Novel Mechanisms of Rotavirus Replication
Published on: 2022-11-28
Rotavirus (RV) cell entry and virion morphogenesis are complex processes that are not yet fully understood even at the molecular level. Slowing down infectious human rotavirus A (RVA) replication in cell culture on ice allowed morphological visualization of the virus particle entry and the assembly of triple-layered particles (virion). Findings suggeste two routes for virus entry and virion assembly. The virus enters the cell by perforating the plasma membrane by a fusion mechanism involving VP5* of the cleaved VP4 protein. After assembling double-layered particles (DLPs) in cytoplasm, they appear to be connected with the endoplasmic reticulum (ER) membrane and become coated with outer capsid proteins (VP4 and VP7) in a coating process. The perforation of the ER membrane is caused by an unknown mechanism following interaction between non-structural protein 4 (NSP4) and the inner capsid protein VP6 of the DLPs. The coating process is closely related to the formation of a hetero-oligomeric complex (NSP4, VP4 andVP7). These lines of evidence suggest the existence of novel mechanisms of RV morphogenesis.
KeywordsMembrane Penetration; Morphogenesis; Rotavirus; Virus Cell Entry
Rotaviruses a (RVAs) were discovered in 1973 as major causative agents of acute gastroenteritis in infants and young children . Rotaviruses (RVs) are nonenveloped viruses, members of the family Reoviridae. RV has a genome consisting of 11 segments of double-strandedRNA surrounded by a triple-layered protein capsid. The RNA segments encode six structural viral proteins (VP1 to VP4, VP6 and VP7) and six non-structural proteins (NSP1 to NSP6). Virus cell entry and virion morphogenesis are complex processes that are not yet fully understoodeven at the molecular level. Findings have suggested two routes for virus entry and virion assembly. Triple-layered particles (TLPs) can enter the cell by either endocytosis or direct cell membrane penetration [2- 4], whereas virion assembly occurs by either a budding process  or membrane perforation . We previously reported a series of morphological investigations of RV using transmission electron microscopy (TEM) [6, 7] and virological investigations . Our knowledge of RV cell entry and virion assembly as explored by these methods has been complemented by other similar studies  and been expanded by numerous more recent investigations [10, 11]. This paper presents the novel RV morphogenesis, presenting findings obtained by TEM and relevant molecular biological studies.
RV particles have a distinctive morphological appearance and three types of particles have been observed by TEM using negative staining (Figure. 1). The RV virions are non-enveloped and triple-layered particles (TLPs) of 100 nm in diameter, including the VP4 that spans the VP6 and VP7 layers from the particle (Fig. 1). Double-layered particles (DLPs) lack the outer layer (VP7 and VP4) and have VP6 of the middle layer exposed on their surface (Figure. 1). Single-layered particles (SLPs), also termed cores, lack the middle layer, resulting in the exposure of VP2. As a unique morphogenic pathway of RV, transiently enveloped particles (TEPs) are formed by the budding of DLPs into the endoplasmic reticulum (ER) lumen , and can be observed by TEM using a thin section method (Figure. 1).
Figure 1: Rotavirus structures.
The panel (1a) indicate one DLP (white arrow) and TLPs in a negative contrast preparation. The panel (1b) indicates three TEPs in the cisternae of the rough endoplasmic reticulum in thin section. TEPs reveale radial bars between the envelope and DLPs, like the spokes of a wheel (1b). These figures are modified from a previous paper (Suzuki et al. 2019). Bars represent 100 nm.
Virus Cell Entry
After RV attachmentto the cell membrane, TLPs can enter the cell by either endocytosis  or direct cell membrane penetration , as evidenced by TEM studies .
To obtain a non-infectious RV with uncleaved VP4, monolayer infected with infectious RV is incubated at 4°C, and 60 min later is washed with cold MEM. Thereafter, the cultures are kept in trypsin-free MEM for 24 h at 37°C. Those non-infectious RVs are capable of being adsorbed on cells at 4°C . VP4 spikes of RV grown in the absence of trypsin are indistinguishable from those of particles grown in the presence of trypsin , suggesting that proteolytic cleavage of VP4 mainly achieves conformational changes enabling viral entry into cells. When human RV grown in a trypsin-free medium is used to infect cells, its mode of penetration into the cytoplasm is mediated by endocytosis (Figure. 2) . Nevertheless, we could not detect any evidence of viral replication. It is suggested that endocytosis of RV is not related to virion assembly.
Figure 2: The endocytosis of non-infectious rotavirus.
The cell is inoculated with non-infectious rotavirus grownin trypsin-free medium and cultivated in trypsin-free medium at 37°C for 24 h. The panel (2a) indicates endocytosis. The panel (2b) indicate DLPs within lysosome after endocytosis of TLPs. These figures are modified from a previous paper (Suzuki et al., 1985, 2019). Bar represents 100 nm.
Direct cell entry
RVs usually grow in vitro in the presence of trypsin, which cleaves the viral VP4 into its subunits VP5* and VP8* to gain maximal infectivity (Estes and Greenberg 2013). The TLP interacts via its VP4 spikes with cellular receptors. The initial cell attachment step is followed by the interactions of viral proteins [VP8* (produced by cleavage of VP4 into VP5* and VP8*) and VP7] with multiple co-receptors .
Direct cell entry
Kinetic studies have revealed that RVs enter cells rapidly . To visualize the cell entry step by TEM, we slowed down the virus entry process at a low temperature (4°C) and successfully showed direct cell membrane penetration by infectious human RV (Figure. 3) (Figure. 4) . The TEM observations of virus cell entry indicated that, at the attachment site of a TLP to the cell membrane, an electron-dense thin line is expelled from the virus core into the cytoplasm through radial space between the capsomere and the cell membrane pore forming its attachment site, suggesting perforation of the cell membrane (Figure.3) (Figure. 4) . This is the first such observation among animal viruses. These results are supported by a paper describing that infectious RVs increase cell membrane permeability, as measured by 51Cr, [14C]choline and [3H]inositol release from prelabelled cells . These early results were supported by subsequent molecular studies. It is conceivable that trypsin plays an important role in dissolution of the outer layer of TLP at the cell membrane to facilitate the entry of viral nucleoids into the cell. VP4 contains discrete functional domains: in vitro treatment of virions with trypsin results in specific cleavage of VP4, and yields subunits consisting of polypeptides VP8* and VP5* with concomitant enhancement of viral infectivity  . The likely membrane-binding surfaces are the set of three hydrophobic loops at the apex of each VP5* β barrel, which is distal to the foot in the spike conformation . Their conformational similarity to the fusion loops of class II and class III viral fusion proteins has been described , and one of them has an amino-acid sequence related to the sequence of the E1 fusion loop of the Semliki Forest virus . Like the fusion loops and peptides of envelopedvirus entry proteins, these hydrophobic surfaces are hidden on the mature virion and exposed only during a conformational change triggered by events that accompany entry .
Uncoating Of Tlps via Perforation of the Cell Membrane
Upon higher-magnification TEM observation using a thin section method, the shapes of TLP and its core change from round to oval (Figure. 3) (Figure. 4), suggesting TLP activation . An electron-dense thin line, probably consisting of nucleic acids of TLPs, is extruded from TLPs into the cytoplasm through perforation of the cell membrane, and finally results in a cotton wool ball-like structure in the cytoplasm, which is visible at a depth of less than 200 nm from the adhering cell surface and soon disappears (Figure. 3), in the same way as in poliovirus cell entry . Our early studies under a TEM using a combination of staining and shadowing methods   indicatedthat EDTA and heat shock treatments cause mild damage to virus particles and viral RNA of TLPs is extruded (Figure. 3) . After discharge of the nucleoprotein under EDTA treatment, empty TLPs with funnel-shaped structures which allow the passage of nucleic acids are shown (Figure. 3), and the same structures are found in culture fluids (Figure. 3). Upon higher-magnification TEM observationusing negative staining, some parts of the nucleocapsid aligned within the funnel-shaped structure are apparently derived from the inner capsid, which consist of a space of radial capsomere sheathed inside by components of the nucleocapsid (Figure.3), probably the class I channel. It appears that the nucleocapsid plays an important role in the process of viral RNA ejection, probably viathe activation of RNA polymerase. However, these early studies had a limitation of not confirming the specific character of the above materials expelled from TLPs. The loss of outer capsid VP7 activates the internal polymerase complex to transcribe capped positive-sense RNA [(+) RNA] from each of the 11 dsRNA genome segments for release into the cytosol ; . The capped transcripts are released through aqueous channels at the five-fold axes of these intact particles . Once these initial transcripts have been translated, RV NSPs then coordinate various stages of genome replication and viral assembly by adapting and modifying the cellular machineryduring this movement of genome ds RNA. VP7 completely covers the tip of VP6, and inhibits transcription by preventing the conformational change of VP6 . This is supported by the findings of a study of transcriptional inhibition by using VP6-specific antibodies . These molecular studies and TEM observations provide grounds for the suggestion that even partial disruption of VP7 protein at the fusion point results in transcriptionally active TLP. However, these key lines of evidence require further detailed investigation. These results led us to conclude that RV uncoating proceedsviatwo steps. At the first step, a change of the outer layer VP4 of TLP occurs by proteolytic digestion by trypsin. The endogenous transcriptase in such particles is in a switched-off state. At the second step, TLP results in fusion of the cell membrane, probably with partial disruption of the VP7 outer layer at the fusion point, followed by conversion into particles with activated transcriptase. It is assumed that this two-step process through the perforation of the cell membrane induces RV uncoating.
Figure 3: Overview of the process of rotavirus cell entry.
For details, see the section on the direct cell membrane penetration. The black arrow indicates the changes of the total and internal shapes of TLP from round to oval (3a). The white arrow indicates the point of perforation of the ER membrane, and the arrowheads indicate the long electron-dense thin line ejected from the core of TLP into the cytoplasm through the space of radial capsomere and the pore of the cell membrane (3a). The series of processes in rotavirus cell entry result in cotton-wool-like structures in the cytoplasm (3b). The panel 3c is TLP visualized by a combination of staining and shadowing: mild disruption of TLP by EDTA. Projection (arrow) is observed on the periphery of TLP, suggesting the release of nucleic acids from active TLP. The panel (d)indicates an empty TLP which some components of the nucleocapsid inside the projection of inner capsid form a funnel-shaped structure (arrow) in trypsin-treated culture fluid. These figures are modified from a previous paper (Suzuki et al., 1986, 2019). Bars represent 100 nm.
Figure 4: The proposed schematic of the rotavirus replication cycle.
The triple-layered particle (TLP) is schematically shown at top. The outer layers of TLP consist of VP4 (magenta bars) and VP7 (yellow). Inner layer of TLP and outer layer of double-layered particle (DLP) consist of VP6 (blue), VP2 (beneath VP6, in black), VP1 and VP3 (greenish brown), and double-stranded RNAs (red bars). Thetransient enveloped particle (TEP) consists of DLP, radial bars (VP4, magenta bars) and envelope (black). VP4of TEPweakly attaches to VP6 via NSP4 and top of VP4 is repressed by envelope, suggesting appearance of shorter size of VP4. For details, see each section on the replication cycle (direct cell membrane penetration, TEP formation, recoating of outer proteins on DLP). The schematic consists of virus entry (top left half), DLP assembly (bottom left half), and TLP assembly (bottom right half), which are divided three areas accordingly by two dotted lines. Top left halfindicatesdirect cell entry and uncoating of transcriptionally active TLP by perforation of the cell membrane. After cell entry of TLP, fine line from inner capsid of TLP results in cotton wool ball-like structure in cytoplasm. Bottom lefthalfindicates the DLP and TEP assembly route viabudding of DLP. DLPs assemble at the periphery of the endoplasmic reticulum(ER) membrane and then TEPs are assembledviabudding of DLP. TEP swell and rupture, resulting in DLPs in the ER lumen. These observations suggest that the budding process serves to create a vehicle to transport DLPs from the cytoplasm to the ER lumen, but does not participate in TLP assembly. Bottom right half indicates the TLP assembly routeviarecoating of outer proteins on DLP in collaboration with hetero-oligomer(orange half balls) at the perforationarea of the ER membrane. The TLP is released from cell through cell lysis. This figure is modified from a previous paper (Suzuki et al., 2019).
B. Triple-Layered Particle (Tlp) Assembly
The review of rotaviruses indicated important points: (a) precise mechanisms of how the envelope on particles was removed, (b) the hetero-oligomeric complexes function in particle budding through the ER, and (c) how the outer capsid was assembled onto the newly . There are two TLP assembly processes in the relationship between DLP and ER membrane, namely, a budding process  and a perforation process . Notably, NSP4 plays an important role in TLP assembly; especially in terms of selecting one of the above two processes (Figure. 4).
Double-Layered Particles (Dlps) Assembly
Role of viroplasm
It is generally accepted that DLPs assembled at the periphery of viroplasm bud across the ER membrane, although no specific mechanism for the exit of DLPs from viroplasm is currently known . Interestingly, DLPs, which appear not to be associated with viroplasm, also show budding (Figure. 5) . This finding contradicts the viroplasm-associated DLP assembly theory as the current model for the assembly of TLP. We generally use the human RV for TEM study, in which viroplasm are relatively rare. The critical concentration of NSP2 outside of viroplasm is far too low for DLPs assembly . It has been shown that primary human RV clinical isolates grow very well in secondary monkey kidney cells, far better than in the permanent cell line MA104 . Furthermore, the simian rotavirus SA11 can be grown at a high titer  and has a high frequency of viroplasm in infected cells . Therefore, SA11 is frequently used for the molecular study of viroplasm in relation to DLP assembly 
Role of Transiently Enveloped Particles (Teps) For Reappearance DLP
TEM study revealed the important finding that TEPs swell and rupture, resulting in DLPs in the ER lumen (Figure. 4) (Figure. 5) . Higher-magnification analysisof TEPs revealed radial bars between the envelope and DLPs, like the spokes of a wheel (Figure. 5) . It is assumed that the spoke structures correspond to VP4 because TEPs contain VP4 . VP4 weakly attaches to VP6, and only the additional assembly of VP7 to the particles lock VP4 proteins in place . Disruption of TEPs is related to viroporin-mediated calcium-activated autophagy . By using an immunogold-labelled mAb to VP4 (K-1532) , the outer capsid layer of TLP is labelled, but neither DLP nor TEP is (Figure. 6) . Serial TEM figures indicated TEP assembly and its disruption to associated with the reappearance of DLP (Figure. 4) (Figure. 5). Since EM studies with viruses are performed with a large number of particles and since virus preparations usually contain a vast excess of noninfectious particles, it is not possible to determine by this method alone whether individual events are part of a pathway leading to productive infection in the case of rotaviruses. It is known that the ratio of physical to infectious viral particles may vary between 100 and 10, 000 . The uncoating efficiency is 20 to 50%; of the uncoated particles, about 10 to 15% synthesized detectable RNA . These results suggest that the high rates of detection of viroplasm and TEPs in infected cells have misled researchers to believe that they play key roles in TLP assembly. Several factors mediate the increase of DLPs. RV-infected cells treated with tunicamycin (TM, N-linked glycosylation inhibitor) are known to accumulate TEPs in the ER lumen and DLPs in the culture medium , . Although TM inhibits the glycosylation of both VP7 and NSP4, studies of the maturation of a variant of SA11 (clone 28), which produces a non-glycosylated VP7, have shown that the glycosylation of NSP4, but not necessarily VP7, is essential for removal of the envelope ; . Viruses produced in calcium free medium, and in the presence of the calcium ionophore A23187, have been found to be exclusively TEPs and DLPs . The structural plasticity of NSP4 is regulated by pH and Ca2+ . Based on these facts, it is conceivable that both the glycosylation of NSP4 and the presence of calcium are indispensable for virion assembly. These observations led us to conclude that the budding process and transiently TEP formation serve to create a vehicle to transport DLPs from the cytoplasm to the ER lumen, but does not participate in TLP assembly (Figure. 4) (Figure. 5). It is known that high rates of detection of TEPs in infected cells and DLP in stool samples from patients. These lead us that as most of human rotavirus are RVA, monovalent rotavirus vaccine (RV1, human rotavirus strain) was highly effective.
Figure 5: Budding process.
The panel (5a) indicates the budding process and TEP formation at the periphery of viroplasm (V). The panel (5b)indicatesTEPsformationvia budding of DLPsat the ER membrane(arrow heads). The panel (4c) showsDLPs formation viar outer membrane disruption (arrowheads). These figures are modified from our previous paper (Suzuki et al., 1984b, 1993, 2019). Bars represent 100 nm.
Triple-Layered Particles (Tlps) Assembly via Perforation System
Recoating Of Outer Proteins on DLP
Arguably, one of the least understood aspects of RV biology is the process by which the assembling particle penetrates the ER membrane to acquire its outer capsid . We successfully and comprehensively characterized the process of recoating of DLPs during their transport across the ER membrane (Figure. 6) (Figure. 7) . At the junction of DLPs and the ER membrane, the lower half of DLPs in the cytoplasm appears to present a smooth surface, which is an original DLP characteristic, whereas the upper half of DLP is an irregular surface with high-density materials in the ER lumen, as in the recoating of VP4 and VP7 (Figure. 6). The outer capsid layer of TLP is labelled by an immunogold-labelled mAb to VP4 (K-1532) (Figure. 6) ; . These results provide evidence that the transport of DLP from the cytoplasm to the ER lumen through the ER membrane results in TLP assembly (Figure. 4) (Figure. 7). However, the perforation of the ER membrane is caused by an unknown mechanism following interaction between NSP4 and the inner capsid protein VP6 of DLP. A number of basic studies in cellular and molecular biology have indicated that the formation of a hetero-oligomeric complex (NSP4, VP4 and VP7) is an important factor for TLP assembly ; . Thus, the TEM observations support the view that, during the recoating process, the VP4 and VP7 outer surface proteins fold onto the DLPs as a hetero-oligomeric complex and result in TLPs, although the correct order of attachment (first VP4 and then VP7) to DLPs is essential for optimal TLP assembly . This is the first direct evidence of outer capsid recoating and TLP assembly as a novel recoating process. The novel mechanism involved in the recoating of outer shell proteins on DLPs remains an important issue in terms of how both DLP and the ER membrane participate in the perforation of the ER membrane. However, no study has specifically focused on this, so further studies are required. In conjunction with these two processes, namely, the budding and perforation processes, TEM observations suggested that the single attachment of VP4 to the ER membrane through NSP4 leads to the budding process, whereas the attachment of a hetero-oligomeric complex to the ER membrane leads to the perforation process (Figure. 2). These results give rise to the view that NSP4 is critical for the selection of the above two processes, and is regulated by NSP4, probably as an ER chaperone. Among the ER chaperones, grp78 (also known as BiP), protein disulphide isomerase, calnexin and calreticulin are important for the formation of infectious virus. Against this background, there is a need for further study to clarify NSP4’s role as the key ER chaperone for virion assembly.
Figure 6: Overview of virion assembly.
For details, see the section on the recoating of outer proteins on DLP and final step of intact virion assembly. The panel (6a) indicates that DLP appears to acquire outer capsid protein during the transport across the ER membrane as a recoating process. The arrowhead indicates the DLP without an outer surface in cytoplasm, whereas the arrow indicates the DLP recoated with outer proteins in the ER lumen (6a). The panels (6b) indicate final step ofTLP assembly at the ER membrane. By using immunogold labelling of KUN-infected cells with mAb to VP4, the outer capsid layers of TLPs are labelled (6c), but not disrupting TEP and its content as DLP are (arrow) (6c). These figures are modified from our previous paper (Suzuki et al., 1993). Bars represent 100 nm.
Figure 7: Various final steps of virion assembly.
For details, see the section on the recoating of outer proteins on DLP and final step of intact virion assembly. The reticular materials of encased TLPs are expanded from the ER membrane (7a to 7f), and finally TLPs are released to the ER lumen as TLP (7d, 7e, 7f). Bars represent 100 nm.
Final step of intact virion assembly
In the ER of SA11-infected cells, there are two pools of VP7, namely, virus-associated VP7 of intact particles and membrane-associated VP7 (Kabcenell et al. 1988). VP7 has a peptidase cleavage site between Ala50 and Gln51, resulting in the removal of a signal peptide . ER retention of VP7, a prerequisite for these steps, requires both the VP7 signal peptide and the first ~31 residues of the mature protein . However, the precise mechanism by which RV particles disconnect from the ER membrane is not well understood. NSP4 has VP6 and VP4 binding domains, and topographically VP4 is localised on the cytoplasmic side of the ER membrane . During the budding process, VP4 (that interacts with the cytosolic domain of NSP4), NSP4 and VP7 are incorporated into TEPs . TEM study has indicated the existence of a final step of virion assembly (Figure. 7) . The outer surface of the TLP stretches from the ER membrane forming bridge, which is eventually disrupted. It is assumed that this process, which corresponds to the scission of stretching VP7 tail from the ER membrane, results in the emergence of virus-associated VP7 of intact particles, as membrane-associated VP7. These TEM observations led us to conclude that the last step of intact virion assembly is key evidence supporting the existence of the perforation pathway for TLP assembly (Figure. 7). NSP4 releases Ca2+ from intracellular stores  by acting as a viroporin . The NSP4-triggered increase of intracellular (Ca2+) activates a kinase-dependent pathway, which leads to autophagy . However, at present, the involvement of unknown factors in the final step of intact virion assembly cannot be ruled out. [42-45].
Although RV is a non-enveloped virus, interestingly, RV utilises cell membrane perforation twice during its life cycle: during cell entry and virion assembly . Further research is needed to fully elucidate the details of these processes. A major challenge for future RV research is to elucidate how viral proteins cooperate during membrane penetration.
I thank Dr. Desselberger U, Department of Medicine, University of Cambridge Addenbrooke’s Hospital, for his valuable advice and constructive discussions on this work.
Conflict of Interest
The author declares no conflict of interest.
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