Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation.

Abstract

Sindbis virus (SIN) is a small positive-strand enveloped RNA The SIN genome consists of a single-stranded mRNA molecule of 11,703 bases with a 5' cap structure and a 3' poly(A) tract. Upon entry of the host cell, the 5' two-thirds of the genomic RNA is translated to produce replicase proteins essential for cytoplasmic RNA synthesis. RNA amplification is initiated by the synthesis of full-length complementary minus-strand RNAs, which serve as templates for synthesis of additional genome-length plus strands. By initiation at an internal promoter sequence, genome-length minus strands are also used for synthesis of 3'-terminal subgenomic mRNAs that encode the SIN structural proteins. In certain vertebrate cell types, the SIN replication machinery can produce an estimated 5 x 105 molecules per cell of the subgenomic mRNA, -W-107-108 molecules per cell of the virion structural proteins, and released virus titers in excess of 103 infectious particles per cell. As a transient expression system for heterologous RNAs and proteins, SIN offers several potential advantages: (i) a broad range of susceptible host cells, including those of insect, avian, and mammalian origin; (ii) high levels of cytoplasmic RNA and protein expression without splicing; and (iii) the facile construction and manipulation of recombinant RNA molecules by using a full-length SIN cDNA clone from which infectious RNA transcripts can be generated by in vitro transcription (2).Previous studies showed that replacement of the SIN structural protein coding region with the bacterial chloramphenicol acetyltransferase (CAT) gene resulted in a selfreplicating RNA, transcription of a subgenomic CAT mRNA, and 108 polypeptides per cell of active CAT (3). Since these RNA constructs do not encode structural proteins, their usefulness is limited by the efficiency of RNA transfection or alternatively requires the use of a helper system for packaging (refs. 3 and 4; B. Pragai and C.M.R., unpublished results). Here, we report the construction of SIN vectors that are both replication-packaging competent and that allow the rapid recovery oftMered infectious recombinant virus stocks.

Alphavirus-derived small RNAs modulate pathogenesis in disease vector mosquitoes

“…aegypti infected with recombinant SIN viruses. Small RNA libraries were prepared from mosquitoes injected with double subgenomic SIN virus (dsSINV) derived from the infectious cDNA clone pTE/3Ј2J (28), from mosquitoes injected with dsSINV expressing the FHV B2 protein [dsSINV-B2 (FHV)], or from uninfected mosquitoes. After sequencing by synthesis, we analyzed the total 18-to 24-nt content in each of the 3 small RNA libraries.…”

Characterization of truncated forms of hepatitis C virus glycoproteins.

“…The sequences of these clones were confirmed by DNA sequencing. A double subgenomic Sindbis virus recombinant (pSINrep\HCV171-311\HCV370-661) expressing truncated E1 and E2 (ending at amino acids 311 and 661, respectively) was constructed as described by Hahn et al (1992). Briefly, the E2-coding sequence from pSINrep\HCV370-661 was subcloned into the phagemid shuttle vector pH3h2J1 and then an ApaI fragment which contained a second copy of the subgenomic promoter driving expression of E2 t''" was subcloned into the ApaI site of pSINrep\HCV171-311.…”

Efficient Induction of Primary and Secondary T Cell-Dependent Immune Responses In Vivo in the Absence of Functional IL-2 and IL-15 Receptors

“…with 5 ϫ 10 7 PFU of recombinant Sindbis virus (SINJS510) expressing the same JHMV S510 epitope and challenging 4 wk later with 5 ϫ 10 7 PFU of vJS510. SINJS510 was generated by inserting hybridized oligonucleotides encoding the S510 epitope (5Ј-CTAGATGTGTTCTCTTTGGAATGGGCCCCATTTGTGA-3Ј and 5Ј-CTAGTCACAAATGGGGCCCATTCCAAAGAGAACACAT-3Ј) into the XbaI site of the parent SIN vector pTE3Ј2J (27). Stocks of SINJS510 virus were generated via transfection of BHK-21 cells with infectious in vitro transcribed mRNA from XhoI linearized pSINJS510 and titered using BHK-21 cells (27).…”

“…SINJS510 was generated by inserting hybridized oligonucleotides encoding the S510 epitope (5Ј-CTAGATGTGTTCTCTTTGGAATGGGCCCCATTTGTGA-3Ј and 5Ј-CTAGTCACAAATGGGGCCCATTCCAAAGAGAACACAT-3Ј) into the XbaI site of the parent SIN vector pTE3Ј2J (27). Stocks of SINJS510 virus were generated via transfection of BHK-21 cells with infectious in vitro transcribed mRNA from XhoI linearized pSINJS510 and titered using BHK-21 cells (27). Mice were sacrificed at indicated time points postchallenge and single-cell suspensions were prepared from the inguinal lymph nodes and spleen.…”