title: Identification of new splice sites used for generation of rev transcripts in human immunodeficiency virus type 1 subtype C primary isolates.
abstract: The HIV-1 primary transcript undergoes a complex splicing process by which more than 40 different spliced RNAs are generated. One of the factors contributing to HIV-1 splicing complexity is the multiplicity of 3' splice sites (3'ss) used for generation of rev RNAs, with two 3'ss, A4a and A4b, being most commonly used, a third site, A4c, used less frequently, and two additional sites, A4d and A4e, reported in only two and one isolates, respectively. HIV-1 splicing has been analyzed mostly in subtype B isolates, and data on other group M clades are lacking. Here we examine splice site usage in three primary isolates of subtype C, the most prevalent clade in the HIV-1 pandemic, by using an in vitro infection assay of peripheral blood mononuclear cells. Viral spliced RNAs were identified by RT-PCR amplification using a fluorescently-labeled primer and software analyses and by cloning and sequencing the amplified products. The results revealed that splice site usage for generation of rev transcripts in subtype C differs from that reported for subtype B, with most rev RNAs using two previously unreported 3'ss, one located 7 nucleotides upstream of 3'ss A4a, designated A4f, preferentially used by two isolates, and another located 14 nucleotides upstream of 3'ss A4c, designated A4g, preferentially used by the third isolate. A new 5' splice site, designated D2a, was also identified in one virus. Usage of the newly identified splice sites is consistent with sequence features commonly found in subtype C viruses. These results show that splice site usage may differ between HIV-1 subtypes.
authors.affiliation: Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Delgado
authors.forename: Cristina
authors.initials: C
authors.lastname: Carrera
authors.forename: Paloma
authors.initials: P
authors.lastname: Nebreda
authors.forename: Aurora
authors.initials: A
authors.lastname: Fernández-García
authors.forename: Milagros
authors.initials: M
authors.lastname: Pinilla
authors.forename: Valentina
authors.initials: V
authors.lastname: García
authors.forename: Lucía
authors.initials: L
authors.lastname: Pérez-Álvarez
authors.forename: Michael M
authors.initials: MM
authors.lastname: Thomson
mesh_terms: D001483:Base Sequence; D005091:Exons / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007438:Introns / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000821:virology; D008969:Molecular Sequence Data; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012984:Software; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000378:metabolism
chemical_list: D022821:RNA Splice Sites; D012333:RNA, Messenger; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus