Protein regions important for plasminogen activation and inactivation of alpha2-antiplasmin in the surface protease Pla of Yersinia pestis.

The plasminogen activator, surface protease Pla, of the plague bacterium Yersinia pestis is an important virulence factor that enables the spread of Y. pestis from subcutaneous sites into circulation. Pla-expressing Y. pestis and recombinant Escherichia coli formed active plasmin in the presence of the major human plasmin inhibitor, alpha2-antiplasmin, and the bacteria were found to inactivate alpha2-antiplasmin. In contrast, only poor plasminogen activation and no cleavage of alpha2-antiplasmin was observed with recombinant bacteria expressing the homologous gene ompT from E. coli. A beta-barrel topology model for Pla and OmpT predicted 10 transmembrane beta-strands and five surface-exposed loops L1-L5. Hybrid Pla-OmpT proteins were created by substituting each of the loops between Pla and OmpT. Analysis of the hybrid molecules suggested a critical role of L3 and L4 in the substrate specificity of Pla towards plasminogen and alpha2-antiplasmin. Substitution analysis at 25 surface-located residues showed the importance of the conserved residues H101, H208, D84, D86, D206 and S99 for the proteolytic activity of Pla-expressing recombinant E. coli. The mature alpha-Pla of 292 amino acids was processed into beta-Pla by an autoprocessing cleavage at residue K262, and residues important for the self-recognition of Pla were identified. Prevention of autoprocessing of Pla, however, had no detectable effect on plasminogen activation or cleavage of alpha2-antiplasmin. Cleavage of alpha2-antiplasmin and plasminogen activation were influenced by residue R211 in L4 as well as by unidentified residues in L3. OmpT, which is not associated with invasive bacterial disease, was converted into a Pla-like protease by deleting residues D214 and P215, by substituting residue K217 for R217 in L4 of OmpT and also by substituting the entire L3 with that from Pla. This simple modification of the surface loops and the substrate specificity of OmpT exemplifies the evolution of a housekeeping protein into a virulence factor by subtle mutations at critical protein regions. We propose that inactivation of alpha2-antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive character of plague.

Adenovirus-activated PKA and p38/MAPK pathways boost microtubule-mediated nuclear targeting of virus.

Nuclear targeting of adenovirus is mediated by the microtubule-dependent, minus-end-directed motor complex dynein/dynactin, in competition with plus- end-directed motility. We demonstrate that adenovirus transiently activates two distinct signaling pathways to enhance nuclear targeting. The first pathway activates integrins and cAMP-dependent protein kinase A (PKA). The second pathway activates the p38/MAP kinase and the downstream MAPKAP kinase 2 (MK2), dependent on the p38/MAPK kinase MKK6, but independent of integrins and PKA. Motility measurements in PKA-inhibited, p38-inhibited or MK2-lacking (MK2(-/-)) cells indicate that PKA and p38 stimulated both the frequency and velocity of minus-end-directed viral motility without affecting the perinuclear localization of transferrin-containing endosomal vesicles. p38 also suppressed lateral viral motilities and MK2 boosted the frequency of minus-end-directed virus transport. Nuclear targeting of adenovirus was rescued in MK2(-/-) cells by overexpression of hsp27, an MK2 target that enhances actin metabolism. Our results demonstrate that complementary activities of PKA, p38 and MK2 tip the transport balance of adenovirus towards the nucleus and thus enhance infection.

The first step of adenovirus type 2 disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol.

Disassembly is a key event of virus entry into cells. Here, we have investigated cellular requirements for the first step of adenovirus type 2 (Ad2) disassembly, the release of the fibers. Although fiber release coincides temporally with virus uptake, fiber release is not required for Ad2 endocytosis. It is, however, inhibited by actin-disrupting agents or soluble RGD peptides, which interfere with integrin-dependent endocytosis of Ad2. Fiber release occurs at the cell surface. Actin stabilization with jasplakinolide blocks Ad2 entry at extended cell surface invaginations and efficiently promotes fiber release, indicating that fiber release and virus endocytosis are independent events. Fiber release is not sufficient for Ad2 escape from endosomes, since inhibition of protein kinase C (PKC) prevents Ad2 escape from endosomes but does not affect virus internalization or fiber release. PKC-inhibited cells accumulate Ad2 in small vesicles near the cell periphery, indicating that PKC is also required for membrane trafficking of virus. Taken together, our data show that fiber release from incoming Ad2 requires integrins and filamentous actin. Together with correct subcellular transport of Ad2-containing endosomes, fiber release is essential for efficient delivery of virus to the cytosol. We speculate that fiber release at the surface might extend the host range of Ad2 since it is associated with the separation of a small fraction of incoming virus from the target cells.

Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus.

Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end-directed movements of cytosolic virus with elementary speeds up to 2.6 micrometer/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end-directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1-10 micrometer/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end-directed vesicular transport, significantly reduced the extent and the frequency of minus end-directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end-directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end- directed cytoplasmic dynein and an unknown plus end- directed activity.

The role of the nuclear pore complex in adenovirus DNA entry.

Adenovirus targets its genome to the cell nucleus by a multistep process involving endocytosis, membrane penetration and cytoplasmic transport, and finally imports its DNA into the nucleus. Using an immunochemical and biochemical approach combined with inhibitors of nuclear import, we demonstrate that incoming viral DNA and DNA-associated protein VII enter the nucleus via nuclear pore complexes (NPCs). Depletion of calcium from nuclear envelope and endoplasmic reticulum cisternae by ionophores or thapsigargin blocked DNA and protein VII import into the nucleus, but had no effect on virus targeting to NPCs. Calcium-depleted cells were capable of disassembling incoming virus. In contrast, inhibitors of cytosolic O-linked glycoproteins of the NPC blocked virus attachment to the nuclear envelope, capsid disassembly and also nuclear import of protein VII. The data indicate that NPCs have multiple roles in adenovirus entry into cells: they contain a virus-binding and/or dissociation activity and provide a gateway for the incoming DNA genome into the nucleus.

Multiple mechanisms for the inhibition of entry and uncoating of superinfecting Semliki Forest virus.

Recombinant Semliki Forest viruses (SFV) that express one or none of the viral structural proteins were used to infect cells and to analyze the fate of incoming superinfecting wild-type viruses. It was found that in addition to the previously described block in replication that superinfecting viruses encounter within 15 min of infection, other mechanisms of superinfection inhibition occurred at later times. Over a 6-hr infection period, inhibition was seen in binding of virus to the cell surface, in acid-activated penetration into the cytoplasm, and in uncoating of nucleocapsids. For each of these processes, the inhibitory mechanism was investigated. In summary, we found that infection evoked several independent mechanisms for blocking the entry and uncoating of superinfecting viruses. The results also offered new insights into the normal processes of penetration and uncoating of SFV.

Efficient multiplication of a Semliki Forest virus chimera containing Sindbis virus spikes.

Using the Semliki Forest virus (SFV) and Sindbis virus (SIN) cDNAs we have constructed recombinants in which the spike genes were exchanged. Analyses of expression showed that the SFV/SIN(spike) RNA directed efficient assembly of infectious virus, whereas the reciprocal SIN/SFV(spike) RNA was completely unable to assemble virus. This was apparently due to a defective capsid-spike interaction.

Targeting of Moloney murine leukemia virus gag precursor to the site of virus budding.

Retrovirus Moloney murine leukemia virus (M-MuLV) matures by budding at the cell surface. Central to the budding process is the myristoylated viral core protein precursor Gag which, even in the absence of all other viral components, is capable of associating with the cytoplasmic leaflet of the plasma membrane and assembling into extracellular virus-like particles. In this paper we have used heterologous, Semliki Forest virus-driven, expression of M-MuLV Gag to study the mechanism by which this protein is targeted to the cell surface. In pulse-chase experiments, BFA, monensin, and 20 degrees C block did not affect incorporation of Gag into extracellular particles thereby indicating that the secretory pathway is not involved in targeting of Gag to the cell surface. Subcellular fractionation studies demonstrated that newly synthesized Gag became rapidly and efficiently associated with membranes which had a density similar to that of plasma membrane-derived vesicles. Protease-protection studies confirmed that the Gag-containing membranes were of plasma membrane origin, since in crude cell homogenates, the bulk of newly synthesized Gag was protease-resistant as expected of a protein that binds to the cytoplasmic leaflet of the plasma membrane. Taken together these data indicate that targeting of M-MuLV Gag to the cell surface proceeds via direct insertion of the protein to the cytoplasmic side of the plasma membrane. Furthermore, since the membrane insertion reaction is highly efficient and specific, this suggests that the reaction is dependent on as-yet-unidentified cellular factors.

Structure-function relation of the NH2-terminal domain of the Semliki Forest virus capsid protein.

The capsid (C) protein of alphaviruses consists of two protein domains: a serine protease at the COOH terminus and an NH2-terminal domain which is thought to interact with RNA in the virus nucleocapsid (NC). The latter domain is very rich in positively charged amino acid residues. In this work, we have introduced large deletions into the corresponding region of a full-length cDNA clone of Semliki Forest virus, expressed the transcribed RNA in BHK-21 cells, and monitored the autoprotease activity of C, the formation of intracellular NCs, and the release of infectious virus. Our results show that if the gene region encoding the whole NH2-terminal domain is removed, the expressed C protein fragment cannot assemble into NCs and virus particles but it is still able to function as an autoprotease. Thus, these results underline the general importance of the NH2-terminal domain in the virus assembly process and furthermore show that the serine protease domain can function independently of the NH2 terminus. Surprisingly, analysis of additional C protein deletion variants showed that not all of the NH2-terminal domain is required for virus assembly, but large deletions involving up to one-third of its positively charged residues are still compatible with NC and virus formation. The fact that so much flexibility is allowed in the structure of the NH2-terminal domain of C suggests that most of this region is involved in nonspecific interactions with the encapsidated RNA, probably through its positively charged amino acid residues.

A significantly improved Semliki Forest virus expression system based on translation enhancer segments from the viral capsid gene.

We recently described a system for heterologous gene expression in a variety of mammalian cell types that is based on an efficiently replicating Semliki Forest virus (SFV) variant in which an RNA encoding a foreign protein replaces the RNA that normally encodes the viruses' structural polyprotein. Although expression levels are sufficiently high for many purposes, in general they are only 10% of the level of the polyprotein in a wild type SFV infection. Here we show that the first 102 bases of the viral capsid gene function as a translational enhancer, and that SFV vectors incorporating this RNA increase heterologous protein synthesis to the level of wild type polyprotein.

Incorporation of homologous and heterologous proteins into the envelope of Moloney murine leukemia virus.

The efficiencies with which homologous and heterologous proteins are incorporated into the envelope of Moloney murine leukemia virus (M-MuLV) have been analyzed by utilizing a heterologous, Semliki Forest virus-driven M-MuLV assembly system and quantitative pulse-chase assays. Homologous M-MuLV spike protein was found to be efficiently incorporated into extracellular virus particles when expressed at a relatively low density at the plasma membrane. In contrast, efficient incorporation of heterologous proteins (the spike complex of Semliki Forest virus and a cytoplasmically truncated mutant of the human transferrin receptor) was observed only when these proteins were expressed at high densities at the cell surface. These results imply that homologous and heterologous proteins are incorporated into the M-MuLV envelope via two distinct pathways.

Assembly and entry mechanisms of Semliki Forest virus.

The alphavirus Semliki Forest (SFV) is an enveloped virus with a positive single-stranded RNA genome. The genome is complexed with 240 copies of a capsid protein into a nucleocapsid structure. In the membrane the virus carries an equal number of copies of a membrane protein heterodimer. The latter oligomers are grouped into clusters of three. These structures form the spikes of the virus and carry its entry functions, that is receptor binding and membrane fusion activity. The membrane protein heterodimer is synthesized as a p62E1 precursor protein which upon transport to the cell surface is cleaved into the mature E2E1 form. Recent studies have given much new information on the assembly and entry mechanism of this simple RNA virus. Much of this work has been possible through the construction of a complete cDNA clone of the SFV genome which can be used for in vitro transcription of infectious RNA. One important finding has been to show that a spike deletion variant and a capsid protein deletion variant are budding-negative when expressed separately but can easily complement each other when transfected into the same cell. This shows clearly that enveloped viruses use different budding strategies: one which depends on a nucleocapsid-spike interaction as exemplified by SFV and another one which is based on a direct core-lipid bilayer interaction as shown before to be the case with retroviruses. Another important finding concerns the activation process of the presumed fusion protein of SFV, the E1 subunit. In the original p62E1 heterodimer E1 is completely inactive. Activation proceeds in several steps. First p62 cleavage activates the potential for low pH inducible fusion. Next the low pH which surrounds incoming virus in endosomes induces dissociation of the heterodimeric structure. This is followed by a rearrangement of E1 subunits into homotrimers which are fusion active.

Alphavirus assembly and entry: role of the cytoplasmic tail of the E1 spike subunit.

The alphavirus Semliki Forest virus (SFV) matures by budding at the cell surface. This process is driven by interactions of its membrane protein heterodimer E2-E1 and the nucleocapsid. The virus penetrates into new cells by an E1-mediated membrane fusion event. The E1 subunit has a short, strongly positively charged cytoplasmic tail peptide (Arg-Arg) which is very conserved among different alphavirus E1 proteins. In this work, we have used in vitro mutagenesis of a full-size cDNA clone of SFV to study the role of the tail peptide of the E1 subunit in virus budding and fusion processes in baby hamster kidney cell culture. Our results suggest that the E1 tail plays no major role in SFV multiplication in animal cell culture.

Spike protein-nucleocapsid interactions drive the budding of alphaviruses.

Semliki Forest virus (SFV) particles are released from infected cells by budding of nucleocapsids through plasma membrane regions that are modified by virus spike proteins. The budding process was studied with recombinant SFV genomes which lacked the nucleocapsid protein gene or, alternatively, the spike genes. No subviral particles were released from cells which expressed only the nucleocapsid protein or the spike proteins. Virus release was found to be strictly dependent on the coexpression of the nucleocapsid and the spike proteins. These results provide direct proof for the hypothesis that the alphavirus budding is driven by nucleocapsid-spike interactions. The importance of the viral 42S RNA for virus assembly and budding was investigated by using the heterologous vaccinia virus-T7 expression system for the synthesis of the SFV structural proteins. The results demonstrate that the viral genome is not absolutely required for formation of budding competent nucleocapsids, since small amounts of viruslike particles were assembled in the absence of 42S RNA.

Alphavirus spike-nucleocapsid interaction and network antibodies.

Vaux et al. (D. J. T. Vaux, A. Helenius, and I. Mellman, Nature (London) 336:36-42, 1988) recently reported the production of network antibodies that were suggested to have reconstructed a specific interaction between the nucleocapsid of Semliki Forest virus and the cytoplasmic tail of the viral E2 spike protein. The F13 anti-idiotype antibody, which was raised against anti-E2 tail antibodies, was claimed to recognize the virus nucleocapsid. In this report, we have used recombinant SFV viruses to demonstrate that the F13 antibody is not nucleocapsid specific but instead most likely recognizes some component of the viral replication machinery.

Intracellular transport of rubella virus structural proteins expressed from cloned cDNA.

The structural proteins of rubella virus consist of a nucleocapsid protein (C) and two membrane-embedded spike glycoproteins (E1 and E2). Since many reports have suggested that rubella virus buds intracellularly, we have examined the intracellular transport of the structural proteins in the absence of virion formation, particularly whether the membrane glycoproteins are retained inside the cell or are transported to the cell surface. We have expressed the structural proteins from cloned cDNA either alone or in different combinations, have examined the intracellular location of the proteins by immunofluorescence and using biochemical methods, and have looked for plasma membrane-localized E1 or E2 using a cell surface biotinylation assay. The C protein was found in the Golgi complex when expressed with E2 and E1; without the membrane glycoproteins, C appeared to remain in the endoplasmic reticulum (ER). When expressed alone, E1 was retained in a pre-Golgi compartment, and was not detected at the cell surface in any cell line. When E2 was expressed alone a small fraction could be detected at the cell surface, but the majority was retained intracellularly, apparently in the ER and the Golgi. Both proteins were transported to the surface when they were expressed together, albeit with low efficiencies in all cell lines. These data suggest that, although neither glycoprotein carries a dominant intracellular retention signal, E2 and E1 are largely retained in the Golgi even when present as a transport-competent heterodimer.

A single amino acid change in E3 of ts1 mutant inhibits the intracellular transport of SFV envelope protein complex.

At 39 degrees the envelope protein complex (E1-p62) of Semliki Forest virus mutant ts1 is arrested in the rough endoplasmic reticulum (RER). When the infected cultures are shifted to 28 degrees, the complex is transported to the cell surface. During the transport p62 is cleaved into E2 under conditions in which no virus budding takes place. We have sequenced the cDNA, which encodes the envelope proteins of ts1. Comparison with the respective wild-type nucleotide sequence shows only one nucleotide change, G----A, causing a replacement of cysteine-58 (TGC) with tyrosine (TAC) in the E3 protein of ts1. A cDNA fragment from the ts1 genome encoding the mutation in E3 was used to replace the respective fragment of prototype SFV in an eukaryotic expression vector. Intracellular arrest of envelope proteins at 39 degrees was seen in transfected BHK21 cells. A shift of the transfected cells to 28 degrees resulted in the appearance of the envelope proteins at the cell surface. We conclude that the single point mutation is solely responsible for the temperature-sensitive transport defect of ts1 envelope glycoproteins.

The E2 signal sequence of rubella virus remains part of the capsid protein and confers membrane association in vitro.

The capsid (C) protein of rubella virus is translated from a 24S subgenomic mRNA as the first part of a polyprotein containing all three structural proteins of the virus. It is separated from the following protein (E2) by signal peptidase, which cleaves after the E2 signal sequence. We raised an antipeptide antiserum directed against the signal sequence and used the antiserum to show that this sequence is still a part of the C protein in the mature virion. Furthermore, we also showed that, when the C protein is synthesized by in vitro transcription and translation, the resultant protein is membrane associated. This association is not seen with a variant C protein which lacks the signal sequence, and a normally soluble protein (dihydrofolate reductase) becomes membrane associated when the signal sequence is placed at its carboxy terminus.

Phosphorylation of the human transferrin receptor by protein kinase C is not required for endocytosis and recycling in mouse 3T3 cells.

We have investigated the role of phosphorylation in the endocytosis of the human transferrin receptor (TR) by replacing its phosphorylation site, Ser24, with Ala through site-directed mutagenesis of the TR cDNA. The TR Ala24 mutant expressed in mouse 3T3 cells was not phosphorylated, even following stimulation of protein kinase C by phorbol ester. However, in spite of this defect the mutant was efficiently endocytosed and recycled back to the plasma membrane with kinetics similar to those of TR and a control mutant TR Ala63. Thus, these results confirm earlier results by Davis et al. (1986, J. Biol. Chem., 261-9034-9041) that Ser24 of human TR is the phosphorylation site for protein kinase C but do not support a role of this modification as a signal for TR endocytosis and recycling.