Remodeling of host membranes during herpesvirus assembly and egress

Many viruses,enveloped or non-enveloped,remodel host membrane structures for their replication,assembly and escape from host cells.Herpesviruses are important human pathogens and cause many diseases.As large enveloped DNA viruses,herpesviruses undergo several complex steps to complete their life cycles and produce infectious progenies.Firstly,herpesvirus assembly initiates in the nucleus,producing nucleocapsids that are too large to cross through the nuclear pores.Nascent nucleocapsids instead bud at the inner nuclear membrane to form primary enveloped virions in the perinuclear space followed by fusion of the primary envelopes with the outer nuclear membrane,to translocate the nucleocapsids into the cytoplasm.Secondly,nucleocapsids obtain a series of tegument proteins in the cytoplasm and bud into vesicles derived from host organelles to acquire viral envelopes.The vesicles are then transported to and fuse with the plasma membrane to release the mature virions to the extracellular space.Therefore,at least two budding and fusion events take place at cellular membrane structures during herpesviruses assembly and egress,which induce membrane deformations.In this review,we describe and discuss how herpesviruses exploit and remodel host membrane structures to assemble and escape from the host cell.     

中国对虾非包涵体型杆状病毒核衣壳的分离纯化

An effective procedure for isolation and purification of nucleocapsids of Penaeus chinensis non-occluded baculovirus (PcNOBV) which has destroyed the Chinese shrimp industry since 1993 was described. Gill, stomach, gut and cuticle epidermis under exoskeleton were excised from cultured P. chinensis diseased with typical white spot syndrome and homogenized in liquid nitrogen with TNE buffer containing PMSF and β-ME. The homogenized mixture was filtered through a 0.45μm millipore filter membrane to remove cell debris and ultracentrifuged to pellet the remaining material.The pellet was suspended in PMTNE buffer and laid onto a handmade CsCl gradient. An obvious viral band was observed in the middle of the gradient. Large amounts of virus nucleocapsids were visualized under electron microscope consistently corresponding to the milk-colored viral band. The viral envelope was all lost after purification. The nucleocapsid was bacilliform averaging 80±13nm×380±24nm in size. The negatively stained PcNOBV nucleocapsids revealed 13-16 conspicuous stripes located periodically perpendicular to the longitudinal axis of the nucleocapsids. Six to seven capsomers of 9 nm in diameter were visualized on each side of the stripe.     

Proteomic Profiling of Purified Rabies Virus Particles

While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus(RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC–MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.     

A new strategy for full-length Ebola virus glycoprotein expression in E.coli

Ebola virus(EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein(GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses.However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 ℃. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.     

Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection

RNA-interference (RNAi) silences gene expression by'guiding mRNA degradation in asequence-specific fashion.Small interfering RNA (siRNA),an intermediate of the RNAi pathway,has beenshown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells.Here,wereport that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) couldinhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-asso-ciated 126 kDa protein in intact plant tissue.Our results indicate that transiently expressed shRNA efficientlyinterfered with TMV infection.The interference observed is sequence-specific,and time-and site-dependent.Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumbermosaic virus (CMV),an unrelated tobamovirus.In order to interfere with TMV accumulation in tobaccoleaves,it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation.Ourresults support the view that RNAi opens the door for novel therapeutic procedures against virus diseases.We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expressioncould be employed as a potent antiviral treatment in plants.     

SARS病毒的形态结构及其在体外培养细胞中的感染装配

Morphological characterization of purified SARS-associated virus(SARS-CoV) from Hubei patient was carried out by negative stain and ultrathin section electronmicroscopy. The spike of isolated SARS-CoV virus is shorter and smaller than Human coronavirus. A large quantity of SARS-CoV particles could be observed in the infected Vero cells. The process of infection, assembly and morphogenesis was observed.     

Nuclear proteome profile of C57BL/6J mouse liver

The liver proteome can serve as a reference to better understand both disease mechanisms and possible therapeutics,since the liver is an important organ in the body that performs a large number of tasks.Here we identify the organelle proteome of C57BL/6J mouse liver nuclei as a promising strategy to enrich low abundance proteins,in the sense that analysis of whole liver cells is rather complex for current techniques and may not be suitable for proteins with low abundance.Evaluation of nucleus integrity and purity was performed to demonstrate the effectiveness of the optimized isolation procedure.The extracted nuclear proteins were identified by 2-DE MS analyses,and a total of 748 proteins were identified.Bioinformatic analyses were performed to demonstrate the physicochemical properties,cellular locations and functions of the proteins.     

Morphological structure and optical properties of the wings of Morphidae

The morphological structure and optical properties of the wings of 14 species of Morphidae have been investigated. Most of the scales of the iridescent species of Morphidae （Lepidoptera） present a very particular structure. The ground scales, responsible for the major part of the optical properties, are covered by a very regular set of longitudinal ridges. The ridges themselves are constituted by a superposition of lamellae that act locally as a multilayered structure. This very specific morphology leads to both interferences and diffraction effects. The first one is responsible of the brilliant blue coloration of the males, while the second one diffracts this colored light at a very large angle. These two phenomena give to the butterfly a very effective long-range communication system. The morphological characteristics of the scales of the various species are presented in detail. Two types of optical measurement were performed on the iridescent wings of 14 different species of Morphidae： spectroscopic measurements under various incidences and gonioscopic measurements for a given incidence angle and wavelength. The first allows a determination of the index of refraction of the cuticular material. The second leads to the drawing of spatial diffraction maps. It shows that most of the reflected light is diffracted laterally over a very large angle （90° 〈 0 〈 120°, according to the different species） and that this repartition depends of the polarization of incident light. As predicted by previous calculations, the dissymmetric structure of the ridge is responsible for the separation of the polarization modes in the various diffraction orders.     

An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% （14 of 101） rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 （DE3） due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B （DE3）, which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.     

Semliki forest virus core protein fragmentation: Its possible role in nucleocapsid disassembly

Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243–255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.     

Isolation and characterization of monopinocytotic vesicles containing polyomavirus from the cytoplasm of infected mouse kidney cells.

Monopinocytotic vesicles containing polyomavirus were isolated from the cytoplasm of mouse kidney cells infected with polyomavirus using sucrose density gradients. Nonenclosed, membrane-associated virions released by the action of neuraminidase separated from vesicle-enclosed virions in the sucrose gradient. Marker enzyme assays indicated the derivation of the vesicle membrane from the plasma membrane of the cell. The 125I-labeled virus enclosed in the vesicle sedimented more slowly in the gradient and was not observed unless infection and endocytosis had occurred. Detergent treatment of virion-containing vesicles caused the release of polyomavirus with sedimentation properties similar to those of purified polyoma virions. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of virion proteins from vesicles containing virions demonstrated patterns of proteins similar to those of purified intact virions. Electron microscopy confirmed the presence of single intact virions inside vesicles. The study of these monopinocytotic virion-containing vesicles represents a further step in elucidating the early events of polyomavirus infection.     

Purification and properties of the Bombyx mori nuclear polyhedrosis virus liberated from polyhedra by dissolution with silkworm gut juice

Occluded virions of the Bombyx mori nuclear polyhedrosis virus were efficiently liberated from polyhedra by dissolution with the silkworm gut juice. The liberated virions were purified by sucrose density gradient centrifugation and the bands of enveloped virions were observed in the gradients. There was no functional difference between the gut juice-liberated and the carbonate-liberated virions. Disruption of enveloped virions by the gut juice was observed, but the formation of nucleocapsids from the degradation of the occluded virions was not detected. High yields of the enveloped virions from the polyhedra dissolved by the gut juice was obtained by separating the virions through sucrose density gradient centrifugation immediately after the dissolution of the polyhedra. Many factors, e.g., rearing seasons, silkworm strains, and rearing conditions, affect the polyhedra-dissolving property of the larval gut juice.     

Isolation and Properties of Newcastle Disease Virus Nucleocapsid

Deoxycholate (DOC) disrupted virions of Newcastle disease virus (NDV), releasing viral nucleocapsids. The nucleocapsids sedimented at about 200S in sucrose gradients and measured from 1.3 to 1.4 mu long by electron microscopy. NDV nucleo-capsids were resistant to pancreatic ribonuclease. These nucleocapsids contained all the 50S ribonucleic acid (RNA) in NDV virions, while virus-associated RNA sedimenting at less than 50S was external to the virions.     

家蚕核型多角体病毒及核衣壳的纯化和其结构蛋白的凝胶电泳分析

 用差速离心和蔗糖密度梯度离心提纯了家蚕核型多角体病毒及其核衣壳,并用SDS聚丙烯酰胺凝胶(均一胶和梯度胶)电泳分析了它们的结构蛋白。测量使用游标卡尺,计算使用微型计算机。凝胶用很染色方法染色。 结果表明,家蚕核型多角体病毒长381.2±17.81纳米,直径88.86±9.605纳米;核衣壳长328.9±5.917纳米,直径41.36±1.167纳米。两种凝胶电泳所得数据显示病毒粒子至少含有20组分子量不同的结构多肽,含量较多的是P31、P40、P28和P20。P45和一些小分子多肽看来主要存于囊膜上。P31的分子量与多角体蛋白相同,我们的实验表明它可能是病毒粒子和核衣壳的结构组分。     

Some properties of influenza virus nucleocapsids

Nucleocapsids released from influenza virions by sodium deoxycholate sedimented heterogeneously in sucrose gradients. Highly infectious virus (complete) preparations yielded nucleocapsids with peak distributions at 64 and 56S; von Magnus type virus (incomplete) lacked 64S nucleocapsids. Treatment of influenza virus nucleocapsids with pancreatic ribonuclease rendered the associated viral ribonucleic acid (RNA) molecules acid-soluble, indicating that capsid proteins do not completely surround the viral RNA's. However, the capsid proteins remained associated after enzymatic hydrolysis of the RNA, as judged by persistently high sedimentation rates. Sedimentation rates of viral nucleocapsids reflected the sedimentation rates of the associated RNA's: 64S nucleocapsids contained 18S RNA, whereas 56S nucleocapsids contained 15S RNA, although in both cases RNA's sedimenting at 4 to 13S were also recovered. Furthermore, just as incomplete virions lacked 64S nucleocapsids, they also lacked 18S RNA. These findings support the hypothesis that the influenza virus genome is divided among several distinct pieces of RNA.     

Interactions of wild-type and mutant M protein of vesicular stomatitis virus with viral nucleocapsid and envelope in intact virions. Evidence from [125I]iodonaphthyl azide labeling and specific cross-linking

Four different temperature-sensitive M protein mutants (tsM) of vesicular stomatitis virus (VSV) were characterized with regard to the association of the mutated M protein either with nucleocapsids or with membranes in the intact virions. Virions were labeled with the photoreactive hydrophobic probe [125I]iodonaphthyl azide (INA) to assess interactions between viral proteins and the lipid envelope. In wild type (wt) virions, the three major structural proteins--G, M, and N--were labeled in the ratio ca. 1.0:0.4:0.2. INA labeled only the membrane-associated peptide of G protein, both in the intact virion and in reconstituted G protein--viral lipid vesicles, demonstrating the specificity of INA for lipid bilayer regions. Labeling of tsM virions with INA resulted in a 2--3-fold greater incorporation into M protein than was found for wt virions, suggesting increased M--membrane associations in the mutant virions. Temperature-stable revertants from tsM possessed wt labeling characteristics. Interaction of the M protein with nucleocapsids was assessed from the abundance of disulfide-linked M--N complexes found after disruption of the virions by sodium dodecyl sulfate solution under nonreducing conditions. The abundance of such complexes was 30--80% less from tsM virions than from wt virions, suggesting decreased M--nucleocapsid interactions in tsM virions. Temperature-stable revertants from tsM resembled wt in the abundance of M--N complex formed. We conclude that the mutations alter M protein in such a way as simultaneously to increase its association with membrane and to decrease its affinity for nucleocapsids in the intact virion.     

Structural Polypeptides of the Granulosis Virus of Plodia interpunctella

Techniques were developed for the isolation and purification of three structural components of Plodia interpunctella granulosis virus: granulin, enveloped nucleocapsids, and nucleocapsids. The polypeptide composition and distribution of protein in each viral component were determined by sodium dodecyl sulfate discontinuous and gradient polyacrylamide slab gel electrophoresis. Enveloped nucleocapsids consisted of 15 structural proteins ranging in molecular weight from 12,600 to 97,300. Five of these proteins, having approximate molecular weights of 17,800, 39,700, 42,400, 48,200, and 97,300, were identified as envelope proteins by surface radioiodination of the enveloped nucleocapsids. Present in purified nucleocapsids were eight polypeptides. The predominant proteins in this structural component had molecular weights of 12,500 and 31,000. Whereas no evidence of polypeptide glycosylation was obtained, six of the viral proteins were observed to be phosphorylated.     

Release of Envelope Glycoprotein from Rabies Virions by a Nonionic Detergent

Treatment of rabies virus with the nonionic detergent Nonidet P-40 resulted in solubilization of viral lipids and in a preferential release of the envelope glycoprotein. The other viral proteins and the viral ribonucleic acid remained associated in "core" particles sedimenting at a rate similar to that of intact virions. After fractionation of treated virus by velocity centrifugation in a sucrose density gradient, the amount of residual glycoprotein recovered in the "core" particle fraction and the extent of contamination of the glycoprotein fraction by other viral components were dependent on the ratio of detergent to viral protein used.