Polarized expression of a chimeric protein in which the transmembrane and cytoplasmic domains of the influenza virus hemagglutinin have been replaced by those of the vesicular stomatitis virus G protein.

In polarized epithelial cells, influenza virus buds exclusively from the apical domain of the plasma membrane, whereas vesicular stomatitis virus (VSV) buds exclusively from the basolateral domain. In virus-infected cells, the envelope proteins, influenza hemagglutinin (HA) and vesicular stomatitis virus G (VSV G), are likewise transported to and localized in the same domain of the plasma membrane from which the viruses bud. Previous studies have shown that influenza HA and VSV G proteins, when expressed from cloned cDNAs, are accumulated preferentially on the proper domains (apical and basolateral, respectively), indicating that the signal(s) for polarized transport resides in the polypeptide backbone of the proteins. To further elucidate the structural features required for apical vs. basolateral transport, we have constructed a gene that encodes a chimeric protein (H1GA) containing the external domain of HA and the transmembrane and cytoplasmic domains of VSV G. When the chimeric protein (H1GA) is expressed in CV1 cells using a simian virus 40 late expression vector, it is transported to the cell surface with kinetics similar to that of the native HA protein. Further, the chimeric protein, when expressed in polarized MDCK cells using a vaccinia virus early expression vector, is transported only to the apical surface, suggesting that the ectodomain of HA contains a signal for apical transport.     

NH2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation.

Influenza virus neuraminidase (NA), unlike the majority of integral membrane proteins, does not contain a cleavable signal sequence. It contains an NH2-terminal hydrophobic domain that functions as an anchor. We have investigated the signal function for translocation of this NH2-terminal hydrophobic domain of NA by constructing chimeric cDNA clones in which the DNA coding for the first 40 NH2-terminal hydrophobic amino acids of NA was joined to the DNA coding for the signal-minus hemagglutinin (HA) of influenza virus. The chimeric HA (N4OH) containing the NH2 terminus of NA was expressed in CV1 cells by using a simian virus 40 late-expression vector. The chimeric HA is synthesized, translocated into the rough endoplasmic reticulum, and glycosylated, whereas HA lacking the signal sequence is present only in small amounts and is unglycosylated. These results clearly show that the NH2 terminus of NA, in addition to its anchor function, also provides the signal function in translocation. However, the acquisition of complex oligosaccharides and the transport of N4OH to the cell surface are greatly retarded. To determine if the presence of two anchor sequences, one provided by NA at the NH2 terminus and the other provided by HA at the COOH terminus of N4OH, was responsible for the slow transport, the NH2 terminus of NA was fused to an "anchorless" HA. The resulting chimeric HA (N4OH482) contains the hydrophobic domain of NA at the NH2 terminus but lacks the HA anchor at the COOH terminus. N4OH482 was synthesized and glycosylated; however, as with N4OH, the acquisition of complex oligosaccharides and the migration to the cell surface are greatly retarded. Immunofluorescence data also support that, compared to the native HA, only a small amount of chimeric HA proteins is transported to the cell surface. Thus, the hydrophobic NH2 terminus of NA, although capable of providing the signal function in translocation across the rough endoplasmic reticulum, interferes with the transport of the chimeric HA to the cell surface.     

Replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells.

The envelope glycoprotein (G protein) of vesicular stomatitis virus (VSV) is transported to the basolateral plasma membrane of polarized epithelial cells, whereas the hemagglutinin glycoprotein (HA protein) of influenza virus is transported to the apical plasma membrane. To determine if the cytoplasmic domain of VSV G protein might be important in directing G protein to the basolateral membrane, we derived polarized Madin-Darby canine kidney cell lines expressing G protein or G protein with its normal cytoplasmic domain replaced with the cytoplasmic domain from an influenza HA protein (GHA protein). Indirect immunofluorescence microscopy showed that G protein was present primarily on basolateral surfaces, whereas the GHA protein was present on the apical and basolateral membranes. These results suggest that the cytoplasmic domain can be an important determinant directing polarized expression of an integral membrane protein.     

A sorting signal for the basolateral delivery of the vesicular stomatitis virus (VSV) G protein lies in its luminal domain: analysis of the targeting of VSV G-influenza hemagglutinin chimeras.

When synthesized in polarized epithelial cells, the envelope glycoproteins hemagglutinin of influenza and G of vesicular stomatitis virus are targeted to the apical and basolateral plasma membranes, respectively. To determine which portions of these transmembrane proteins contain information necessary for their sorting, the behavior of two different G-hemagglutinin chimeric polypeptides, consisting of all or nearly all the luminal portion of the vesicular stomatitis virus G protein linked to C-terminal segments of influenza hemagglutinin that included its transmembrane and cytoplasmic domains, was studied in MDCK cells transformed with the corresponding cDNAs. Both chimeras were transported from the endoplasmic reticulum to the Golgi apparatus and from there to the cell surface with the same rapid kinetics as the intact G protein. By using a cell surface immunoprecipitation assay with monolayers cultured on permeable filters that allows the recovery of labeled protein molecules present in each cell surface domain, it was found that both chimeric proteins as well as the intact G protein were delivered almost exclusively to the basolateral surface. This polarized distribution of the polypeptides did not change during a subsequent 90-min chase period, although during this time a large fraction of the glycoprotein molecules underwent degradation. In addition, a small fraction of the cell surface-associated glycoprotein molecules shed their ectoplasmic segments into the basolateral compartment, apparently as a result of a proteolytic cleavage. Immunofluorescence on transverse frozen sections and immunoelectron microscopy revealed a prominent accumulation of the chimeric polypeptides in the lateral cell membranes, with lesser amounts on the basal and apical surfaces. These results indicate that information specifying the basolateral transport of the G glycoprotein is located within the first 426 N-terminal amino acids of its ectoplasmic portion.     

Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus

We have determined the COOH-terminal and NH(2)-terminal amino acid sequences of the vesicular stomatitis virus (VSV) glycoprotein (G). A sequence of 122 COOH-terminal amino acids was deduced from the complete sequence of a cloned DNA insert carrying 470 nucleotides derived from the 3' end of the G mRNA. Evidence presented indicates that this portion of the polypeptide includes the domains of G that reside inside the virion and span the lipid bilayer of the virion. This seems clear because a partial amino acid sequence of a fragment of G that remains associated with the membrane of the virion after exhaustive proteolytic digestions can be located unambiguously in the predicted sequence. This predicted sequence contains an uninterrupted hydrophobic domain beginning 49 amino acids and ending 30 amino acids from the COOH terminus. This region presumably spans the lipid bilayer. The COOH-terminal portion of 29 amino acids contains a high proportion of basic residues and resides inside the virion. The COOH-terminal portion of the VSV G protein therefore resembles in structure that of glycophorin, an erythrocyte membrane protein well characterized previously. The configuration of G in the viral membrane demonstrated here is probably similar for other viral glycoproteins, although this has not been tested as directly in any other case. From the sequence of a DNA primer extended on the RNA genome from the adjacent M protein gene into the G protein gene, we have deduced an NH(2)-terminal G protein sequence of 53 amino acids, including the leader sequence of 16 amino acids. Our sequence confirms, extends, and corrects two partial amino acid sequences reported for this region previously.     

Conditional expression of the vesicular stomatitis virus glycoprotein gene in Escherichia coli.

Bacterial plasmids that directed expression of the vesicular stomatitis virus glycoprotein (G-protein) gene under control of the tryptophan operon regulatory region were constructed. A plasmid directing the synthesis of a G-protein-like protein (containing the NH2-terminal segment of seven amino acids encoded by the trpE gene fused to the complete G-protein sequence lacking only its NH2-terminal methionine) could be transformed into trpR+ (repressed) but not into trpR- (derepressed) cells. This result suggested initially that derepressed synthesis of the G-protein-like protein encoded by this plasmid was lethal in Escherichia coli. Deletion of the sequence encoding the large hydrophobic segment near the COOH terminus of G-protein did not overcome this lethality. Lethality of derepressed synthesis was overcome by deletion of the G-protein gene region encoding 10 amino acids in the hydrophobic NH2-terminal domain (signal peptide). Tryptic peptide mapping demonstrated that the G-protein-like protein and some truncated proteins encoded by the plasmid contained G-protein protein sequences. Antisera to vesicular stomatitis virus precipitated the G-protein-like protein, showing that it shares antigenic determinants with the authentic G-protein protein.     

Purification of the fusion protein of Sendai virus: analysis of the NH2-terminal sequence generated during precursor activation.

The two glycoproteins of Sendai virus, the hemagglutinin-neuraminidase and the fusion protein (F), were separated and purified by affinity chromatography on a Lens culinaris lectin-Sepharose column. F was shown to consist of two disulfide-bonded glycopolypeptide chains, F1 and F2, of molecular weights 51,000 and 11,000, each of which contained 15% carbohydrate by weight. Amino-terminal sequence analysis showed that F2 was blocked and that the hydrophobic sequence NH2-Phe-Phe-Gly-Ala-Val-Ile-Gly-Ile-Ile-Ala-Leu-Gly-Pro-Ala-Thr- was at the amino terminus of F1. This sequence shows identity at six positions with the hydrophobic amino-terminal sequence of the smaller glycopolypeptide chain, HA2, of the hemagglutinin of influenza virus. Both F1 and HA2 are formed by proteolytic cleavage of precursor glycoproteins (Fo, Sendai virus; HAo, influenza virus). Since these cleavages confer infectivity upon both Sendai and influenza viruses and the ability to induce cell-to-cell fusion upon Sendai virus, the hydrophobic NH2-terminal sequences on F1 and HA2 may play a role in fusion of viral and host-cell membranes.     

Rewiring the RNAs of influenza virus to prevent reassortment

Influenza viruses contain segmented, negative-strand RNA genomes. Genome segmentation facilitates reassortment between different influenza virus strains infecting the same cell. This phenomenon results in the rapid exchange of RNA segments. In this study, we have developed a method to prevent the free reassortment of influenza A virus RNAs by rewiring their packaging signals. Specific packaging signals for individual influenza virus RNA segments are located in the 5′ and 3′ noncoding regions as well as in the terminal regions of the ORF of an RNA segment. By putting the nonstructural protein (NS)-specific packaging sequences onto the ORF of the hemagglutinin (HA) gene and mutating the packaging regions in the ORF of the HA, we created a chimeric HA segment with the packaging identity of an NS gene. By the same strategy, we made an NS gene with the packaging identity of an HA segment. This rewired virus had the packaging signals for all eight influenza virus RNAs, but it lost the ability to independently reassort its HA or NS gene. A similar approach can be applied to the other influenza A virus segments to diminish their ability to form reassortant viruses.     

Active influenza virus neuraminidase is expressed in monkey cells from cDNA cloned in simian virus 40 vectors.

We have replaced the late genes of simian virus 40 (SV40) with a cloned cDNA copy of the neuraminidase (NA; EC 3.2.1.18) gene of the WSN (H1N1) strain of human influenza virus. When the SV40-NA recombinant virus was complemented in a lytic infection of monkey cells with a helper virus containing an early region deletion mutant, influenza NA was expressed and readily detected by immunofluorescence as well as by immunoprecipitation of in vivo labeled proteins with monoclonal antibodies against NA. In addition, the expressed NA exhibited enzymatic activity by cleaving the sialic acid residue from alpha-2,3-sialyllactitol. The expressed protein was glycosylated and transported to the cell surface, and it possessed the same molecular weight as the NA of WSN virus grown in monkey cells. Because the structure of NA is quite different from that of other integral membrane proteins and includes an anchoring region at the NH2 terminus consisting of hydrophobic amino acids, we also constructed deletion mutants of NA in this region. Replacement of DNA coding for the first 10 NH2-terminal amino acids with SV40 and linker sequences had no apparent effect on NA expression, glycosylation, transport to the cell surface, or enzymatic activity. However, further deletion of NA DNA encoding the first 26 amino acids abolished NA expression. These data suggest that the hydrophobic NH2-terminal region is multifunctional and is important in biosynthesis and translocation of NA across the membrane as well as in anchoring the protein.     

Studies on the primary structure of the influenza virus hemagglutinin.

The amino-terminal sequence and composition of the subunits of the hemagglutinin (HA) of influenza virus has been determined. The hemagglutinin has been isolated by two techniques. (1) as the intact hemagglutinin after disruption of the virus in sodium dodecyl sulfate, giving 2 subunits of 58,000 daltons (HA1) and 26,000 daltons (HA2), and (2) after treatment of the virus with bromelain, giving 2 subunits of 58,000 daltons (BHA1) and 21,000 daltons (BHA2). In both preparations these subunits are linked by disulfide bonds. The aminoterminal sequences of HA1 and BHA1, and HA2 and BHA2 are the same. The composition of the 50 residue peptide associated with the membrane, which is removed from the C-terminus of HA2 by bromelain, is deduced and shown to be hydrophobic and contain 50% of the serine residues of HA2. The biosynthetic precursor of the hemagglutinin has been purified from the membranes of abortively infected chick fibroblasts and shown to have the same amino terminus as HA1. Thus the order of biosynthesis is NH2-HA1-HA2-COOH. The amino-terminal sequence of BHA2--at the cleavage site of the precursor--is shown to be a palindrome: NH2-Gly-Leu-Phe-Gly-Ala-Ile-Ala-Gly-Phe-Ile-. This sequence is conserved in representative viruses from each of the major pandemics. A region of homologous sequence is described between the hemagglutinins of influenza type A and B viruses.     

Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization.

We have studied the intracellular compartmentalization in yeast of Escherichia coli beta-galactosidase bearing heterologous amino acid sequences at its amino terminus. Chimeras containing as few as 74 NH2-terminal amino acids of GAL4, a yeast positive regulatory protein, at the amino terminus accumulate in the cell nucleus. This and other results are consistent with the proposal that the GAL4 gene product mediates positive control by binding to DNA and that the information for nuclear localization resides in its amino terminus. The amino acid sequence of the GAL4 amino terminus does not agree with the previously proposed consensus sequences responsible for nuclear localization. The beta-galactosidase activity in cells bearing the non-nuclear chimeric proteins is 10-fold greater than in cells bearing chimeric proteins that specifically concentrate in the nucleus.     

Sequence of a RNA templated by the 3''-OH RNA terminus of defective interfering particles of vesicular stomatitis virus.

We have sequenced the endogenous RNA polymerase product produced by disrupted purified virions of vesicular stomatitis virus defective interfering particles by using the newer one-dimensional rapid gel sequencing techniques and confirming this with a modified two-dimensional gel vectoring technique. The sequence of this 46-nucleotide RNA is: 5'(pp)pACGAAGACCACAAAACCA-GAUAAAAAAUAAAAACCACAAGAGGG(U)COH3'. We infer that this sequence is identical to the sequence at the 5' end of infectious vesicular stomatitis virus RNA and is complementary to the sequence of the 3'-OH terminus of this defective interfering particle genome RNA.     

Synthesis and glycosylation in vitro of glycoprotein of vesicular stomatitis virus.

Coupling of ribonucleoprotein particles from L cells infected with vesicular stomatitis virus to a pre-incubated ribosomal system obtained from uninfected HeLa cells allowed synthesis of two proteins. G1 (molecular weight 63,000) and G2 (molecular weight 67,000), and all other proteins of vesicular stomatitis virus except the spike protein G (molecular weight 69,000). Analyses of the tryptic peptides showed that G1, G2, and G had identical peptide sequences. The synthesis of G2 required the presence of membranes; only G1 was synthesized in the absence of any membranes. G2 but not G1 was shown to be a glycoprotein by affinity chromatography on a concanavalin A-Sepharose column. Removal of sialic acid residues from G by neuraminidase resulted in a product having an identical mobility to G2. Digestion of G2 or G with a mixture of neuraminidase (EC 3.2.1.18), beta-galactosidase (EC 3.2.1.23), and beta-N-acetylglucosaminidase (EC 3.2.1.30), however, produced a protein of molecular weight 65,000. These data suggest that G2 is the desialated G and is formed by glycosylation of G1, which is the unglycosylated polypeptide backbone of G.     

Coated vesicles transport newly synthesized membrane glycoproteins from endoplasmic reticulum to plasma membrane in two successive stages.

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Clathrin-coated vesicles have been purified from CHO cells infected with vesicular stomatitis virus and shown to contain G protein in amounts nearly stoichiometric with clathrin. Pulse-chase experiments have demonstrated that this G protein is a transit form and have revealed that G is transported to the cell surface in two successive waves of coated vesicles. The oligosaccharides of G1 protein carried in the early wave are of the "high-mannose" variety which can be cleaved by the enzyme endoglycosidase H; the oligosaccharides of G2 protein in the second, later wave are resistant to endoglycosidase H. The early wave is therefore proposed to correspond to transport of G protein in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, where the oligosaccharides are processed and resistance to endoglycosidase H is conferred; the succeeding wave would represent transport from the Golgi apparatus to the plasma membrane.     

Synthesis and assembly of membrane glycoproteins: presence of leader peptide in nonglycosylated precursor of membrane glycoprotein of vesicular stomatitis virus.

Translation of mRNA encoding vesicular stomatitis virus envelope glycoprotein G by as membrane-free ribosomal extract obtained from HeLa cells yielded a nonglycosylated protein (G1 (Mr 63,000). In the presence of added microsomal membranes, G1 was converted to the glycosylated protein (G2 (Mr 67,000) which is inserted in the membrane vesicles as a transmembrane protein. Labeling with methionine donated by wheat germ initiator tRNA1Met showed that G1 but not G2 contains methionine in the NH2-terminal position. Determination of the NH2-terminal sequence of G1, G2, and G showed that a leader peptide of 16 amino acids is present in G1 but absent from the glycosylated proteins G2 and G. This leader peptide contains at least 62% hydrophobic amino acids and is removed presumably during insertion of G1 into the membrane.     

A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection

To investigate the importance of the rabies virus (RV) glycoprotein (G) in protection against rabies, we constructed a recombinant RV (rRV) in which the RV G ecto- and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein (rRV-VSV-G). We were able to recover rRV-VSV-G and found that particle production was equal to rRV. However, the budding of the chimeric virus was delayed and infectious titers were reduced 10-fold compared with the parental rRV strain containing RV G. Biochemical analysis showed equal replication rates of both viruses, and similar amounts of wild-type and chimeric G were present in the respective viral particles. Additional studies were performed to determine whether the immune response against rRV-VSV-G was sufficient to protect against rabies. Mice were primed with rRV or rRV-VSV-G and challenged with a pathogenic strain of RV 12 days later. Similar immune responses against the internal viral proteins of both viruses indicated successful infection. All mice receiving the rRV vaccine survived the challenge, whereas immunization with rRV-VSV-G did not induce protection. The results confirm the crucial role of RV G in an RV vaccine.     

2018-2020年克拉玛依市甲型H3N2流感病毒分子特征分析北大核心CSCD

目的了解克拉玛依市甲型H3N2流感病毒HA和NA基因特征,为防控提供科学依据。方法收集2018-2020年克拉玛依市甲型H3N2流感毒株进行HA和NA基因序列测定,运用Mega软件与疫苗株进行序列比对和分析。结果 18株毒株HA基因与A/Hong Kong/4801/2014同源性高于A/Kansas/14/2017,NA基因与A/Hong Kong/4801/2014同源性低于A/Kansas/14/2017。相对疫苗株,克拉玛依市毒株已出现氨基酸同时在2个及以上抗原决定簇发生替换;HA蛋白均发生了糖基化位点的改变;未发现与耐药相关位点突变。结论 2019-2020年克拉玛依市毒株与当年度疫苗推荐株匹配度降低且HA已发生抗原漂移,可能导致本地区H3N2毒株流行,神经氨酸酶抑制剂类药物依旧能够有效治疗流感,应加强对流感病毒HA和NA基因进行监测。     

Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles.

In a previous study we demonstrated that vesicular stomatitis virus (VSV) can be used as a vector to express a soluble protein in mammalian cells. Here we have generated VSV recombinants that express four different membrane proteins: the cellular CD4 protein, a CD4-G hybrid protein containing the ectodomain of CD4 and the transmembrane and cytoplasmic tail of the VSV glycoprotein (G), the measles virus hemagglutinin, or the measles virus fusion protein. The proteins were expressed at levels ranging from 23-62% that of VSV G protein and all were transported to the cell surface. In addition we found that all four proteins were incorporated into the membrane envelope of VSV along with the VSV G protein. The levels of incorporation of these proteins varied from 6-31% of that observed for VSV G. These results suggest that many different membrane proteins may be co-incorporated quite efficiently with VSV G protein into budding VSV virus particles and that specific signals are not required for this co-incorporation process. In fact, the CD4-G protein was incorporated with the same efficiency as wild type CD4. Electron microscopy of virions containing CD4 revealed that the CD4 molecules were dispersed throughout the virion envelope among the trimeric viral spike glycoproteins. The recombinant VSV-CD4 virus particles were about 18% longer than wild type virions, reflecting the additional length of the helical nucleocapsid containing the extra gene. Recombinant VSVs carrying foreign antigens on the surface of the virus particle may be useful for viral targeting, membrane protein purification, and for generation of immune responses.