C. Yong Kang

Nucleotide sequence analysis of the s genomic segment of prospect hill virus: Comparison with the prototype hantavirus

The genomic S RNA segment of Prospect Hill virus (PHV), a member of the Hantavirus genus, was molecularly cloned and the nucleotide sequence of the cDNA determined. The PHV S RNA segment is 1675 nucleotides long. A long open reading frame was identified in the viral complementary-sense RNA that could encode a 433 amino acid (49K) nucleocapsid (N) protein. Comparison with the sequence of the related Hantavirus (Hantaan 76-118) S RNA segment indicated that there was 57% nucleotide sequence homology between the two S RNA segments. A higher degree of conservation in amino acid sequence homology (62%) was observed in the N proteins of these viruses. At the N-terminus 147 of 225 amino acids are homologous, while approximately 82% of the 124 amino acids at the C-terminus are homologous between the two N proteins. The longest stretch of homologous amino acid sequence is found in this region, and is 17 amino acids in length. Also, many of the differences in amino acid sequence between the two N proteins resulted from conservative substitutions. Hydropathy plots of the two N proteins also reveal many similarities including a conserved potential antigenic site. Unlike Hantaan virus, a second smaller overlapping open reading frame was observed in the viral complementary-sense RNA of PHV and could potentially encode a 90 amino acid (10.5K) protein. Our data indicate that the N proteins of PHV and Hantaan virus are closely related despite divergence in the nucleotide sequence of their S RNA segments.

The fusion and hemagglutinin-neuraminidase glycoproteins of human parainfluenza virus 3 are both required for fusion

Recombinant vaccinia viruses, VF and VHN, expressing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of human parainfluenza virus 3 (HPIV3) were constructed. Infection of HeLa T4 cells with VF and VHN led to the synthesis of glycoproteins, with the correct apparent molecular weights, that were recognized by monoclonal antibodies specific for HPIV3F and HN. The HN glycoprotein was present on the surface of cells infected with VHN and these cells demonstrated both hemadsorbing and neuraminidase activities. The F glycoprotein was present in cleaved and uncleaved forms and was also expressed on the surface of VF-infected cells. Fusion activity, however, as evidenced by syncytium formation and lysis of human erythrocytes, could only be demonstrated when HeLa T4 cells were coinfected with VF and VHN. Fusion events that are mediated by HPIV3, therefore, require both the F and HN glycoproteins.

Comparison of the deduced gene products of the L, M and S genome segments of hantaviruses

The amino acid sequences deduced from all currently available nucleotide sequences of hantaviruses are compared. Comparisons of three large (L), eight medium (M) and five small (S) genome segments are included. A consensus sequence is provided, allowing easy identification of conserved and unique gene regions. The viruses included in this report represent four serologically distinct hantaviruses which are capable of causing severe, moderate, mild or no human disease.

Expression of gag precursor protein and secretion of virus-like gag particles of HIV-2 from recombinant baculovirus-infected insect cells

A recombinant baculovirus carrying the gag gene but lacking the protease coding sequences of human immunodeficiency virus type 2 (HIV-2) has been constructed. When this recombinant baculovirus is used to infect insect cells, a high level of gag precursor protein, gag pr41, is expressed. Electron microscopy showed that the majority of gag pr41 was budding through the plasma membrane and being released into the culture medium in spherical virus-like particles with a diameter of approximately 100 nm. Metabolic labeling demonstrates that gag pr41 is myristylated. Our results demonstrated that HIV-2 gag pr41 can be assembled into virus-like particles in the absence of other HIV proteins. Rabbits immunized with purified gag pr41 particles produced high-titer antibody and Western blot analysis showed that anti-gag pr41 rabbit sera recognize p17, p24, and p55 gag proteins of HIV-1. These results show that gag pr41 particles are highly immunogenic and that gag proteins of HIV-1 and HIV-2 have similar antigenic epitopes.

Molecular characterization of the Prospect Hill virus M RNA segment: a comparison with the M RNA segments of other hantaviruses

Complementary DNA representing the genomic M RNA segment of the Prospect Hill (PH) Hantavirus was cloned and its nucleotide sequence determined. The PH virus M RNA segment is 3707 nucleotides in length and has a long open reading frame in the viral complementary-sense RNA with a coding capacity of 1142 amino acids. The predicted gene product of the PH virus M segment was compared with the corresponding gene products of Hantaan virus strain 76-118 (Hantaan), Sapporo rat virus strain SR-11 (SR) and Puumala virus strain Hällnäs B1 (Hällnäs). The amino acid sequence identities between the G1 and G2 proteins of PH virus and Hällnäs virus are respectively 74% and 79%. In contrast, the amino acid sequence similarities between the G1 proteins of PH virus and SR virus or Hantaan virus are only 50%. However the G2 proteins of SR and Hantaan viruses were more closely related to the G2 protein of PH virus with amino acid sequence similarities of approximately 62%. The G1 proteins of all four viruses had three potential asparagine-linked glycosylation sites conserved and there was one conserved site in the G2 proteins. Hydrophilicity plots of the four virus glycoproteins were very similar. The region of greatest hydrophilicity was conserved in the Hällnäs, SR and Hantaan viruses, and was located near the C terminus of the G1 protein, whereas the region of greatest hydrophilicity in the PH virus glycoprotein precursor is located closer to the N terminus of the G1 protein. Our data demonstrate that despite differences in the serotypic profiles and virulence of PH and Hällnäs viruses, their G1 and G2 proteins are closely related. We conclude that PH and Hällnäs viruses may have evolved along a separate evolutionary pathway in the Hantavirus genus from that of SR and Hantaan viruses.

Nucleotide sequence analysis of the L gene of vesicular stomatitis virus (New Jersey serotype): identification of conserved domains in L proteins of nonsegmented negative-strand RNA viruses.

We have determined the nucleotide sequence of the L gene of vesicular stomatitis virus (VSV), New Jersey serotype (Ogden strain) by primer extension dideoxy sequencing of the genomic RNA with reverse transcriptase. This analysis completes the entire genomic sequence of the VSVNJ (Ogden). Comparison of the deduced amino acid sequence of this L protein with those reported for L proteins of Indiana serotype and Hazelhurst strain of New Jersey serotype revealed an extensive sequence similarity among all three proteins. The comparison was further extended to the L proteins of other nonsegmented negative-strand RNA viruses, namely the rabies virus and four members of the paramyxovirus family: measles, Newcastle disease, human parainfluenza 3, and Sendai viruses. Our findings confirmed the existence of conserved as well as unique domains in the L proteins, suggesting an evolutionary relationship among these viruses.

Typing of Hantaviruses from five continents by polymerase chain reaction

Hantavirus, a genus in the family Bunyaviridae, is comprised of at least four serologically distinct types: Hantaan, Seoul, Puumala and Prospect Hill. The present communication reports the use of polymerase chain reaction (PCR) for typing 27 independently isolated Hantaviruses from 5 different continents. Total cellular RNA was extracted from virus-infected Vero E6 cell monolayers by the acid guanidium thiocyanate-phenol-chloroform method. We have utilized 5 different sets of oligonucleotide primers ranging from 18 to 22 nucleotides in length; one set was specific for a conserved region of the S genomic segment and used as genus-specific primers, the other 4 sets of primers were designed from unique sequences of the M genomic segment such that each primer set was specific to only one serological type of Hantavirus. The PCR products were analyzed by restriction endonuclease digestion for further confirmation. We typed 10, 12, 3 and 1 isolates into Hantaan, Seoul, Puumala and Prospect Hill respectively. The results of PCR were 100% agreeable with that of serological typing, and thus, PCR can be used as an adjunct test with serological method(s) or an independent test for diagnosis and for typing of new isolates of Hantaviruses.

Maturation of hantaan virus glycoproteins G1 and G2

Hantaan virus-infected Vero E6 cell lysates were used for immunoprecipitation with monoclonal antibodies against glycoprotein G1 (MAbG1) or G2 (MAbG2). When cell lysates were prepared with buffer containing nonionic detergent, both G1 and G2 glycoproteins were precipitated with either MAbG1 or MAbG2. In contrast, when cell lysates were prepared with a buffer containing ionic detergents MAbG1 precipitated only glycoprotein G1 and MAbG2 precipitated only glycoprotein G2. Heterodimers and possibly higher oligomeric forms of the glycoproteins were detected on nonreducing SDS-polyacrylamide gels only after chemical cross-linking and immunoprecipitation with either MAbG1 or MAbG2. In order to determine the sites of Hantaan virus glycoproteins maturation and the G1-G2 complex formation, infected cells were treated with inhibitors that prevent specific steps of oligosaccharide processing. Furthermore, glycoproteins G1 and G2 immunoprecipitated from infected cell lysates or from isolated virus particles were tested for sensitivity to endoglycosidase H, endoglycosidase F, and endoglycosidase D. The results of these experiments show that maturation of both G1 and G2 takes place in the endoplasmic reticulum (ER). Furthermore, G1-G2 complex formation occurs in the ER as well, since the two glycoproteins co-precipitated with either MAbG1 or MAbG2 from infected cell lysates treated with brefeldin A and prepared with buffer containing nonionic detergent.

Genetically modified VSVNJ vector is capable of accommodating a large foreign gene insert and allows high level gene expression

It is desirable to develop a RNA virus vector capable of accommodating large foreign genes for high level gene expression. Vesicular stomatitis virus (VSV) has been used as a gene expression vector, especially Indiana serotype (VSV(Ind)), but less with New Jersey serotype (VSV(NJ)). Here, we report constructions of genetically modified rVSV(NJ) vector carrying various lengths of human hepatitis C virus (HCV) non-structural (NS) protein genes, level of inserted gene expression and characterization of rVSV(NJ). We modified the M gene of VSV(NJ) by changing methionine to arginine at positions 48 and 51 (rVSV(NJ)-M) (Kim and Kang, 2007) for construction of rVSV(NJ) with various lengths of HCV non-structural genes. The NS polyprotein genes of HCV were inserted between the G and L genes of the rVSV(NJ)-M vector, and recombinant VSV(NJ)-M viruses with HCV gene inserts were recovered by the reverse genetics. The recombinant VSV(NJ)-M vector with the HCV NS genes express high levels of all different forms of the NS proteins. The electron microscopic examination showed that lengths of recombinant VSV(NJ)-M without gene of interests, VSV(NJ)-M with a gene of HCV NS3 and NS4A (VSV(NJ)-M-NS3/4A), VSV(NJ)-M with a gene of HCV NS4AB plus NS5AB (VSV(NJ)-M-NS4AB/5AB), and VSV(NJ)-M carrying a gene of HCV NS3, NS4AB, and NS5AB (VSV(NJ)-M-NS3/4AB/5AB) were 172±10.5 nm, 201±12.5 nm, 226±12.9 nm, and 247±18.2 nm, respectively. The lengths of recombinant VSVs increased approximately 10nm by insertion of 1kb of foreign genes. The diameter of these recombinant viruses also increased slightly by longer HCV gene inserts. Our results showed that the recombinant VSV(NJ)-M vector can accommodate as much as 6000 bases of the foreign gene. We compared the magnitude of the IFN induction in mouse fibroblast L(Y) cells infected with rVSV(NJ) wild type and rVSV(NJ) M mutant viruses and show that the rVSV(NJ) M mutant virus infection induced a higher level of the IFN-β compare to the wild type virus. In addition, we showed that the NS protein expression level in IFN-incompetent cells (Mouse-L) infected with rVSV(NJ)-M viruses was higher than in IFN-competent L(Y) cells. In addition, we confirmed that HCV NS protein genes were expressed and properly processed. We also confirmed that NS3 protein expressed from the rVSV(NJ)-M cleaves NS polyprotein at junctions and that NS4A plays an important role as a co-factor for NS3 protease to cleave at the NS4B/5A site and at the NS5A/5B site.

Evolution of the fusion protein gene of human parainfluenza virus 3.

The nucleotide sequences of the fusion (F) gene of 15 clinical strains of human parainfluenza virus 3 (HPIV3) isolated between 1959 and 1987 were compared with the F gene sequence of the prototype strain, Wash/47885/57. Nucleotide sequence diversity was greatest in the noncoding regions of the F gene; however, regions believed to function as transcriptional signals were completely conserved. Amino acid sequences were highly conserved and all but a few amino acid substitutions were conservative in nature. Sequence comparisons indicate heterogeneity in HPIV3 F genes; however, a significant proportion of nucleotide changes are maintained after they first appear and seem to be accumulating with time. Phylogenetic analysis suggests that there are 2 lineages of HPIV3 in North America. The two lineages can be distinguished by specific amino acid differences in the F protein, which correlate with differences in antigenic properties and neutralization patterns of HPIV3. The pattern of HPIV3 evolution, based on the analysis of F gene sequences, most closely resembles that of influenza virus B, vesicular stomatitis virus and Newcastle disease virus.