Nipah Virus Entry and Egress from Polarized Epithelial Cells

Highly pathogenic Nipah virus (NiV) infections are transmitted via airway secretions and urine, commonly via the respiratory route. Epithelial surfaces represent important replication sites in both primary and systemic infection phases. NiV entry and spread from polarized epithelial cells therefore determine virus entry and dissemination within a new host and influence virus shedding via mucosal surfaces in the respiratory and urinary tract. To date, there is no knowledge regarding the entry and exit sites of NiV in polarized epithelial cells. In this report, we show for the first time that NiV can infect polarized kidney epithelial cells (MDCK) from both cell surfaces, while virus release is primarily restricted to the apical plasma membrane. Substantial amounts of basolateral infectivity were detected only after infection with high virus doses, at time points when the integrity of the cell monolayer was largely disrupted as a result of cell-to-cell fusion. Confocal immunofluorescence analyses of envelope protein distribution at early and late infection stages suggested that apical virus budding is determined by the polarized sorting of the NiV matrix protein, M. Studies with stably M-expressing and with monensin-treated cells furthermore demonstrated that M protein transport is independent from the glycoproteins, implying that the M protein possesses an intrinsic apical targeting signal.     

Epstein-Barr Virus Transcytosis through Polarized Oral Epithelial Cells

Although Epstein-Barr virus (EBV) is an orally transmitted virus, viral transmission through the oropharyngeal mucosal epithelium is not well understood. In this study, we investigated how EBV traverses polarized human oral epithelial cells without causing productive infection. We found that EBV may be transcytosed through oral epithelial cells bidirectionally, from both the apical to the basolateral membranes and the basolateral to the apical membranes. Apical to basolateral EBV transcytosis was substantially reduced by amiloride, an inhibitor of macropinocytosis. Electron microscopy showed that virions were surrounded by apical surface protrusions and that virus was present in subapical vesicles. Inactivation of signaling molecules critical for macropinocytosis, including phosphatidylinositol 3-kinases, myosin light-chain kinase, Ras-related C3 botulinum toxin substrate 1, p21-activated kinase 1, ADP-ribosylation factor 6, and cell division control protein 42 homolog, led to significant reduction in EBV apical to basolateral transcytosis. In contrast, basolateral to apical EBV transcytosis was substantially reduced by nystatin, an inhibitor of caveolin-mediated virus entry. Caveolae were detected in the basolateral membranes of polarized human oral epithelial cells, and virions were detected in caveosome-like endosomes. Methyl β-cyclodextrin, an inhibitor of caveola formation, reduced EBV basolateral entry. EBV virions transcytosed in either direction were able to infect B lymphocytes. Together, these data show that EBV transmigrates across oral epithelial cells by (i) apical to basolateral transcytosis, potentially contributing to initial EBV penetration that leads to systemic infection, and (ii) basolateral to apical transcytosis, which may enable EBV secretion into saliva in EBV-infected individuals.     

Role of DC-SIGN in Lassa Virus Entry into Human Dendritic Cells

The arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with high mortality in humans. Antigen-presenting cells, in particular dendritic cells (DCs), are early and preferred targets of LASV, and their productive infection contributes to the virus-induced immunosuppression observed in fatal disease. Here, we characterized the role of the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in LASV entry into primary human DCs using a chimera of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that differentiation of human primary monocytes into DCs enhanced virus attachment and entry, concomitant with the upregulation of DC-SIGN. LASV and rLCMV-LASVGP bound to DC-SIGN via mannose sugars located on the N-terminal GP1 subunit of LASVGP. We provide evidence that DC-SIGN serves as an attachment factor for rLCMV-LASVGP in monocyte-derived immature dendritic cells (MDDC) and can accelerate the capture of free virus. However, in contrast to the phlebovirus Uukuniemi virus (UUKV), which uses DC-SIGN as an authentic entry receptor, productive infection with rLCMV-LASVGP was less dependent on DC-SIGN. In contrast to the DC-SIGN-mediated cell entry of UUKV, entry of rLCMV-LASVGP in MDDC was remarkably slow and depended on actin, indicating the use of different endocytotic pathways. In sum, our data reveal that DC-SIGN can facilitate cell entry of LASV in human MDDC but that its role seems distinct from the function as an authentic entry receptor reported for phleboviruses.     

The Viral Spike Protein Is Not Involved in the Polarized Sorting of Coronaviruses in Epithelial Cells

Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCK_MHVR cells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCK_MHVR cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO J. 15:2020–2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed.     

Role of the C Terminus of Lassa Virus L Protein in Viral mRNA Synthesis

The N terminus of arenavirus L protein contains an endonuclease presumably involved in “cap snatching.” Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.     

A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells

Chikungunya virus (CHIKV) has resulted in several outbreaks in the past six decades. The clinical symptoms of Chikungunya infection include fever, skin rash, arthralgia, and an increasing incidence of encephalitis. The re-emergence of CHIKV with more severe pathogenesis highlights its potential threat on our human health. In this study, polarized HBMEC, polarized Vero C1008 and non-polarized Vero cells grown on cell culture inserts were infected with CHIKV apically or basolaterally. Plaque assays, viral binding assays and immunofluorescence assays demonstrated apical entry and release of CHIKV in polarized HBMEC and Vero C1008. Drug treatment studies were performed to elucidate both host cell and viral factors involved in the sorting and release of CHIKV at the apical domain of polarized cells. Disruption of host cell myosin II, microtubule and microfilament networks did not disrupt the polarized release of CHIKV. However, treatment with tunicamycin resulted in a bi-directional release of CHIKV, suggesting that N-glycans of CHIKV envelope glycoproteins could serve as apical sorting signals.     

Preferential Budding of Vesicular Stomatitis Virus from the Basolateral Surface of Polarized Epithelial Cells Is Not Solely Directed by Matrix Protein or Glycoprotein

Vesicular stomatitis virus has been shown to bud basolaterally, and the matrix protein, but not glycoprotein, was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface.     

辛德毕斯病毒6K蛋白在病毒成熟释放中的作用

应用多种细胞生物学技术,对辛德毕斯病毒(Sindbis Virus,SbV)的6K蛋白在细胞中的分布和转运动态及在病毒释放过程中的作用进行了探讨,结果表明:6K蛋白不仅存在于成熟病毒粒子中,而且在粗面内质网合成后及在向细胞质膜的转运过程中均与囊膜蛋白E2形成复合物。对6K蛋白酰基化位点突变株的研究结果表明,它与E2突变株在病毒形态发生方面有惊人的相似之处,说明SbV的出芽释放过程不仅仅是靠E2蛋白C端与病毒核衣壳的相互作用,6K蛋白的酰基化修饰可能在这个过程中也起着十分重要的作用。根据6K蛋白的二级结构的预测及综合上述实验结果,对SbV出芽释放的模式提出了新的补充。     

Echovirus 7 Entry into Polarized Caco-2 Intestinal Epithelial Cells Involves Core Components of the Autophagy Machinery

Echovirus 7 enters polarized Caco-2 intestinal epithelial cells by a clathrin-mediated endocytic process and then moves through the endosomal system before releasing its genome into the cytoplasm. We examined the possible role in virus entry of core components of the autophagy machinery. We found that depletion of Beclin-1, Atg12, Atg14, Atg16, or LC3 with specific small interfering RNAs inhibited echovirus 7 infection upstream of uncoating but had little or no effect on virus attachment to the cell surface. These data indicate that multiple autophagy-related proteins are important for one or more events that occur after the virus has bound its receptor on the cell surface but before RNA is released from the virus capsid. Although we have not determined the mechanism by which each protein contributes to virus entry, we found that stable depletion of Atg16L1 interfered with virus internalization from the cell surface rather than with intracellular trafficking. Autophagy gene products may thus participate in the endocytic process that moves virus into polarized Caco-2 cells.     

Human Cytomegalovirus Glycoprotein B Contains Autonomous Determinants for Vectorial Targeting to Apical Membranes of Polarized Epithelial Cells

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells grown on permeable filter supports. Laser scanning confocal immunofluorescence microscopy and domain-selective biotinylation of surface membrane domains showed that CMV gB was transported to apical membranes independently of other envelope glycoproteins and that it colocalized with proteins in transport vesicles of the biosynthetic and endocytic pathways. Determinants for trafficking to apical membranes were located by evaluating the targeting of gB derivatives with deletions in the lumen, transmembrane (TM) anchor, and carboxyl terminus. Derivative gB(Δ717-747), with an internal deletion in the luminal juxtamembrane sequence that preserved the N- and O-glycosylation sites, retained vectorial transport to apical membranes. In contrast, derivatives that lacked the TM anchor and cytosolic domain (gBΔ646-906) or the TM anchor alone (gBΔ751-771) underwent considerable basolateral targeting. Likewise, derivatives lacking the entire cytosolic domain (gBΔ772-906) or the last 73 amino acids (gBΔ834-906) showed disrupted apical transport. Site-specific mutations that deleted or altered the cluster of acidic residues with a casein kinase II phosphorylation site at the extreme carboxyl terminus, which can serve as an internalization signal, caused partial missorting of gB to basolateral membranes. Our studies indicate that CMV gB contains autonomous information for apical targeting in luminal, TM anchor, and cytosolic domain sequences, forming distinct structural elements that cooperate in vectorial transport in polarized epithelial cells.     

牛病毒性腹泻病毒的成熟和释放

试验中用电镜观察了牛病毒性腹泻病毒ＯｒｅｇｏｎＣ２４Ｖ株在感染新生牛睾丸细胞中的形态发生。成熟的病毒颗粒是直径约为５０ｎｍ的球形颗粒,内含直径约为３０ｎｍ的核心。病毒在宿主细胞的胞质内复制,通过糙面内质网膜出芽成熟。病毒可以通过外排或在细胞死亡后含有病毒颗粒的空泡崩溃而释放到胞外。     

Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif

Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.Hepatitis E virus (HEV) remains enigmatic in spite of recent advances (see references 7 and 16 for reviews). HEV is a major cause of acute hepatitis in numerous developing countries, but hepatitis E is infrequently detected in industrialized countries even though seroprevalence rates of anti-HEV as high as 20% in these countries have been reported. Although hepatitis E normally is a self-limited acute disease, recent studies have identified it as an emerging cause of chronic hepatitis in immunocompromised patients. Whereas contaminated drinking water is the source of most infections in developing countries, the sources in industrialized countries are not fully evaluated, but many, if not most, infections appear linked to eating undercooked meat, especially pork. These differences in epidemiology may reflect the fact that most infections in developing countries are caused by genotypes 1 and 2 while those in industrialized countries are mainly due to genotypes 3 and 4.HEV was initially classified as a calicivirus, but subsequent sequence analysis suggested that it was more closely related to the enveloped rubella virus. However, although HEV may be associated with lipids under some conditions (22), HEV virions do not possess an envelope. Four genotypes of HEV that infect humans have been identified (4). Genotypes 1 and 2 infect primates exclusively, whereas genotypes 3 and 4 are zoonotic and commonly also infect swine and rarely other nonprimates. Recent identification of a strain infecting farmed rabbits in China suggests that other reservoirs may exist (32).The capsid protein encoded by open reading frame 2 (ORF2) is able to form infectious virus particles, but these particles remain cell associated. The crystal structure of a truncated recombinant protein has been solved, but the size of the protein in mature virions is unknown (11, 15, 28, 31). The virus is not cytopathic, and it is unclear how it gets out of cells.The 7.2-kb genome of HEV is a capped mRNA that contains three ORFs that encode proteins involved in replication (ORF1), a capsid protein (ORF2), and a small protein of only 113 to 114 amino acids (ORF3). All but the 5′ terminus of ORF3 is overlapped by ORF2, and both proteins are translated from the same bicistronic subgenomic RNA (10). When overexpressed in cell culture, ORF2 is glycosylated, and ORF3 is phosphorylated (26); this phosphorylated ORF3 protein binds to nonglycosylated ORF2 protein in cell culture, but phosphorylation is not required for infection of macaques (9). The virus has been exceedingly difficult to propagate in cell culture, but recently Okamoto and colleagues reported the successful adaptation of both a genotype 3 and a genotype 4 strain to efficient growth in cultures of PLC/PRF/5 hepatoma or A549 lung cells (23, 24).The tiny ORF3 protein is particularly intriguing because it has a significant impact on virus propagation through mechanisms that have yet to be defined. Data from experiments performed with overexpressed ORF3 protein have suggested that, among other things, ORF3 may interact with cellular proteins, including signaling proteins containing Src homology 3 domains (14), bikunin (27), hemopexin (21), and microtubule proteins (13), and it may function to modulate the acute-phase disease response (3), protect cells from mitochondrial depolarization (18), and enhance expression of glycolytic pathway enzymes (17). Yet within transfected hepatoma cells in culture, virions of an ORF3 null mutant of genotype 1 were assembled in the absence of ORF3 protein and were infectious for naïve hepatoma cells (6) although this same ORF3 null mutant was unable to mount a detectable infection in rhesus monkeys (8). Also, swine transfected with genotype 3 mutant genomes encoding a truncated ORF3 protein did not get infected, indicating that an intact ORF3 protein is needed for infectivity in vivo (12). This lack of infectivity in vivo is possibly explained by the recent demonstration that the ORF3 protein of genotype 3 virus is important for export of virions out of cultured cells in vitro (30); however, this dependence on ORF3 for virion egress has not been confirmed in vivo or for strains of the other three genotypes.The four major genotypes of human HEV appear to segregate naturally into two distinct groups. One group contains genotype 1 and 2 strains that lack a zoonotic component and are spread mainly via contaminated water; in contrast, the second group contains genotype 3 and 4 strains which are able to cross species boundaries and are zoonotic since humans have been infected as a result of eating undercooked meat (16, 25). The molecular basis for the two groupings is unknown, and much more extensive comparative analyses are required to determine which variables are epidemiologically relevant. Here, for lack of an efficient cell culture system for genotype 1 or 2 strains, we have utilized an infectious cDNA clone of a genotype 1 strain in order to explore the role of the ORF3 protein in this group.     

The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)₂ motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.