Glycoproteins G1 Research Articles

Persistent infection of mammalian cells by Rift Valley fever virus.

Infection of mammalian cells with Rift Valley fever virus (RVFV) leads generally to the production of virus and cell death. In this paper we examined the fate of Vero cells infected with three strains of RVFV and observed that, while a large proportion of cells exhibited a clear cytopathic effect (CPE), a small but significant fraction did not undergo a lytic infection but was able to proliferate and establish a persistent infection. Several independent RVFV persistently infected cell lines have been established and passaged for more than 1 year after infection with a virulent strain (ZH548) and two attenuated strains (C13 and MP12). Although the viruses used for the primary infection were plaque-purified, we do not know whether defective-interfering particles were responsible for the establishment of the persistent infection. The persistently infected cells became resistant to superinfection with RVFV but not with other viruses and shed low amounts of infectious, lytic and non-lytic virus during a limited number of passages. In all the passages tested, the three genomic segments or related products were synthesized as well as the structural nucleoprotein N and glycoproteins G1 and G2. Abnormal defective RNAs were detected, migrating faster or slower than their respective counterparts. The faster-migrating RNAs were internally deleted, some of them possessing only the very terminal part of the 5' genomic end.

The Extracellular Domain of La Crosse Virus G1 Forms Oligomers and Undergoes pH-Dependent Conformational Changes

The La Crosse virus G1 glycoprotein plays a critical role in virus binding to susceptible cells and in the subsequent fusion of viral and cellular membranes. A soluble form of the G1 glycoprotein (sG1) prepared in a recombinant baculovirus system mimics the cell-binding pattern of La Crosse virus and inhibits La Crosse virus infection (A. Pekoszet al., Virology214, 339–348, 1995), presumably by competing for a cellular receptor, a finding that implies that sG1 can perform some functions absent G2, the smaller of the two bunyavirus glycoproteins. We have performed experiments to determine whether sG1 is present as an oligomer and whether it undergoes the conformational changes associated with fusion (F. Gonzalez-Scarano,Virology140, 209–216, 1985). Our results indicate that both sG1 and native G1 undergo similar changes in conformation after exposure to an acidic environment, as detected by reactivity with monoclonal antibodies. Furthermore, using chemical cross-linking, both proteins were detected as oligomers (most likely dimers). Sucrose density gradient analysis of sG1 verified that it was present in monomeric and oligomeric forms. These results demonstrate that the isolated G1 glycoprotein can undergo a pH-dependent change in conformation in the absence of its transmembrane and cytoplasmic tail domains and that the extracellular portion of the glycoprotein can oligomerize.

Analysis of the Intracellular Transport Properties of Recombinant La Crosse Virus Glycoproteins

The G1 and G2 glycoproteins of La Crosse virus, a member of theBunyavirusgenus of the Bunyaviridae, are encoded as a single open reading frame (ORF) in the viral middle-sized RNA segment. The primary product from this ORF is processed, either cotranslationally or shortly after translation, into the two glycoproteins and a nonstructural protein, NSm, of unknown function. We have expressed La Crosse glycoproteins using vaccinia vectors and studied their processing and localization. When expressed in the native G2-NSm-G1 configuration, both G1 and G2 targeted to the Golgi apparatus as shown by their colocalization with wheat germ agglutinin and acquired resistance to endoglycosidase H. When expressed independently, G2 was targeted to the Golgi apparatus but G1 was retained in the endoplasmic reticulum, indicating that a G1–G2 association is required for Golgi targeting of G1. In contrast to results with other members of the Bunyaviridae, we found that expression of G1 and G2 from separate vectors did not lead to the transport of the G1–G2 complex to the Golgi. However, disruption of the NSm region with a foreign sequence did not interfere with transport of the complex. When a portion of the β-galactosidase gene was inserted in frame into NSm, the glycoproteins derived from this construct were processed and targeted properly and were capable of mediating cell-to-cell fusion.

Unique and conserved features of Tula hantavirus M gene encoding envelope glycoproteins G1 and G2.

The sequence of the genomic M segment encoding the surface glycoproteins G1 and G2 of wild-type Tula hantavirus (TUL) has been determined. Analyses of M nucleotide sequences show that TUL is genetically distinct from other hantaviruses. Comparison to ten currently known hantavirus G1G2 amino acid sequences points out several, presumably functional, regions with substantial homology indicating a common ancestry and similar evolutionary pathways for all members of the Hantavirus genus.

Baculovirus Expression and Processing of Tomato Spotted Wilt Tospovirus Glycoproteins

The open reading frame encoding the polyprotein precursor to the tomato spotted wilt tospovirus (TSWV) G1 and G2 glycoproteins was cloned as cDNA and used to produce a recombinant baculovirus. Protein expression was monitored in recombinant baculovirus-infected Spodoptera frugiperda IPLB-SF21 (SF21) cells with monoclonal antibodies raised to TSWV G1 and G2. Western blot analysis demonstrated expression, proteolytic processing, and glycosylation of the G1G2 polyprotein to yield proteins with serological specificities and electrophoretic mobilities similar to the G1 and G2 proteins found in TSWV virions. Indirect immunofluorescence localization of G1 and G2 in infected SF21 cells revealed accumulation of the glycoproteins in the plasmalemma. These experiments establish that the glycoproteins of TSWV, a plant-infecting virus in the family Bunyaviridae, can be processed in the absence of other viral proteins.

Tropism of Bunyaviruses: Evidence for a G1 Glycoprotein-Mediated Entry Pathway Common to the California Serogroup

The California serogroup is composed of antigenically and biologically related viruses within theBunyavirusgenus of the Bunyaviridae. We used a large panel of murine cells to study their tissue tropisms and found virtually identical patterns of viral replication among all of the members of this serogroup, in contrast to other members of the family (Bunyamwera, Cache Valley, and Punta Toro viruses). By analyzing the nonpermissive infections with both an RNA dot-blot and a virus binding assay, we determined that tropism for cultured cells was determined at the level of entry. A truncated soluble form of the La Crosse G1 glycoprotein (sG1) was expressed in a baculovirus system and, despite slight differences in glycosylation, was shown to resemble native G1 by immunoprecipitation with six monoclonal antibodies. sG1 bound to permissive but not to nonpermissive cell lines, as demonstrated by flow cytometry. The sG1 effectively blocked infection of permissive cell lines with all of the California serogroup viruses, but did not block infection of two other bunyaviruses. These results indicate that the California serogroup bunyaviruses share a common receptor on vertebrate cells which may differ from the receptor used by other Bunyaviridae and demonstrate that the G1 glycoprotein is the virus attachment protein. sG1 will be a useful reagent in the search for a putative receptor molecule.

Isla Vista virus: a genetically novel hantavirus of the California vole Microtus californicus.

Prospect Hill virus (PH) was isolated from a meadow vole (Microtus pennsylvanicus) in 1982, and much of its genome has been sequenced. Hantaviruses of other New World microtine rodents have not been genetically characterized. We show that another Microtus species (the California vole M. californicus) from the United States is host to a genetically distinct PH-like hantavirus, Isla Vista virus (ILV). The nucleocapsid protein of ILV differs from that of PH by 11.1% and a portion of the G2 glycoprotein differs from that of PH by 19.6%. ILV antibodies were identified in five of 33 specimens of M. californicus collected in 1975 and 1994-1995. Enzymatic amplification studies showed that 1975 and 1994-1995 ILV genomes were highly similar. Secondary infection of Peromyscus californicus was identified in Santa Barbara County, California. A long-standing enzootic of a genetically distinct hantavirus lineage is present in California voles.

Genetic characterization of a human isolate of Puumala hantavirus from France

PUU90-13 is a strain of Puumala (PUU) virus (family Bunyaviridae: genus Hantavirus) isolated from a human in northeastern France (Rollin et al., 1995) This report describes the full-length sequences of the small (S) and medium (M) genomic RNAs of PUU90-13. The terminal sequences of both the S and M genomic RNAs were found to be conserved and imperfectly complementary. The S RNA of PUU90-13 is 1847 nt in length and contains the nucleocapsid (N) protein gene and a potential overlapping open reading frame (ORF-2) previously described in other hantaviruses. Statistical analysis of the third base substitution frequency in the N ORFs of PUU90-13 and other PUU viruses suggests that the ORF-2 is functional. The M RNA is 3681 nt in length and encodes the glycoprotein precursor. Both genomic segments share the highest degree of nucleotide and amino acid sequence identity with PUUBerkel, a PUU virus from Germany. Phylogenetic analyses of sequences from both segments indicate that PUU90-13 occupies a distinct Western European PUU virus lineage that it shares with PUUBerkel. Both PUU90-13 and PUUBerkel lack a potential N-linked glycosylation site found on the G2 glycoprotein of other PUU viruses.

Homodimeric Association of the Spike Glycoproteins G1 and G2 of Uukuniemi Virus

We have studied the interactions of the G1 and G2 membrane glycoproteins of Uukuniemi virus, a bunyavirus, in virus particles and in Triton X-100-solubilized virus. The G1 glycoprotein in intact virus or in Triton solution could be oxidized into a covalent homodimer using Cu 2+ ion as a catalyst. Immunoprecipitations of the glycoproteins from Triton-solubilized virus lysates showed that G1 and G2 do not form a stable heterodimeric or heterooligomeric complex. The oligomeric association of G1 and G2 was further analyzed using centrifugation in sucrose gradients in the presence of Triton X-100. The results indicate that G1 exists as a Triton-resistant pH-insensitive homodimer. This is in contrast to the behavior of G2, which exists as a homodimer and partially as a monomer at pH 6.4 or above and is dissociated completely into a monomer at pH 6.0 or below. The threshold for the dimer-monomer shift of G2 is between pH 6.2 and pH 6.0. Electron microscopy studies show that the surface structure of the virus particle undergoes a pH-dependent change. Studies on the kinetics of virus entry suggest that pH below 6.2 is necessary for the penetration of Uukuniemi virus.

Nucleotide Sequence Analysis of the M Genomic Segment of El Moro Canyon Hantavirus: Antigenic Distinction from Four Corners Hantavirus

El Moro Canyon hantavirus (ELMC, previously known as HMV-1) is associated with the western harvest mouse Reithrodontomys megalotis. The interpretation that ELMC is a novel hantavirus was based upon comparisons of the nucleotide sequence of the S genomic segment with those of other hantaviruses. We now show that the ELMC M genome, like the S genome, is genetically similar to but distinct from that of Four Corners hantavirus (FC). The ELMC M genome is 3801 nt in length and encodes a glycoprotein precursor of 1139 amino acids. The G1 and G2 genes are 71 and 73.1% identical to those of FC and the corresponding glycoproteins are 73.5 and 82.3% identical. A portion of the G1 glycoprotein of ELMC that is homologous to an important linear epitope of FC differs from the FC epitope by 10 of 31 residues. That domain and flanking sequences were expressed in Escherichia coli. G1 antibodies from 3 of 19 FC-infected patients showed cross-reactivity with ELMC by Western blot assay. The portion of the ELMC G1 antigen recognized by those antibodies is the region of homology to the FC epitope. These data support our previous interpretation that ELMC is distinct from FC and other hantaviruses.

Comparison of the M RNA genome segments of two human isolates of La Crosse virus

The M genomic RNA segments of La Crosse (LAC) virus isolates from the brains of two children autopsied 18 years apart in Wisconsin were molecularly cloned using a reverse transcriptase-PCR assay and the nucleotide sequences of the cDNAs determined. The M RNA of each virus contains 4526 nucleotides, similar to that reported previously for a New York mosquito isolate of LAC. There were 20 nucleotide differences between the two human isolates, which results in the prediction of 7 amino acid changes in the proteins encoded in the single, long open reading frame of the M segment. One of these predicted differences occurs in the G2 glycoprotein and six in the G1 glycoprotein. The two viruses were identical in terms of predicted amino acid sequence in the region believed to represent a nonstructural protein. These data have been further compared to those available for two other California serogroup isolates.

Detection of California serogroup viruses using universal primers and reverse transcription-polymerase chain reaction.

Universal primers have been identified and a protocol developed that are suitable for rapid detection of California encephalitis (CE) complex viruses in a reverse transcription-polymerase chain reaction (RT-PCR) assay. These primers correspond to sequences in the coding regions of the G2 glycoprotein of the middle-size RNA segment. The identities of the amplified products were confirmed by sequencing on the clones or PCR products. The technique is capable of detecting 40 plaque-forming units (PFU) directly on an ethidium bromide-stained agarose gel and the sensitivity increases to 0.4-1 PFU when a radiolabeled probe is used as the detector.

The membrane glycoprotein G1 of Uukuniemi virus contains a signal for localization to the Golgi complex

Members of the Bunyaviridae family acquire their envelopes by budding into the Golgi complex (GC). The accumulation of the membrane glycoproteins G1 and G2 in the GC probably determines the site of maturation. Here we have studied the intracellular transport and targeting to the GC of G1 and G2 of Uukuniemi virus, a member of the Phlebovirus genus, and report on their expression from cloned cDNAs either together or separately by using a T7 RNA polymerase-driven vaccinia virus expression system. When G1 and G2 were expressed together from a full-length cDNA as the p110 precursor, both proteins were localized to the Golgi complex, as evidenced by colocalization with the Golgi marker enzyme mannosidase II. Immunofluorescent staining indicated that G1 expressed alone also localized to the GC. However, pulse-chase experiments showed that G1 remained endoglycosidase H sensitive. G2 expressed alone remained associated with the endoplasmic reticulum (ER). G2 could be rescued from the ER and transported to the GC by coexpression with G1 from separate mRNAs. Coexpression also increased the efficiency of G1 transport to the GC. With none of the constructs could the glycoproteins be observed on the cell surface. These results show that efficient export of G1 and G2 from the ER requires coexpression of both proteins, in conformity with our previous results showing that G1 and G2 from heterodimeric complexes in the ER. Since G1 expressed alone is retained in the GC, we conclude that G1 contains a retention signal for localization to the GC. G2 might thus become associated with the GC indirectly via its interaction with G1.

Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm

The Bunyamwera bunyavirus (BUN) M RNA genome segment encodes three proteins, two glycoproteins termed G1 and G2 and a non-structural protein called NSm, in the form of a polyprotein precursor that is co-translationally cleaved to give the mature proteins. Indirect immunofluorescence experiments have shown that these proteins localize to the Golgi complex in BUN-infected cells. We have used a recombinant vaccinia virus (vTF7-3), which expresses bacteriophage T7 RNA polymerase, to drive the expression of plasmids containing either the entire BUN M segment cDNA or fragments that encode the G1, G2 and NSm proteins separately under control of the T7 promoter. After transfection of these plasmids into vTF7-3-infected cells, correctly sized and processed proteins were detected by immunoprecipitation with BUN-specific antibodies. Immunofluorescence experiments showed that G1, G2 and NSm localized to the Golgi when transiently expressed from the full-length cDNA. When G2 or NSm were expressed separately they also localized to the Golgi, but when G1 was expressed alone a staining pattern typical for the endoplasmic reticulum was obtained. However coexpression of G2 and G1 from independent plasmids resulted in G1 localizing to the Golgi. In contrast translocation of G1 to the Golgi was not observed when G1 was coexpressed with NSm, although NSm itself was still detected in the Golgi. Similar results were obtained when the proteins were expressed from transfected plasmids containing the G2-, NSm- or G1-coding sequences under control of the cytomegalovirus immediate-early promoter. The localization of G1 to the Golgi when coexpressed with G2 was confirmed by the loss of endoglycosidase H (endo H) sensitivity of G1 after approximately 60 min in a pulse-chase experiment; G1 remained sensitive to endo H when expressed either alone or in combination with NSm. These results suggest that G2 contains the Golgi targeting and/or retention signals and that G1 has to interact with this protein to localize to this cellular compartment.

A signal for Golgi retention in the bunyavirus G1 glycoprotein.

The G1 and G2 glycoproteins of Punta Toro virus, a member of the bunyaviruses, are targeted to the Golgi complex, where viral budding occurs. We found that the G1 protein, when expressed in the absence of G2, is also targeted to the Golgi complex. A series of G1 proteins truncated at the carboxyl-terminal region was constructed, and the localization of the expressed proteins was examined. It was found that the proteins expressed from constructs with partial deletions in the cytoplasmic domain were transported to the Golgi complex at a significantly slower rate than G1. Although a major fraction of these proteins was eventually transported to the Golgi complex, they did not exhibit as clearly defined a pattern of accumulation as G1, but rather appeared to be distributed throughout the endoplasmic reticulum as well as the Golgi complex. The proteins expressed from constructs lacking most of the cytoplasmic domain and, in some cases, part of the transmembrane domain sequences as well were transported to the cell surface. We have also constructed chimeric proteins with the envelope protein of a murine leukemia virus (MCFenv), which is efficiently transported to the plasma membrane. A MCF-G1 chimera that contained the G1 transmembrane and cytoplasmic domains was found to be efficiently retained in the Golgi complex, and a construct that contained only the G1 transmembrane domain was also partially retained in the Golgi complex. Thus, the transmembrane domain as well as a portion of the cytoplasmic domain adjacent to the transmembrane domain are apparently crucial for Golgi retention of the G1 protein.

Herpes Simplex Virus Type 2 Antibody in Patients Attending Antenatal or STD Clinics

Objective To determine the prevalence of antibody to herpes simplex virus type 2 (HSV-2) in patients attending a general public antenatal clinic and three public sexually transmitted disease (STD) clinics in Sydney. Background Highly specific tests for herpes simplex type 2 antibody, using the glycoprotein G2, have been recently introduced, allowing determination of past asymptomatic infection. Overseas studies have confirmed the long held suspicion that asymptomatic infection is more common than clinical genital herpes. The seroprevalence of HSV-2 in antenatal and STD clinic patients varies markedly in different countries. These are the first data available for Australia by means of this highly specific test. Design Cross-sectional study of seroprevalence in these two patient groups. Sera used in the antenatal study were those submitted for routine antenatal screening for viral markers. Participants Two hundred and twenty-nine consecutive patients attending the Westmead Hospital antenatal clinics, and 107 consecutive patients attending three public STD clinics. Hypotheses That Australian populations show a relatively high prevalence of past asymptomatic infection with HSV-2; and that higher rates of infection will be found in patients attending STD clinics and with past or current histories of STDs. Main outcome measures Comparison of HSV-2 seroprevalence between antenatal clinic patients and STD clinic patients; and associations of HSV-2 antibody with age, sex, occupation, country of birth, a history of current or past STDs and antibody to HSV-1. Results Antibody to HSV-2 was found in 14.5% of antenatal clinic patients and 40% of STD clinic patients. None of the antenatal patients and less than half of the seropositive STD clinic patients reported clinical genital herpes. Associations with age, socioeconomic status and previous HSV-1 infection were less marked than in studies from the United States. Female STD clinic patients had a significantly higher seroprevalence than males and three times the seroprevalence of age-matched antenatal clinic patients. The correlation between HSV-2 antibody and current gonorrhoea was more marked than that between HSV-2 and other STDs. Conclusion Asymptomatic infection with HSV-2 is quite common in Australian antenatal patients and more common in patients with STDs, who have higher rates of sexual exposure.

Hantaan virus nucleocapsid N protein or G1, G2 glycoprotein precursor cDNA DNA sequence; vector e.g. plasmid pGEM1, plasmid pBR322 or phage M13, expression in e.g. Escherichia coli; application as a recombinant vaccine

Hantaan virus nucleocapsid N protein or G1, G2 glycoprotein precursor cDNA DNA sequence; vector e.g. plasmid pGEM1, plasmid pBR322 or phage M13, expression in e.g. Escherichia coli; application as a recombinant vaccine

Epitope Mapping Studies with Neutralizing and Non-neutralizing Monoclonal Antibodies to the G1 and G2 Envelope Glycoproteins of Hantaan Virus

Epitopes recognized by three G1-specific and two G2-specific neutralizing monoclonal antibodies to Hantaan virus were mapped by sequence analyses of the complete M genome segments of neutralization escape variant viruses. For each variant, we detected nucleotide sequence substitutions which resulted in a single amino acid change in either the G1 or G2 protein. Serological properties of the variant viruses correlated with changes identified by nucleotide sequence analyses. To map epitopes recognized by three G1-specific and six G2-specific, non-neutralizing monoclonal antibodies, we prepared genes, truncated at the carboxy terminal coding regions of G1 or G2, and expressed them with baculovirus recombinants or transiently in a vaccinia/T7 RNA polymerase system. Reactivities of the monoclonal antibodies with the truncated proteins were monitored by immune precipitation of the radiolabeled, truncated glycoproteins. We determined that all three of the G1-specific antibodies reacted with truncated proteins, which retained the amino terminal one-third of G1, but lost reactivity with shorter G1 proteins. The G2-specific antibodies only recognized G2 proteins, which retained approximately 80% of the G2 gene.

Ribonucleotide reductase-deficient mutants of pseudorabies virus are avirulent for pigs and induce partial protective immunity.

We have mutagenized and mapped the gene encoding the large subunit of ribonucleotide reductase (RR1) in pseudorabies virus (PRV; synonyms Aujeszky's disease virus, suid herpesvirus type 1). PRV strains carrying an oligonucleotide that leads to termination of translation of the RR1 gene are avirulent for mice. We subsequently constructed a PRV strain carrying a deletion in the RR1 gene and also a PRV strain carrying both the deletion in the RR1 gene and a deletion in the glycoprotein g1 gene, which is a marker for PRV virulence. Both PRV strains were assayed for virulence and immunogenicity in pigs, the natural host for PRV. In contrast to a marker-rescued PRV strain, these RR1-deleted mutants were avirulent, were shed in very low titres in the oropharyngeal fluid by the animals, and induced low titres of neutralizing antibodies. However, protection against clinical signs after infection with virulent PRV was induced by both RR1-deleted mutants. The relative importance of viral RR and thymidine kinase enzymes for deoxynucleotide synthesis in viral replication is discussed. In addition, we discuss the potential use of RR as a target for anti-herpesviral drugs and the use of PRV strains, deleted for the RR1 gene, as vaccine strains.

The nucleotide sequence of the M RNA segment of tomato spotted wilt virus, a bunyavirus with two ambisense RNA segments.

The complete sequence of the tomato spotted wilt virus (TSWV) M RNA segment has been determined. The RNA is 4821 nucleotides long and has an ambisense coding strategy similar to that of the S RNA segment. The M RNA segment contains two open reading frames (ORFs), one in the viral sense which encodes a protein with a predicted size of 33.6K, and one in the viral complementary sense which encodes the precursor to the G1 and G2 glycoproteins, with a predicted size of 127.4K. Both ORFs are expressed via the synthesis of subgenomic mRNAs that possibly terminate at a stable hairpin structure, located in the intergenic region. The precursor for the glycoproteins contains a sequence motif (RGD) which is characteristic of cellular attachment domains. Significant sequence homology was found between the G1 glycoproteins of members of the genus Bunyavirus and a corresponding region in the glycoprotein precursor of TSWV, indicating a close evolutionary relationship between these viruses. With the elucidation of the M RNA sequence, the complete nucleotide sequence of TSWV has been determined. TSWV represents the first member of the Bunyaviridae shown to contain two ambisense RNA segments.