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.     

Interspecies aminopeptidase-N chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus, and transmissible gastroenteritis virus

We report that cells refractory to canine coronavirus (CCV) and feline infectious peritonitis virus (FIPV) became susceptible when transfected with a chimeric aminopeptidase-N (APN) cDNA containing a canine domain between residues 643 and 841. This finding shows that APN recognition by these viruses is species related and associated with this C-terminal domain. The human/canine APN chimera was also able to confer susceptibility to the porcine transmissible gastroenteritis virus (TGEV), whereas its human/porcine homolog failed to confer susceptibility to CCV and FIPV. A good correlation was observed between the capacity of CCV, FIPV, and TGEV to recognize the different interspecies APN chimeras and their ability to infect cells derived from the relevant species. As an exception, TGEV was found to use a human/bovine APN chimera as a receptor although itself unable to replicate in bovine cells.     

Expression of the recombinant anchorless N-terminal domain of mouse hepatitis virus (MHV) receptor makes hamster of human cells susceptible to MHV infection

Mouse hepatitis virus (MHV) receptor, the receptor for the murine coronavirus MHV, was expressed in MHV-resistant hamster and human cells as a series of mutant, recombinant glycoproteins with carboxy-terminal deletions lacking the cytoplasmic tail, transmembrane domain, and various amounts of the immunoglobulin constant-region-like domains. The soluble receptor glycoproteins containing the N-terminal virus-binding domain were released into the supernatant medium and inactivated the infectivity of MHV-A59 virions in a concentration-dependent manner. Surprisingly, some of the anchorless glycoproteins were found on the plasma membranes of transfected cells by flow cytometry, and these cells were rendered susceptible to infection with three strains of MHV. Thus, in the cells in which the anchorless, recombinant receptor glycoprotein is synthesized, some of the protein is bound to an unidentified moiety on the plasma membrane, which allows it to serve as a functional virus receptor.     

Discrimination between transmissible gastroenteritis virus isolates

Twenty TGEV isolates were compared by sequencing a 393-414 nucleotide stretch near the 5' end of the S gene, after amplification by RT-PCR. This part of the S gene is known to show considerable variation between porcine, canine and feline coronaviruses and is completely deleted from porcine respiratory coronaviruses. The discrimination achieved by nucleotide sequence analysis was compared with that obtained by monoclonal antibody typing. The viruses could be split into several clusters, and recent isolates of TGEV from England, The Netherlands and Belgium showed the greatest differences compared to earlier reference types. However, not all viruses with unique isolation histories were distinct, suggesting either genetic stability over many years, laboratory cross-contaminations or repeated introductions of similar viruses into the field. Firm conclusions on evolutionary trends cannot be drawn without obtaining a larger number of isolates, preferably from outbreaks with known epidemiological links. The sequences of some field isolates from the 1980s contained both nucleotide deletions and insertions. The latter included a short sequence of fourteen nucleotides with identity to a region of the TGEV polymerase gene.     

Isolation of porcine respiratory coronavirus from pigs affected with porcine reproductive and respiratory syndrome

Four cytopathogenic viruses were isolated in CPK cells derived from porcine kidneys from tonsils and lungs of 3 of 15 pigs affected with porcine reproductive and respiratory syndrome virus. Physicochemically and morphologically, the isolates were similar to a coronavirus. The isolates were not distinguished from transmissible gastroenteritis virus (TGEV) by a neutralization test using polyclonal antibodies, but differentiated from TGEV by monoclonal antibodies capable of discriminating between TGEV and porcine respiratory coronavirus (PRCV), indicating that the isolates were PRCV. In a serological survey of 30 serum samples each collected from about 50 days old pigs in the 2 affected farms, 29 (97%) and 15 (50%) sera were positive for neutralizing antibody against the isolate with the titers ranging from 2 to 64, respectively.     

Polarized budding of measles virus is not determined by viral surface glycoproteins

For viruses that mature by a budding process, the envelope glycoproteins are considered the major determinants for the site of virus release from polarized epithelial cells. Viruses are usually released from that membrane domain where the viral surface glycoproteins are transported to. We here report that measles virus has developed a different maturation strategy. Measles virus was found to be released from the apical membrane domain of polarized epithelial cells, though the surface glycoproteins H and F were transported in a nonpolarized fashion and to the basolateral membrane domain, respectively.     

Soluble free-fermion model in d dimensions

In addition to the spike (S) glycoprotein that binds to carcinoembryonic antigen-related receptors on the host cell membrane, some strains of mouse coronavirus (mouse hepatitis virus [MHV]) express a hemagglutinin esterase (HE) glycoprotein with hemagglutinating and acetylesterase activity. Virions of strains that do not express HE, such as MHV-A59, can infect mouse fibroblasts in vitro, showing that the HE glycoprotein is not required for infection of these cells. The present work was done to study whether interaction of the HE glycoprotein with carbohydrate moieties could lead to virus entry and infection in the absence of interaction of the S glycoprotein with its receptor glycoprotein, MHVR. The DVIM strain of MHV expresses large amounts of HE glycoprotein, as shown by hemadsorption, acetylesterase activity, and immunoreactivity with antibodies directed against the HE glycoprotein of bovine coronavirus. A monoclonal anti-MHVR antibody, MAb-CC1, blocks binding of virus S glycoprotein to MHVR and blocks infection of MHV strains that do not express HE. MAb-CC1 also prevented MHV-DVIM infection of mouse DBT cells and primary mouse glial cell cultures. Although MDCK-I cells express O-acetylated sialic acid residues on their plasma membranes, these canine cells were resistant to infection with MHV-A59 and MHV-DVIM. Transfection of MDCK-I cells with MHVR cDNA made them susceptible to infection with MHV-A59 and MHV-DVIM. Thus, the HE glycoprotein of an MHV strain did not lead to infection of cultured murine neural cells or of nonmurine cells that express the carbohydrate ligand of the HE glycoprotein. Therefore, interaction of the spike glycoprotein of MHV with its carcinoembryonic antigen-related receptor glycoprotein is required for infectivity of MHV strains whether or not they express the HE glycoprotein.     

Enterotropic mouse hepatitis virus

Mouse hepatitis virus [MHV], the coronavirus of the mouse, is the most common viral pathogen in contemporary laboratory mouse colonies throughout the world. It is highly contagious with variable clinical manifestations. The majority of infections are subclinical, but can still significantly influence biological responses, thus interfering with research, mainly in the field of immunology. MHV has been intensively studied from a number of research perspectives and has become the prototype for studying the molecular biology of coronaviruses. MHV contains a single-stranded, positive-sense RNA genome ranging from 27 to 31 kb, which is divided into seven genes. Virions consist of four to five structural proteins. There are many MHV strains that vary in virulence, organotropism and cell tropism, and are constantly evolving by naturally occurring mutation and recombination. Based on pathogenesis studies MHV strains are usually grouped according to their primary tissue tropism into two biotypes: polytropic and enterotropic. Enterotropic strains of MHV replicate in the intestinal mucosa and only rarely spread to other tissues. No morphological structure of the virion has as yet been identified that is responsible for enterotropism. The course of an MHV infection is dependent on the virus strain and host factors. Generally, MHV causes an acute, self-limiting infection which is inapparent in adult mice. Neonates are highly susceptible to disease and show high mortality. In an enzootically infected colony, however, they are protected by maternally derived passive immunity. Detection of MHV infections depends on serological screening of colonies. MHV is controlled by culling and rederivation of the affected colony using hysterectomy or embryo transfer or by elimination through cessation of breeding.     

Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5

PURPOSE: To determine the polarity of fibroblast growth factor 5 (FGF5) secretions from retinal pigment epithelium (RPE) cells and to examine the viability and utility of the ARPE-19 cell line as a model for the study of RPE polarity. METHODS: Influenza infection and adenovirus-mediated gene transfer were used to deliver and express genes encoding influenza hemagglutinin (HA), p75-NTR (a neurotrophin receptor), low-density lipoprotein (LDL) receptor (LDLR), and FGF5 in confluent monolayers of ARPE-19 cells. The localization of HA, p75-NTR, and LDLR was determined by confocal microscopy. Domain selective biotinylation assays were used to quantitatively determine the polarities of p75-NTR and LDLR. The secretion of FGF5 into the apical and basal media of ARPE-19 cultures was examined by immunoblot analysis of conditioned media. RESULTS: Hemagglutinin and p75-NTR were found to be localized on the apical surface of infected and transduced ARPE-19 cells. In contrast, LDLR was associated preferentially with the basolateral membrane of ARPE-19 cells. Biotinylation studies indicated that 84% of p75-NTR was present on the apical surface, and 79% of LDLR was basolaterally polarized. Over the course of 6 hours, more than 90% of the total secreted FGF5 protein accumulated in the basolateral media. CONCLUSIONS: ARPE-19 cells exhibit a polarized distribution of cell surface markers when examined by either confocal microscopy or surface-labeling assays. This indicates that the ARPE-19 cell line is a valid model for studies of RPE cell polarity. FGF5, a secreted protein normally produced by RPE cells, is accumulated preferentially in the basal media after only 6 hours, suggesting that it is vectorially secreted from the basolateral surface of ARPE-19 cells.     

A helical coat protein recognition domain of the bacteriophage P22 scaffolding protein

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.     

Vectorial release of poliovirus from polarized human intestinal epithelial cells

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.     

Immunization of pregnant gilts with PRCV induces lactogenic immunity for protection of nursing piglets from challenge with TGEV

The level of passive protection against transmissible gastroenteritis virus (TGEV) was evaluated by experimentally infecting 12 pregnant gilts with different doses of porcine respiratory coronavirus (PRCV) and challenging their litters at 4 days of age. An overall survival rate of 70% was found for piglets nursing the 12 PRCV-infected gilts, compared to a 16% survival rate for piglets of nine uninfected control gilts. Six of the PRCV-infected gilts had adequate levels of immunity to resist infection with TGEV following the challenge of their litters. These six completely immuned gilts also solidly protected their litters from TGEV as shown by a 96% piglet survival rate through weaning at 3 weeks of age. The results suggest that respiratory infection with PRCV induces a substantial degree of protective lactogenic immunity against TGEV.     

Polarized secretion of apoA-I and apoA-II by transfected MDCK cells

Apolipoproteins (apo) are secreted preferentially from the basolateral surface of hepatocytes and enterocytes. The polarized secretion of proteins is either mediated by a protein-dependent sorting signal or by a cell-dependent default pathway. In order to determine the mechanism for the polarized secretion of apolipoproteins, we examined the secretion of apoA-I and apoA-II in transfected Madin-Darby canine kidney (MDCK) cells. Transfected MDCK cells and Caco-2 cells were grown as a polarized monolayer on tissue culture inserts, which separate an upper apical compartment from the lower basolateral compartment, and the secretion of apoA-I and apoA-II into the apical and basolateral compartments was quantitated by immunoprecipitation. Caco-2 cells almost exclusively secreted apoA-I and apoA-II basolaterally, with an apical to basolateral ratio of 18:82 for apoA-I, and 11:89 for apoA-II. In contrast, transfected MDCK cells secreted significant amounts of apoA-I and apoA-II into both compartments, but with a bias toward apical secretion and an apical to basolateral ratio of 66:34 and 68:32, respectively. The polarized secretion of MDCK cells was not due to transcytosis, diffusion, or differential recovery. As assessed by density gradient ultracentrifugation, apoA-I and apoA-II secreted from either the apical or basolateral surface were relatively lipid-poor. Overall, these results suggest that the polarized secretion of apoA-I and apoA-II does not occur by a protein-dependent sorting signal, but by a cell-dependent default pathway that leads to preferential basolateral secretion by Caco-2 cells and both apical and basolateral secretion in MDCK cells, but with a bias toward apical secretion.     

Contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs

OBJECTIVE: To determine the ability of porcine respiratory coronavirus (PRCV) infections to induce passive immunity in suckling pigs to transmissible gastroenteritis virus (TGEV) challenge exposure. DESIGN AND ANIMALS: 4 TGEV seronegative sows and their litters (group A) served as controls, whereas 2 other groups (B and C) of sows (also TGEV seronegative) were oronasally inoculated with live PRCV during 1 or 2 subsequent pregnancies, respectively. PROCEDURE: Effectiveness of passive immunity provided to pigs via colostrum and milk was assessed after TGEV challenge exposure, and TGEV antibody responses in colostrum and milk were analyzed. RESULTS: Mortality in the 3 groups of young pigs correlated with severity of clinical signs of TGEV infection and was highest in control litters (86% in group-A pigs) and lowest in litters of sows inoculated with PRCV in 2 subsequent pregnancies (14% in group-C pigs). Virus-neutralization and IgA and IgG TGEV antibody titers of milk collected from sows at challenge exposure had significant positive correlation with litter survival. Significantly higher numbers of TGEV-specific IgA and IgG antibody-secreting cells were found in group-A pigs than in group-C pigs, suggesting that high titer of maternal antibodies (induced in group-C sows multiply exposed to PRCV) may interfere with active antibody responses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that, in PRCV-infected pig herds, multiple exposures of pregnant sows are associated with higher IgA and IgG antibody titers to TGEV in milk, and these titers contribute to protection against TGEV infection.     

Apical polarity of N-CAM and EMMPRIN in retinal pigment epithelium resulting from suppression of basolateral signal recognition

Retinal pigment epithelial (RPE) cells apically polarize proteins that are basolateral in other epithelia. This reversal may be generated by the association of RPE with photoreceptors and the interphotoreceptor matrix, postnatal expansion of the RPE apical surface, and/or changes in RPE sorting machinery. We compared two proteins exhibiting reversed, apical polarities in RPE cells, neural cell adhesion molecule (N-CAM; 140-kD isoform) and extracellular matrix metalloproteinase inducer (EMMPRIN), with the cognate apical marker, p75-neurotrophin receptor (p75-NTR). N-CAM and p75-NTR were apically localized from birth to adulthood, contrasting with a basolateral to apical switch of EMMPRIN in developing postnatal rat RPE. Morphometric analysis demonstrated that this switch cannot be attributed to expansion of the apical surface of maturing RPE because the basolateral membrane expanded proportionally, maintaining a 3:1 apical/basolateral ratio. Kinetic analysis of polarized surface delivery in MDCK and RPE-J cells showed that EMMPRIN has a basolateral signal in its cytoplasmic tail recognized by both cell lines. In contrast, the basolateral signal of N-CAM is recognized by MDCK cells but not RPE-J cells. Deletion of N-CAM's basolateral signal did not prevent its apical localization in vivo. The data demonstrate that the apical polarity of EMMPRIN and N-CAM in mature RPE results from suppressed decoding of specific basolateral signals resulting in randomized delivery to the cell surface.     

A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus

Mouse hepatitis virus (MHV), a murine coronavirus known to cause encephalitis and demyelination, uses murine homologues of carcinoembryonic antigens as receptors. However, the expression of these receptors is extremely low in the brain. By low-stringency screening of a mouse brain cDNA library, we have identified a member of the pregnancy-specific glycoprotein (PSG) subgroup of the carcinoembryonic antigen gene family. Unlike other PSG that are expressed in the placenta, it is expressed predominantly in the brain. Transfection of the cDNA into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to infection by some MHV strains, including A59, MHV-2, and MHV-3, but not JHM. Thus, this is a virus strain-specific receptor. The detection of multiple receptors for MHV suggests the flexibility of this virus in receptor utilization. The identification of this virus in receptor utilization. The identification of a PSG predominantly expressed in the brain also expands the potential functions of these molecules.     

Branch migration through DNA sequence heterology

We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells. We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER. The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface. In the TGN caveolin-1/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-1 homooligomers are targeted to the apical side. Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present. This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane. Anti-caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein. Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.     

The detection and characterization of multiple hemagglutinin-esterase (HE)-defective viruses in the mouse brain during subacute demyelination induced by mouse hepatitis virus

It has previously been shown that passive immunization with antibodies specific for the hemagglutinin-esterase (HE) protein of mouse hepatitis virus (MHV) prevents acute lethal encephalitis, resulting in a subacute and chronic demyelination in mice infected with JHM(2), an isolate of the neurotropic JHM strain of MHV. To determine possible genetic changes occurring during infection, viruses were isolated from the brain of infected mice at various time points after infection and examined for the patterns of their structural proteins. The results showed that the sizes and expression levels of the viral spike, membrane, and nucleocapsid proteins were constant among 161 virus isolates throughout the infection. In contrast, most of the viruses isolated later in infection did not synthesize HE protein. This finding suggests that the HE gene expression is extremely variable and is preferentially lost during prolonged viral infections. In contrast, when viruses were passaged in tissue culture, no significant accumulation of HE protein-defective mutants was observed, suggesting that the accumulation of HE protein-defective mutants in infected animals was most likely the result of the positive selection for these mutants during the subacute and chronic infection. The genetic defects of HE gene in these mutants were characterized by cloning, sequencing, and in vitro translation of HE genes. Most of the mutations in the HE protein-defective mutants consisted of deletions of various lengths at different sites within the HE-coding region, resulting in the change of the reading frame and early termination. However, most of the truncated HE proteins were not detected in the infected cells. Since viruses from different mice exhibited different types of defects, the HE mutations probably occurred de novo in the brain. These results demonstrated the exceptionally rapid selection of HE-defective mutants during viral infection of mice and suggest that their selection may be related to the neuropathogenicity or persistence of MHV.     

Selective perturbation of apical membrane traffic by expression of influenza M2, an acid-activated ion channel, in polarized madin-darby canine kidney cells

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin-Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.