Entry of Bombyx Mori Cypovirus 1 Into Midgut Cells in Vivo

____________________________________________________________________________www.paper.edu.cn Letter Entry of Bombyx mori cypovirus 1 into midgut cells in vivo Yu-Rong Tan, Jing-Chen Sun, Xin-Ying Lu, De-Ming Su and Jing-Qiang Zhang* Division of Structural Biology, State Key Laboratory for Biocontrol, College of Life Sciences, Zhongshan University, Guangzhou 510275, China * Corresponding author: Prof. Jing-Qiang Zhang Division of Structural Biology, State Key Laboratory for Biocontrol, College of Life Sciences, Zhongshan University, #135 Xingang West Road, Guangzhou 510275, China Telephone number: 86-20-84112286 Fax number: 86-20-84110108 E-mail address: [email protected] Running title: Entry of BmCPV-1 Keywords: Bombyx mori cypovirus 1, in vivo, intact virion, direct penetration, electron microscopy The total number of pages is nine and the total number of figures is three. 1 ____________________________________________________________________________中国科技论文在线 www.paper.edu.cn Abstract In vivo entry of Bombyx mori cypovirus 1 (BmCPV-1) into silkworm midgut cells was studied by electron microscopy. Virions were observed adhering to the plasma membrane of microvilli of the columnar cells, embedding in the membrane, and settling themselves intact inside the microvilli. These behaviors suggested that intact BmCPV-1 virions enter columnar cells by means of direct penetration through the cell membrane. In addition, goblet cells, muscle cells and the hemocoele were also involved in early events of the virus infection. However, no replication of the virus had ever been detected in these invaded cells except for columnar cells. Keywords: Bombyx mori cypovirus 1, in vivo, intact virion, direct penetration, electron microscopy 2 ____________________________________________________________________________中国科技论文在线 www.paper.edu.cn Cytoplasmic polyhedrosis viruses (cypoviruses, CPVs), the second major group of insect pathogens, belong to the genus Cypovirus in the family Reoviridae [1] These viruses mainly infect midgut epithelial cells of host larvae, and are characterized by the production of huge numbers of virion-occluded polyhedra in the cytoplasm [2]. Viruses belonging to Reoviridae are nonenveloped, double-stranded RNA viruses with segmented genomes. Two modes of virus entry, viropexis and direct penetration, have been proposed for mammalian reovirus, a member of the Reoviridae family [3]. Direct penetration is the major mode of entry for the intermediate subviral particles (ISVPs), which lose all of the outer capsid protein except for the spike protein [4], while intact virions enter host cells primarily by viropexis. ISVPs induce leakage of radioactive 51Cr from host cells and are in close proximity to regions of apparent discontinuity of the plasma membrane. As for CPVs, the mode of virus entry into host cells is not finally resolved. According to Kobayashi [5], the core material of BmCPV-1 is injected via the viral spikes into the cytoplasm of primary cultured midgut cells. Whereas the study on Euxoa scandens CPV (EsCPV) showed that the virions enter the cultured cells by viropexis [6-7]. We herein report the results of electron microscopic observation which suggests direct penetration of intact BmCPV-1 virions into midgut cells of silkworm Bombyx mori. Purified polyhedra of BmCPV-1 were generously donated by Prof. Xinyao Xu (South China Agriculture University, Guangzhou, China). Second-instar silkworm larvae were fed with fresh mulberry leaves at room temperature. Prior to infection, the leaves were smeared with a high concentration of polyhedron preparation (2.3h107 polyhedra/ml) and air-dried. Newly molted second-instar larvae were fed with the virus-contaminated leaves for 6 hours and then replaced with virus-free fresh leaves. Larvae fed only with virus-free fresh leaves were set as the control. For electron microscopy, the posterior half of the midguts from both infected and control larvae were dissected at the following time intervals post infection (p.i.): 1 h, 2 h, 3 h, 8 h, 12 h, 24 h, and 48 h (the start 3 ____________________________________________________________________________中国科技论文在线 www.paper.edu.cn of administration of virus-contaminated leaves is set as zero time). The midguts were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h, washed with phosphate buffer, and postfixed with 1% osmium textroxide in phosphate buffer for 1 h. Then washed again with phosphate buffer, dehydrated in graded series of ethanol, and embedded in Spurr low viscosity epoxy mixture. Ultrathin sections (<60-90 nm) were obtained with an LKB-Nova Ultrotome, stained with uranyl acetate for 30 min, and then with lead citrate for 20 min. Sections were examined under a JEOL electron microscope at 100 kV. During the first 8 hours of virus infection, no formation of the virogenic stroma, the first morphological sign of CPV replication perceivable by electron microscopy, was detected in the midgut cells. The virions observed during this period should occur as the result of invasion and not as the result of replication. At 3 h p.i., virions were observed outside and inside midgut cells. As seen in Fig. 1a, virions released from polyhedra were detected in the midgut lumen, within the peritrophic membrane, and in association with microvilli of columnar cells. Apparently the fibrous peritrophic membrane did not prevent the invasion of virions. Virions adhered to the surface of microvilli, embedded in the plasma membrane, or settled themselves inside the microvilli (Fig. 1b-d). The plasma membrane with embedded virion was discontinuous only at the virus penetration site, and no obvious disruption of the membrane was observed. Some virions were seen close to the proximal end of microvillus, or partially inside it, and some were found free in the cytoplasm (Fig. 1d). It was noted that all these virions retained their integrity of an icosahedron, and seemed not impaired by the entry. Since none of plasma membrane invaginations containing virions, or cytoplasmic vesicles containing virions had been detected, we excluded the possibility of virion internalization by means of viropexis. Therefore, we summarized the process of entry of BmCPV-1 virions into columnar cells as follows: first the virions recognized and attached themselves to 4 ____________________________________________________________________________中国科技论文在线 www.paper.edu.cn the plasma membrane of microvilli by viral spikes; then the virions continued to interact with the membrane, resulting in a barely discernible membrane disruption at the site; and eventually, the virions penetrated intact through the microvilli. Now the invading virions had settled in the cytoplasm of columnar cells, and it is of great interest to know how they would behave later on. In addition to columnar cells, goblet cells, muscle cells and the hemocoele were also involved in early events of BmCPV-1 infection at 3h p.i.. Intact virions were seen in the goblet chamberˈand in the cytoplasmic projections and cytoplasm of goblet cells (Fig. 2a). Occasionally, virions were caught penetrating the membrane of cytoplasmic projections. These observations suggested that virions in the midgut lumen could enter the goblet chamber through the apical opening, and penetrate the cytoplasm of goblet cells through these cytoplasmic projections. Virions were also detected in midgut muscle cells adjacent to the basal lamina and in the hemocoele (Fig. 2b). These observations indicated that some of the virions that entered the cytoplasm of midgut epithelial cells continued to migrate toward the basal portion of the cell, penetrated through the basal plasma membrane, and entered muscle cells or the hemocoele. Still, we should not rule out the possibility that some virions might reach their destination through the intercellular space between epithelial cells. Unfortunately, no virions or virus-like particles were observed at 1 h, 2 h and 8 h p.i.. The reason may be as following: (ν) It is very difficult to catch the transient image of virus entry which occurred within split second, the image of BmCPV-1 entry was not observed except in 3 h samples; (ξ) In vivo, the entry of the virus is asynchronous which makes our work tough and hazardous; (ο) During the eclipse phase of virus infection no virus-like particles or virions could be found yet within the cells. The virogenic stroma and progeny virions were first seen in infected columnar cells at 12h p.i.. The mode of in vivo BmCPV-1 replication was the same as described previously for other CPV infections [8]. 5 ____________________________________________________________________________中国科技论文在线 www.paper.edu.cn The virus particles first appeared in the virogenic stroma with a micronet structure, and many of mature virions were then occluded into crystalline protein forming polyhedra (Fig. 3a). Occasionally, empty virus particles (i.e., viral capsids without the core) were produced, together with partially filled and mature virus particles (Fig. 3b), suggesting that the viral capsid was produced and was subsequently filled with the core material via the spike. In this work, we did not find any signs of virus replication in non-columnar cells of the midgut. The present study provides the first in vivo morphological evidence that intact BmCPV-1 virions enter B. mori midgut cells by direct penetration, as was the case with ISVPs of reovirus [3]. Morphologically, CPV virions are similar to ISVPs in that they lack the outer coat of reovirus except for the spike protein

Load more