Hiroshi Shibuta

Production of human immunodeficiency virus (HIV)-like particles from cells infected with recombinant vaccinia viruses carrying the gag gene of HIV

We constructed a recombinant vaccinia virus carrying the entire gag and pol genes of human immunodeficiency virus type 1 (HIV-1). The main gene product detected in the lysates of infected CV-1 and SW480 cells was the gag precursor protein. However, in the culture fluid of infected SW480 cells, but not of infected CV-1 cells, reverse transcriptase (RT) activity was detected. The highest RT activity was found at a density of 1.15 g/ml and this fraction contained many round particles with diameters of 100-150 nm. In contrast to the infected cell lysates, the particles contained the processed gag and pol proteins, suggesting that particle formation may be a prerequisite for efficient processing of the gag precursor by the HIV protease encoded in the pol gene. Particles were also recovered from the culture fluid of SW480 cells infected with another recombinant vaccinia virus carrying only the gag gene. These particles contained the unprocessed gag precursor, indicating that the gag precursor alone was sufficient for particle production.

RNA packaging signal of human immunodeficiency virus type 1

Cells infected with a recombinant vaccinia virus carrying the gag and pol regions of the human immunodeficiency virus type 1 genome (Vac-gag/pol) released human immunodeficiency virus (HIV)-like particles containing HIV-specific RNA. However, cells infected with another recombinant vaccinia, Vac-gag/pol-dP, derived through the deletion of an 85-base region (nucleotide positions 679-763) of the HIV genome between the primer binding site and the gag initiation codon of Vac-gag/pol, produced HIV-like particles devoid of the HIV-specific RNA. This 85-base deletion was suggested to cause the collapse of a stable stem-loop structure of 46 bases (751-796) around the gag initiation codon. To examine the role of the stem-loop structure in the packaging of RNAs, we constructed a vaccinia vector plasmid that carried this 46-base sequence followed by the Sendai virus nucleocapsid (NP) gene. When both Vac-gag/pol-dP and this plasmid were introduced into cells, HIV-like particles released from the cells contained the NP gene RNA. However, another vaccinia vector plasmid, which carried the 46-base sequence in the midst of the NP gene, could not supply RNA for incorporation into HIV-like particles. Computer analysis of this plasmid sequence suggested that the 46-base sequence cannot form the stem-loop structure. These findings suggest that the stem-loop structure formed by the 46-base sequence is crucial as a packaging signal.

The induction of cataracts by HIV-1 in transgenic mice

ObjectiveTo elucidate the tissue specificity of the expression of HIV-1 genes in an animal and its pathological effects on these tissues. Design and methodsTransgenic mice carrying a defective HIV-1 genome were bred in order to overcome the host–range barrier of this virus. ResultsmRNA specific to the transgene was detected in the eyes and the spleen, and, in smaller quantities, in the thymus and the brain. Interestingly, many of the transgenic mice developed cataracts at 3–6 months of age. Swelling and vacuolation of the lens fiber cells were marked, but the epithelial cells of the lens were less affected. HIV antigens were detected in the lens fiber cells and the retina by immunological staining. Accumulation of large amounts of p24 Gag antigen was demonstrated in the affected lens by immunoblot analysis, while negligible Env or other viral proteins was detected. Although accumulation of the Gag protein was also detected in the skin and the brain, no apparent abnormality was observed in these tissues. ConclusionsPreferential expression of the HIV genes in the eyes, skin, brain and lymphoid tissues was demonstrated. The accumulation of the Gag protein is suggested to have detrimental effects on lens fiber cells, causing cataracts.

Characterization of bovine parainfluenza virus type 3.

Bovine parainfluenza virus type 3 (PIV‐3) has a buoyant density of 1.197. The RNA of PIV‐3, like that of Sendai virus, is a single continuous chain which lacks polyadenylic acid sequences and tends to self‐anneal to a marked extent. It has a sedimentation coefficient of 42S and a molecular weight of 4.5 × 106, being slightly smaller than Sendai virus RNA (47S, 5.3 × 106).

Varicella virus isolation from spinal ganglion

There is strong support for the view that herpes zoster of skin represents a reactivation of varicella virus which has remained latent in one or more of the spinal or cranial ganglia following an episode of varicella infection (5, 6). However, information regarding the presence of virus in nervous tissues is extremely meager despite the regular demonstration of virus in the skin lesions. The present paper describes the isolation of varicella virus from a zoster patient not only from the skin lesion but also from the corresponding spinal ganglion which had typical histological lesions and varicella antigens demonstrable by immunofluorescence. A 71-year-old Japanese female developed vesicles of herpes zoster on the right chest 3 days before her death due to bacterial pneumonia. Autopsy revealed pneumonia in the left lung and the right lower lobe. Multiple fresh vesicles covered an area of the right lateral chest corresponding roughly to the 7th and 8th thoracic dermatomes. The 7th, 8th and 9th thoracic ganglia on both sides, the corresponding segments of the spinal cord and the affected skin region were removed at autopsy. The skin specimen was then subjected to virus isolation. The other specimens were cut into 2 portions, one for virus isolation and the other for histological and immunofluorescent examinations, and stored at 8 0 ~ in Eagles minimum essential medium (MEM) containing 10 per cent fetal calf serum (FCS) and 7 per cent dimethylsulfoxide until use. The conventional histological examination revealed tha t the right 7th thoracic ganglion was severely affected, while the other ganglia and the spinal cord showed no changes. The central portion of the affected ganglion showed complete hemorrhagic necrosis, which was surrounded by a less damaged zone showing degeneration of ganglion cells, satellite cells, Schwann cells and nerve fibers, accompanied by infiltration of lymphocytes and polymorphonuelear leukoeytes and congestion of blood vessels. Ghosts of ganglion cells were identified on account

Single amino acid substitution of Sendai virus at the cleavage site of the fusion protein confers trypsin resistance.

Amino acid sequences of fusion (F) proteins of two trypsin-resistant mutants of Sendai virus, TR-2 and TR-5, were deduced from nucleotide analysis of cDNA encoding the F gene and were compared with that of the trypsin-sensitive wild-type Sendai virus. In both mutants, amino acid substitutions were found at residues 116 (Arg----Ile), the cleavage site of the F protein, and 109 (Asn----Asp). Two trypsin-sensitive revertants, TSrev-52 and TSrev-58, derived from TR-5 were both activated by trypsin similarly to the wild-type virus and had a single amino acid reversion from Ile to Arg at residue 116, leaving Asp as before at residue 109. These results indicate that the trypsin sensitivity of Sendai virus can be changed by a single amino acid substitution at the cleavage site of the F protein and a mutation from Arg to Ile is responsible for the acquisition of resistance to trypsin.

Antigenic variation of human and bovine parainfluenza virus type 3 strains.

Three human and six bovine parainfluenza virus type 3 (PIV3) strains were examined by the use of 60 monoclonal antibodies (MAbs). Fifty-three MAbs to the human C243 strain were directed against six, four, nine and seven epitopes of the haemagglutinin-neuraminidase (HN), fusion (F), nucleocapsid (N) and matrix proteins, respectively. Seven MAbs to the bovine strain were directed against three epitopes of the HN protein and three epitopes of the F protein. Each strain was characterized in ELISA and immunofluorescence tests with all MAbs and in a haemagglutination inhibition assay with the anti-HN MAbs. There were marked differences between human and bovine viruses, primarily in the HN protein where five epitopes differed. One epitope of the F and one of the N protein also differed. Bovine PIV3 was found to be a homogeneous subtype and distinct from human PIV3.

Rescue of Sendai virus from viral ribonucleoprotein-transfected cells by infection with recombinant vaccinia viruses carrying Sendai virus L and P/C genes.

The Sendai virus ribonucleoprotein (RNP) showed only very low plaque-forming titers upon transfection and the virus yields after one-step growth were quite limited. We tried to enhance the Sendai virus yield by supplying the viral L and P/C gene products through vaccinia vectors. A combination of the recombinant vaccinia viruses carrying the L gene (Vac-HL) and the P/C gene (Vac-HPC), both of which were driven by the promoter of the vaccinia virus 7.5K protein gene, enhanced the yield only a little whereas another combination of Vac-HLd7.5, the L gene insert of which was driven by the promoter of the vaccinia virus thymidine kinase gene in place of the 7.5K promoter, and Vac-HPC greatly enhanced the Sendai virus yield. This seemed to correlate with the fact that the Vac-HL interfered with Sendai virus growth markedly while the Vac-HLd7.5 did not. These results strongly suggest that the L and P/C gene products act in cooperation as the RNA polymerase, and overproduction of the L protein is inhibitory for Sendai virus growth. This system seems to be of value as a tool for analyzing the functions of L and P/C genes of Sendai virus.

Augmentation of c-fos and c-jun expression in transgenic mice carrying the human T-cell leukemia virus type-I tax gene

To analyze the effect of human T-cell leukemia virus type I (HTLV-I) on cellular gene expression and its relation to tumorigenesis, two lines of transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR regions of the HTLV-I genome were produced. The transgene was expressed in many organs, including the brain, salivary gland, spleen, thymus, skin, muscle, and mammary gland. We found that the expression of the c-fos and c-jun genes, but not of thelyn and c-myc genes, was augmented 2- to 20-fold in histologically normal skin and muscle of these mice. The augmentation was tissue specific, suggesting the involvement of a cellular factor in the transgene action. In these mice, a three to seven times higher incidence of tumors was seen as compared with the control mice. These tumors included mesenchymal tumors, such as fibrosarcoma, neurofibroma, and lipoma, and adenocarcinomas of the mammary gland, salivary gland, and lung. The c-fos and c-jun genes were also activated in these tumors. The possible roles of elevated c-fos and c-jun gene expression in tumorigensis are discussed.

Molecular analysis of structural protein genes of the yamagata-1 strain of defective subacute sclerosing panencephalitis virus. II. Nucleotide sequence of a cDNA corresponding to the P plus M dicistronic mRNA

The nucleotide sequence of a cloned cDNA corresponding to the P+M dicistronic mRNA of a subacute sclerosing panencephalitis (SSPE) virus was determined and compared with data of measles virus (MV). The dicistronic mRNA of the SSPE virus consisted of the 3′ proximal 626 nucleotides of P mRNA, intercistronic trinucleotides, a full length of M mRNA, and 75 poly A nucleotides. The part encoding the P protein had a high homology to MV, except at the noncoding region. The terminating consensus sequence of the P gene and the intercistronic trinucleotides of the SSPE virus were CTAC(A)6 and CCT; in MV they are TTAT(A)6 and CTT, respectively. In the M gene, the starting consensus sequence was exactly the same as MV, but at the 5′ proximal end, one third of this gene was different: The first ATG codon of the MV M gene signaling opening of the reading frame was changed to ACG in the SSPE virus and one long open reading frame started from the third ATG codon. The stop codon (TAG) of the MV M gene was also changed to CAG in the SSPE virus. Thus, the deduced SSPE-virus M protein lacked 50 amino acids at the amino terminal and had 15 extra amino acids at the carboxyl end when compared with the MV M protein.