evolution of packaging mechanisms and role of small viruses in eukaryogenesis.

Genome packaging in small viruses infecting plant: Plant virus assembly takes place by the nucleation of CP onto nucleic acids instead of direct translocation of genome inside the empty prohead (Rao et al., 2005; Chelikani et al., 2014a) (Figure 1A & Figure 2). During infection, viruses enter, disassemble and release their genetic material inside the cytoplasm (Figure 1A &B) (Rao, 2006). Early translation synthesizes many viral encoded proteins such as movement protein (MP) (Figure 1C.II) and simultaneously during the progress, a vesicular-like structure called the ‘replication factory’ assembles in the cytoplasm due to perturbation of inner membranes (Figure 1C.I) (Rao et al., 2005). The replication factory is equipped with different proteins involve in multiplication of viral genome. After amplification of genomic copy number, the CP and MP synthesis takes place in a large amount at late translation stage (Figure 1D, E, F& 1E.I) (Francki et al., 1985; Buck, 1996; Catalano, 2005). The CP has the ability to discriminate the viral nucleic acids from the pools of host cytoplasmic RNA (Francki et al., 1985). It seems that viruses have evolved with efficient machinery for selective packaging of their genomes inside coat (Ranjan et al., 2021; Fox et al., 1998). The stringency towards selective packaging of genome is accomplished by many factors viz., configuration of nucleic acid, packaging signals, interaction between nucleic acid-protein, protein-protein and some additional factors for example tRNA (Rao, 2006). Finally, CP recognizes the signals at the genomic end and start co-condensing over it (with or without hydrolyzing ATP), lead to the generation of mature virus particles (Figure 1G&H, Figure 2). Our extensive bioinformatics data has found a novel ATP binding and hydrolysis motifs on the polypeptide chain of CP, suggesting their role during the viral maturation process (Kumari et al., 2020; Kumar et al., 2020; Kumar et al., 2022; Ranjan et al., 2021). Apart from this, enough studies have been done by our lab which further confirms the role of ATP in genome packaging and virus assembly process (Kumar et al., 2022; Ranjan et al., 2021). Co-condensation is usually arranged in a helical fashion throughout the RNA genome in TMV (Figure 2). Finally, MP helps in spreading of mature virions to the neighbor cells (Figure 1I). MP either transport virus particles through the enlarged pore of plasmodesmata (Figure 1I) or directly allow the genome as nucleoprotein complex to the adjoining cells (Figure 1C.III & IV) (Kumari et al., 2021; Sunpapao, 2013). At last, newly assembled viral particles exit from the cell and are transmitted to another host by the help of insect and other means (Figure 1J).

Factors governing genome encapsidation in plant viruses

Genomic Organization: Genome of plant viruses exhibits different forms ranging from segmented to non-segmented. Most of the spherical plant virus possesses partite genome. Viruses (such as Tymovirus, Sobemovirus, Luteovirus, and Tombus virus) with monopartite genome (Table 1) encode an extra sub-genomic RNA (sgRNA), which is packaged into a separate particle. Bipartite viruses with two RNA copies either package or translocate their RNAs into a single virion particle (Diantbovirus) or in two separate virions particle (Comovirus and Nepovirus) (Table 1). Bromovirus, Cucumovirus and Alfamovirus are typical example of multipartite virus (Koev and Miller, 2000). These Icosahedral plant viruses encode more than two segments of RNA (Table 1). In Bromovirus and Cucumovirus, first two segments of RNA package into two separate virion particles but third RNA segment package in a separate compartment along with their sgRNA. Interestingly, in case of Alfamovirus, all the three segments and one sgRNA are package inside four distinct particles (Table 1). Study suggests that packaging of different segments into a single or separate capsid may be co-operative and in other words, packaging of one segment further helps other segments to be encapsidated (Koev and Miller, 2000). Apart from genomic RNA, many of the plant virus needs some secondary RNA such as sgRNA, satRNA during encapsidation/or genome packaging. Although these RNAs does not have direct role during infection process but their involvement during gene regulation has been reasonably well understood (Koev and Miller, 2000; Catalano, 2006). In case of plant and animal icosahedral virus (Polio virus and Hepatitis A virus), (+) ssRNA arranged in stem-loop like secondary structure and helps in nucleation of capsid protein with high affinity. Studies have shown that disruption of such kind of structure leads to the abruption of mature virions production (Frolova et al., 1997). Other secondary structure like 5’-UTR with single stranded RNA dimer in case of HIV allows Gag protein (a major capsid protein) to interact with each other and oligomerize over it. And later Gag protein docks the genome to the membrane raft of host where assembly takes place by the aiding of other structural proteins (Momany et al., 1996).

Table 1: Characteristic features of majority of plant viruses with their typical example.

The sum of nucleic acid that can be packaged inside the spherical/icosahedral/helical virion depends upon the interior capacity or volume of protein shell and this may differ from virus to virus. For example, Cowpea mosaic virus package their genome segments I and II (3.2 & 5.8kb, respectively) in to the two different capsids with an inner capsid volume of 4.32×10^-6 Å. The nucleic acid density in human picornavirus is higher than that of crystalline duplex RNA (Johnson et al., 1997). The extent of distribution and arrangement of hydrogen bonding inside virions can be solved by several techniques like solution X-ray scattering, Raman spectroscopy and neutron diffraction (Schmidt et al., 1983; Li et al., 1992; Timmins et al., 1994).

Molecular Interactions: Elimination of host RNA and precise viral genome packaging inside the capsid coat is crucially governed by molecular interactions, which could be either protein-nucleic acid interaction or protein-protein interaction (Catalano, 2005). Contribution of above two molecular interactions varies from genera to genera. These interactions operate while capsid-capsid oligomerization and capsid-nucleic acid cross talk during viral assembly and morphogenesis. In the members of genera Tymovirus and Comovirus, virion assembly is predominantly stabilized by protein-protein interaction and oligomerization of capsid protein is not dependent on the presence of RNA (Schneemann, 2006; Rao, 2006). Assembly of several plant viruses is guided by mostly nucleic acid-protein interaction, where oligomerization of CP is critically initiated in the presence of genome only as seen in Alfamovirus, Bromovirus and Cucumovirus etc (Schneemann, 2006; Rao, 2006). This kind of physical interaction ensures the encapsidation of viral genome exclusively into the progeny virions. One of the structural proteins of picornavirus viz., VP3 with ATPase activity behaves like chaperone and helps in condensation of genome with the icosahedral symmetry (Zhu et al., 2015).

We divide protein-RNA interactions into two subtypes, which could be either sequence specific or non-specific. A specific interaction between nucleic acid and capsid protein leads to the nucleation of other capsid proteins and ensure the exclusive encapsidation of viral genome. Non-specific interactions provide stability or compactness of packaged genome inside the envelope through interaction between N-terminal region of capsid protein (rich in basic amino acids) and RNA molecules (Vriend et al., 1986; Johnson, 2003). Apart from these interactions, genome trans-encapsidation mechanism, where capsid protein of one virus package nucleic acid of other one has also been reported in viruses with similar taxonomic groups (Creamer and Falk, 1990; Hull, 2002).

Impacts of molecular interactions on selective genome packaging: The CP of plant viruses usually do not package the genomes of different viruses after co-infection of the same host. To increase the stringicity of selective packaging, the CP has capability to distinguish between viral genomes. However, studies also suggest nucleocapsid-independent viral RNA packaging in case of Coronavirus (Catalano, 2005, Masters et al., 2019). Genome packaging mechanism is very simple in non-segmented genomic viruses, where only one segment has to go inside a single compartment (Cuillel et al., 1979; Damayanti et al., 2000). In case of bipartite and tripartite viruses, the packaging mechanism is relatively complex as CP has to distinguish gRNA from sgRNA prior to their segregation into individual virions. Virus has to act smartly in distribution of segmented genome to the multiple virion particles. In multipartite virus, genome packaging seems to be co-operative, where RNA binding capability of CP facilitates RNA-RNA interaction. The binding of CP to nucleic acids exposes the packaging signal, which further allows their recruitment and oligomerization (Lazinski et al., 1989; Qu et al., 1997).

The CP with both sequence non-specific and specific nucleic acid binding activity, plays a vital role while selective packaging of viral genome via specific RNA-protein interaction from a large pool of cellular RNAs (Duggal and Hall, 1993). The specific RNA-protein interaction in plant and other viruses is mainly governed by ‘arginine rich RNA binding motif’ (ARM) of CP, which resulted in nucleation of CP onto genome while packaging (Ford et al., 2013). The ARM is found in CP of several plant (such as CMV and BMV) and animal (such as HIV) viruses and first time it was discovered in lambda bacteriophage (Lazinski et al., 1989; Weeks et al., 1991; Wei et al., 1991; Zeltins, 2018). The ARM is consisting of small stretch of basic amino acids with the α-helix, β hairpin and extended chains conformations (Dreher, 1999; Catalano, 2005). This ARM is a hallmark of CP, which lowers the chance of co-packaging of cellular RNAs. It seems that during the course of evolution, viruses have developed stringency towards selective packaging of nucleic acids and minimized the chances of encapsidation of high backgrounds of cellular RNAs (Catalano, 2005; Lazinski et al., 1989; Burd and Dreyfuss, 1994). The CP of sobemovirus has T=3 symmetry, made up of highly flexible basic amino acids rich R-domain and jelly-roll containing motif S-domain (Choi et al., 2000; Tamm and Truve, 2000). Studied have shown that removal of R-domain leads to the capsid T=1 symmetry without RNA, indicates flexibility of N-terminal R-domain has role in protein-nucleic acid interaction and ultimately in packaging. Apart from ARM, R-domain contains another motif which has beta-annulus like structure and thought to impart a critical role in transition of capsid symmetry from T=1 to T=3 (Satheshkumar et al., 2004; Basnak et al., 2009; Sarah et al., 2011).

It has been hypothesized that ARM specifically interacts with RNA in their alpha-helical conformation, which makes them highly flexible prior to communication between CP and RNA (Vriend et al., 1986). This flexibility of N-terminal ARM has a great impact in term of degree of freedom, which increases the probability of interaction to nucleic acids. In case of majority of plant viruses and few animal viruses such as HIV, the helical configuration of ARM makes more accessible and suitable to interact with nucleic acids via salt bridge interaction between basic amino acids (ARM region of CP) and phosphate backbone of nucleic acid (Tan and Frankel, 1995). Interestingly, alpha configuration of ARM is essentially not required for genome packaging in case of Bromovirus, where proline (a helix destabilizer) residues are prominent in CP (Catalano, 2005). Mutational study indicates the contribution of N-terminal ARM (specifically amino acids from 9 to 19) in cooperative recognition and packaging of genome in multipartite viruses such as BMV. Duggal and Hall in 1993 also suggests that amino acids between 9 to 20 residues of ARM critically required for recognition of 5’ 943 nucleotides of RNA1 in case of BMV. Evidences suggest that N-ARM also contains critical amino acids which are more specific to sgRNA (Duggal and Hall, 1993). The N-terminus of CP is highly flexible which tends to be located inside the internal cavity of capsid coat and encapsidate the genome via several kind of interaction with them (Ni and Kao, 2013). Recent high resolution de novo atomic model indicates that not only N-terminal region of CP is solely responsible for genome encapsidation but C-terminal region also critically imparts in selective viral genome packaging. Similarly, C-terminal region is also highly basic in nature and rich with arginine amino acids particularly. Mutagenesis study by replacing arginine residues (R193, R195) with other amino acid shows complete absence of mature virion particle production (Hesketh et al., 2015; Ni and Kao, 2013).

Structure of RNA: RNA can adopt several secondary and tertiary conformations such as 5’ UTR, 3’ tRNA-like structure, and stem-loop and these unique conformations are very crucial for assembly and nucleation of CP (Barry and Miller, 2002; Momany et al., 1996; Wang et al., 1999). Studies indicate that stem loop-like structure at the end of genomes act like a nucleation center for CP during packaging in BMV. Not only these higher secondary structure at the genomic end but overall structure is also important for interaction between CP and nucleic acid. For example, packaging of 300 nucleotide satRNA of CMV is efficiently regulated by the overall structure of RNA rather than any specific secondary configuration at the end (Qu and Morris, 1997; Choi et al., 2000; Choi et al., 2003; Catalano, 2005). Thus, a correlation between structural features of RNA and its interconnection to replication, translation and genome packaging needs to be explored in details and this could be done by the help of advancement in several techniques like x-ray and others. Small angle X-ray can offer us for direct anticipation of shape and size of RNA molecules inside envelope and their interaction with capsid protein.

Based on specific sequence on genomic RNA, three different models were found to operate in monopartite, bipartite and tripartite viruses (Catalano, 2005). Study in monopartite spherical Turnip crinkle virus (TCV) indicates that there are three high affinity CP-binding sites in genomic RNA; one in polymerase gene about 700 nucleotides from 5’ end, second in CP gene about 700 nucleotides from 3’ end and third one and third one in 186 nucleotides fragment at 3’ end. For the efficient and selective packaging of genomic and sgRNA inside confined space of capsid coat, contribution of these CP binding sites is very critical (Wei et al., 1990; Qu and Morris, 1997). The nucleotide region starting from 1410 to 1438 has been found to attain stem-loop like secondary structure in case of Southern beam mosaic virus (SBMV), where CP interact, oligomerize and nucleate to initiate the packaging process (Bink et al., 2003). Similarly, in Turnip yellow mosaic virus (TYMV), packaging is initiated on to the two hairpins like structures at 5’UTR region in genomic RNA (Hacker, 1995; Bink et al., 2003).

In case of bipartite virus, CP recognizes and encapsidate segmented genomes either within the same virion or into a separate virion particle containing each segment of RNA. Experiments suggest that both of the above strategies are being operated in bipartite virus. Irradiation and heat treatment experiment suggest formation of high molecular weight genome complex (RNA heterodimer and multimers) inside capsid after packaging. Based on this observation another strategy, called hybrid model for virus assembly (hybrid of both above strategies discussed) was proposed (Basnayake et al., 2005). Importantly, most these partite viruses have evolved with a mechanism of co-operativity while their genome encapsidation (Hollings and Stone, 1977; Gould et al., 1981; Hamilton et al., 1996). For example, in Dianthovirus, cross-talk between RNAI and II is very essential for expression of CP and genome packaging. Thus the arrangement of genome and interaction of both RNA while CP expression makes their encapsidation into two independent virion particles interlinked (Red dover necrotic mosaic virus and Sweet clover necrotic mosaic virus) (Okuno et al., 1983; Sit et al., 1998). Although in Red clover necrotic mosaic virus (RCNMV), both RNA I & II have their independent origin of assembly (OAS) but RNA II usually possess a minimal OAS site. Most of the viruses have evolved themselves in such a way to encapsidate particular amount of their genome for optimum packaging and slight deviation may lead to the very low packaging efficiency (Basnayake et al., 2005).

Experiments suggest that gRNA I and II package independently into two different particles, but gRNA III and sgRNA IV co-package together form separate virion particles in the tripartite BMV with icosahedral symmetry (Rao, 2000). Despite the fact that gRNA I, II, III, and sgRNA IV have different sizes, all three virion particles are morphologically and physically indistinguishable. This could be the one of the reason of tight regulations during genome distribution into three independent particles. Several experimental studies suggest a critical role of cis-acting elements/signals in assortment as well as packaging. At the end of each of the eight segments of the influenza A virus genome encodes a unique packaging signal (Rao, 2000; Catalano, 2005). The panhandle structures created by base-pairing at the ends of all 11 segments of the rotavirus genome may serve as a site for genome assortment and packing (Sarah et al., 2011).

Transfer RNA: Apart from their primary involvement in protein synthesis and biomolecule formation (chlorophyll, Heme), tRNA has been linked to a variety of additional processes such as replication. In the case of retroviruses, host tRNA acts as a primer during reverse transcription. (Jahn et al., 1992; Marquet et al., 1995). The 3' transfer RNA like sequence (TLS) serves as the origin of replication and primitive telomeres in plant viruses such as Bromovirus, Cucumovirus, and others. According to the findings, cellular tRNA is also detected in various plant icosahedral viruses, such as the Eggplant mosaic virus (Rao et al., 1989; Hema et al., 2005). Till date, we do not have any clue on co-packaging and active selection of cellular tRNA inside the capsid. For the first time, Choi et al in 2005 has suggested the role of tRNA in genome packaging in case of BMV (Choi et al., 2002).

All four RNAs in BMV have a 200-nucleotide-long 3'UTR that folds into a tRNA-like structure (TLS) by imitating themselves. According to the findings, the highly organized structure (such as TLS) enhances CP nucleation and genome packaging. The deletion of 3’UTR leads to inhibition of virus assembly and infectivity of virus can be restored by adding 200 nucleotides (Dreher, 1999; Catalano, 2005). Thus TLS mediated assembly is strictly dependent on its ordered structure and this hypothesis further can be supported by the evidence that TLS from CMV, TMV and tRNA as well as from yeast supports BMV assembly (Zlotnick et al., 2000). This could explain the fact of how viruses take advantage of host tRNA in their genome replication, translation and packaging. The TLS basically act as a scaffold for oligomerization of CP which could be dimers to multimers (Choi et al., 2000; Barry and Miller, 2002; Choi et al., 2002).

Unlike BMV (Infect to monocotyledonous) where packaging of RNA III is dependent on bipartite signal, CCMV (infect dicotyledonous) RNA III does not depend on CP ORF, MP ORF and 3’UTR (Rao, 1997). Apart from this CCMV differ in several aspects from the BMV. For example, CCMV transfer their genetic material between cells through plasmodesmata without utilizing CP but BMV has to ensure assembly of virion particles before movement to neighbor cells (Figure 1). This might be the reason of different mode of genome packaging operating in these two viruses (Schmidt et al., 1983). The genomic RNA of alfalfa virus has 3’ non-coding 112 nucleotide region, which can attain either stem-loop or TLS type structure. The switching between these two secondary structures regulates the transition between translations to replication in these viruses. Like BMV, genomic RNA III of CCMV encodes TLS, which plays a trans-acting role during the packaging of genome (Olsthoorn et al., 1999).

Interestingly, in 2003, Choi and Rao reported that apart from TLS, OAS and UTR, a cis-acting position dependent 180 nucleotide present in the MP ORF is critically required in genome packaging of BMV. Study also suggests that deletion of this region from MP ORF leads to the disruption of assembly but replication and translation was observed efficiently (Wang et al., 1999).

Viral replicase: Apart from multiplication of copy number, a role of viral replicase in their genome encapsidation has also been demonstrated in plant viruses (Annamalai and Rao, 2005).The role of viral replicase in genome encapsidation in many of animal viruses such as Poliovirus, Floch house virus (FHV), and Kunjin virus has already been well understood. Although the precise role of replicase in genome packing is still unknown, but investigations suggest that its knockdown may cause early disruption in genome condensing and encapsidation process inside the capsid coat. Replicase and CP work together to distinguish the viral genome from the cytoplasmic pool and package it (Traynor et al., 1991; Nugent et al., 1999). According to the findings, the lack of a functional replicase results in generation of genome deficit virus particle (Annamalai and Rao, 2005). Size/volume of viral capsid is determined by the interaction between identical CP subunits and conformational changes induced due to these interactions. Interaction between multiple identical subunits of CP sometimes leads to the spherical and icosahedral head conformation. In case of adenovirus and herpesvirus, scaffold protein is known to regulate the correct procapsid assembly. Evidences suggest that absence of scaffold protein leads to the generation of abnormal procapsid which is unable to package the genome (Rixon, 1973). In case of some animal viruses, host encoded histone proteins has also an impact on genome packaging and condensation inside capsid coat. For example, SV40 has minichromosome like structure wrapped with the nucleosome formed with the help of host histone proteins (Polisky et al., 1975).

Conclusion: Mechanisms of genome packaging and encapsidation vary from simple coating of the genome with capsid proteins as in most-small viruses to the highly complex genome packaging mechanisms of larger DNA viruses that resemble prokaryotic chromosome segregation and pumping mechanisms. Here, we have made an attempt to revisit and understand the mechanism of genome packaging in small plant viruses (type I packaging system). Finding of a novel ATP hydrolysis motif in CP of several plant viruses has raised questions about their place under passive packaging system. Using potexvirus and geminivirus as a model, our group is attempting to decipher the mystery of genome packaging in plant viruses. The details mechanism of viral packaging systems will help us in better understanding the viral packaging mechanisms. We believe that the molecular machinery that carries out the complex operation of genome encapsidation in plant viruses with remarkable fidelity remain to be discovered and understood in detail.

Acknowledgement: This work was supported by grants from the Science and Engineering Research Board (SERB), Department of Science and Technology, Govt. of India, New Delhi (India) (File No. SRG/2019/002223) to TR. We would also like to thanks BAU, Sabour for providing the communication number. This article bears BAU COMMUNICATION NO. 712/2019.

Conflict of interest: The authors declare that they have no competing interests.

Compliance with ethical standards: The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

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