Tatsuo Shioda

Sendai virus C proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis

The P/C mRNA of Sendai virus (SeV), a prototypic member of the family Paramyxoviridae in the Mononegavirales superfamily comprising a large number of nonsegmented negative strand RNA viruses, encodes a nested set of accessory proteins, C′, C, Y1 and Y2, referred to collectively as C proteins, initiating, respectively, at ACG/81 and AUGs/114, 183, 201 in the +1 frame relative to the ORF of phospho (P) protein, the smaller subunit of RNA polymerase. Among them, C is the major species expressed in infected cells at a molar ratio which is several‐fold higher than the other three. However, their function has remained an enigma. It has not even been established whether or not the C proteins are essential for viral replication. Many other viruses in Mononegavirales encode C‐like proteins, but their roles also remain to be defined.

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.

IL-4 and a glucocorticoid up-regulate CXCR4 expression on human CD4+ T lymphocytes and enhance HIV-1 replication.

CXCR4 is a key co‐receptor required for the infection of T‐tropic HIV‐1 strain of CD4+ T lymphocytes. The regulation of this chemokine receptor was therefore studied. Th2 polarized cells expressed more CXCR4 than Th1 cells. Among a panel of cytokines and stimulants, a Th2 type cytokine interleukin‐4 (IL‐4) selectively up‐regulated the mRNA level as well as surface protein expression of CXCR4 within 16 h. In addition, CXCR4 was also up‐regulated by a glucocorticoid, dexamethasone. These treated cells became more responsive in transendothelial migration assays to the specific CXCR4 ligand, SDF‐1α. Furthermore, up‐regulation of CXCR4 was also associated with the enhancement of HIV replication in human CD4+ T lymphocytes. This study indicates the enhanced T‐tropic HIV‐1 infection to CD4+ T lymphocytes through up‐regulation of CXCR4 by several immunomodulating agents, IL‐4, and a glucocorticoid. These findings may explain the shift to T‐tropic HIV‐1 dominance during AIDS progression when Th2 comes to predominate. J. Leukoc. Biol. 64: 642–649; 1998.

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.

Accommodation of foreign genes into the Sendai virus genome: sizes of inserted genes and viral replication

Sendai virus (SeV) is an enveloped virus with a negative sense genome RNA of about 15.3 kb. We previously established a system to recover an infectious virus entirely from SeV cDNA and illustrated the feasibility of using SeV as a novel expression vector. Here, we have attempted to insert a series of foreign genes into SeV of different lengths to learn how far SeV can accommodate extra genes and how the length of inserted genes affects viral replication in cells cultured in vitro and in the natural host, mice. We show that a gene up to 3.2 kb can be inserted and efficiently expressed and that the replication speed as well as the final virus titers in cell culture are proportionally reduced as the inserted gene length increases. In vivo, such a size‐dependent effect was not very clear but a remarkably attenuated replication and pathogenicity were generally seen. Our data further confirmed reinforcement of foreign gene expression in vitro from the V(−) version of SeV in which the accessory V gene had been knocked out. Based on these results, we discuss the utility of SeV vector in terms of both efficiency and safety.

Negative regulation of the anti-human immunodeficiency virus and chemotactic activity of human stromal cell-derived factor 1α by CD26/dipeptidyl peptidase IV

Stromal cell‐derived factor 1α (SDF‐1α) is a chemokine that has been shown to prevent infection of T‐tropic HIV strains and is a possible substrate of CD26/dipeptidyl peptidase IV (DPPIV). In this study, we show that SDF‐1α was cleaved at the N‐terminal region by CD26/DPPIV and as a result the inhibitory activity of SDF‐1α against HIV infection disappeared. Moreover, the chemotactic activity of SDF‐1α also disappeared specifically by DPPIV activity of recombinant soluble CD26. These results suggested that dissemination of T‐tropic HIV strains in vivo may be facilitated by CD26/DPPIV via inactivation of functional SDF‐1α.

Importance of the N-glycan in the V3 loop of HIV-1 envelope protein for CXCR-4- but not CCR-5-dependent fusion

The V3 region of HIV‐1 envelope protein possesses a single N‐linked sugar chain, which is conserved in most HIV‐1 strains. We studied its role in the life cycle of HIV‐1 strains with different co‐receptor usage. Removal of the glycan appeared to cause a marked reduction of CXCR‐4‐ but not CCR‐5‐dependent virus entry. A basic amino acid substitution at the 11th position of V3 markedly compensated for the removal of the N‐glycan. These results indicate that the N‐glycan plays an important role for CXCR‐4‐dependent virus entry and that this role is exerted in a particular context of the peptide backbone.

Chimeric viruses between SIVmac and various HIV-1 isolates have biological properties that are similar to those of the parental HIV-1.

Objective:To examine the biological properties of HIV-1/SIVmac chimeric viruses from HIV-1 isolates that have different replication rates, cell tropisms and cytopathicities. Design and methods:Four chimeric viruses with gag, pol, vif, vpx, nef and long terminal repeats of SIVmac and vpr, tat, rev, vpu and env of various HIV-1 isolates were constructed and compared in vitro. Cynomolgus monkeys were inoculated with two chimeras that were replicative in monkey peripheral blood mononuclear cells (PBMC). Results:The type-specific neutralization of the chimeras by monoclonal antibodies 0.5β and μ5.5, which recognize V3 of HIV-1IIIB and HIV-1MN respectively, was observed to be similar to those of the parental viruses, HIV-1NL432, HIV-1HAN2 and HIV-1SF13. The chimeras constructed from HIV-1SF2 and HIV-1SF13, which were isolates from the same individual but from different disease stages, reflected their parental properties, that is, the isolate from the later stage was rapid–high replicating, was more cytopathic and had a wider host range. Chimeras constructed from HIV-1HAN2, HIV-1SF13 and HIV-1NL432 were infectious to macaque monkeys, although the monkeys infected with the chimera from HIV-1SF13 showed lower virus loads and shorter viremic periods than those infected with the others. Conclusions:Chimeras have in vitro properties that are similar to those of their parental HIV-1 isolates, but their growth in macaque PBMC was dependent on which HIV-1 isolate was used. Evaluation of a vaccine by challenging with viruses possessing different antigenicities has become possible in macaque monkeys using newly constructed chimeras.

Enhanced anti-HIV-1 activity of CC-chemokine LD78β, a non-allelic variant of MIP-1α/LD78α

We compared the anti‐HIV‐1 activity of CC‐chemokine LD78β with that of MIP‐1α, another CC‐chemokine which shows 94% sequence homology with LD78β. Despite its close similarity to MIP‐1α, the anti‐HIV‐1 activity of LD78β appeared to be nearly 10 times higher than that of MIP‐1α. Mutagenesis of MIP‐1α showed that the N‐terminal additional tetrapeptide, which was present in LD78β and absent in MIP‐1α, is responsible for enhanced anti‐HIV‐1 activity. The N‐terminal structure‐function relationship of LD78β described here will be of value in understanding the chemokine‐receptor interactions and designing anti‐HIV‐1 compounds based on LD78β.

Large quantity production with extreme convenience of human SDF‐1α and SDF‐1β by a Sendai virus vector

We describe a robust expression of human stromal cell‐derived factor‐1α (SDF‐1α) and SDF‐1β, the members of CXC‐chemokine family, with a novel vector system based upon Sendai virus, a non‐segmented negative strand RNA virus. Recombinant SDF‐1α and SDF‐1β were detected as a major protein species in culture supernatants, reached as high as 10 μg/ml. This remarkable enrichment of the products allowed us to use even the crude supernatants as the source for biological and antiviral assays without further concentration nor purification and will thus greatly facilitate to screen their genetically engineered derivatives.