Nucleocytoplasmic export of type D simian retrovirus genomic RNA: identification of important genetic subregions and interacting cellular proteins.

The simian retrovirus (SRV) genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. The serogroup 2 SRV CTE is predicted to form a stable stem-loop structure containing two major internal loops exhibiting 180 degrees inverse symmetry, with loop face sequences A, A', B, and B' and additional minor internal and terminal loops. To begin the identification of potential CTE-interacting proteins and to assess structural requirements for protein interaction, we conducted RNA mobility shift assays using IR fragments that obliterated this region's known stable stem-loop structure. Using immunoblotting assays, we have determined that RNA helicase A, implicated in the nuclear export of unspliced SRV genomic RNA, does not appear to interact directly with either the complete serogroup 2 SRV 3' IR or the subregion RNAs and that formation of RNA-protein complexes is conferred by interaction with other novel proteins. UV crosslinking of RNA-protein complexes, coupled with RNase T1/A digestion, indicates that a novel protein of 120 kDa molecular weight interacts with the complete CTE or with individual subregion RNAs. Transfection analyses indicate that SRV recombinants containing A, A', B, or B' sequences forming the faces for two open loops undergo RNA export; only the complete sense CTE recombinant or a second recombinant containing two subregions in sense orientation that reconstitute the 3' two-thirds of the 3' IR, and contain only A' and B that form the faces for two terminal loops, are capable of SRV RNA export. These experiments indicate that secondary structural determinants of the 3' IR and multiple protein interactions may be important factors in the nuclear export of unspliced SRV RNA.

Base-pairings within the RNA pseudoknot associated with the simian retrovirus-1 gag-pro frameshift site.

Frameshift and readthrough sites within retroviral messenger RNAs are often followed by nucleotide sequences that have the potential to form pseudoknot structures. In the work presented here, NMR methods were used to characterize the base-pairings and structural features of the RNA pseudoknot downstream of the gag-pro frameshift site of simian retrovirus type-1 (SRV-1) and a functional mutant of the SRV-1 pseudoknot. Evidence is presented that these pseudoknots contain two A-form helical stems of six base-pairs each, connected by two loops, in a classic H-type pseudoknot topology. A particularly interesting feature is that the shorter of the two connecting loops, loop 1, consists of only a single adenosine nucleotide that spans the major groove of stem 2. In this respect, the frameshift-associated pseudoknots are structurally similar to the pseudoknot within the gene 32 mRNA of bacteriophage T2, previously characterized by NMR methods. Despite having similar nucleotide sequences, the solvent exchange rates of the imino protons at the junction of the helical stems in the wild-type and mutant frameshifting pseudoknots differ from each other and from the bacteriophage T2 pseudoknot. The implications of this finding are discussed.

Screening for simian type-D retrovirus infection in macaques, using nested polymerase chain reaction.

A nested polymerase chain reaction (PCR) assay was developed to detect proviral DNA in peripheral blood mononuclear cells (PBMC) of macaques infected with simian type-D retrovirus (SRV/D). Primers were designed to amplify gag gene sequences of SRV/D serotype 1, 2, and 3 viral genomes and were used in a single assay for simultaneous detection of infection with SRV/D-1, SRV/D-2, or SRV/D-3. Results of plasmid dilution studies indicate sensitivity of nested PCR in the range of 1 to 10 genomic copies. The PBMC samples from 395 macaques of unknown SRV/D status, obtained from several primate facilities, were tested in parallel by Western blot (immunoblot) analysis, virus isolation, and nested PCR. Infection was detected in 60 (15.2%) animals by nested PCR, in 40 (10.1%) animals by virus isolation, and in 28 (7.1%) animals by immunoblot. All 40 culture-positive samples were positive by nested PCR. In addition, 11 of 23 immunoblot-positive/virus isolation-negative samples, 2 of 20 immunoblot-indeterminate/virus isolation-negative samples, and 7 of 312 immunoblot-negative/virus isolation-negative samples were identified as positive by nested PCR. Nested PCR is a sensitive and specific assay for simultaneous screening for infection with serotypes 1, 2, and 3 of simian type D retrovirus, and is a powerful tool for rapid screening and surveillance in macaque colonies.

Characterization of rhesus macaque B-lymphoblastoid cell lines infected with simian type D retrovirus.

A simian type D retrovirus designated SRV induces a fatal immunosuppressive disease in rhesus macaques. This syndrome shows many clinical similarities to acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus-infected individuals. To investigate the mechanisms of immune dysfunction in SRV infection, we have focused on the interactions of SRV serotype 1 (SRV-1) with macaque B-lymphoblastoid cell lines (B-LCL). Procedures were optimized for establishing B-LCL by immortalization of macaque B lymphocytes with rhesus Epstein-Barr virus (EBV). These cell lines express B-cell surface markers, secrete immunoglobulins of the IgG or IgM isotypes, and release EBV which transforms monkey B cells. In vitro cultures of B-LCL supported replication of SRV-1. Several B-LCL infected with SRV-1 showed downregulation of major histocompatibility complex (MHC) class II antigen expression whereas levels of MHC class I antigen remained unchanged. Infection of B-LCL with SRV-1 did not alter the level of secreted immunoglobulin. Rhesus EBV was also used to obtain B-LCL from macaques infected with SRV-1; these cell lines were found to release infectious SRV-1. Investigations on the interactions of SRV-1 with B cells will be useful for elucidating mechanisms involved in the immunopathogenesis of primate retroviruses.