Encapsidation Determinants Located Downstream of the Major Splice Donor in the Maedi-Visna Virus Leader Region

Abstract

We investigated the role of the 5-untranslated region between the primer binding site and the gag initiation codon in ovine lentivirus maedi-visna virus (MVV) genomic RNA encapsidation. We identified five computerpredicted stem-loops, three of which were highly conserved in primary sequence and structure. One stable 83-nucleotide (nt) stem-loop (SL4) was not conserved in the primary sequence, but phylogenetic analysis revealed several base pair covariations. The deletion of individual stem-loops did not markedly affect the relative encapsidation efficiency (REE). Only one mutant, carrying a disruption of a 31-nt stem-loop (SL5), had 58% REE in fetal ovine synovial (FOS) cells. A 168-nt deletion (⌬3MSD) downstream of the major splice donor (MSD) which removed three stem-loops, including SL5, resulted in 24% and 20% REE in FOS and 293T cells, respectively. A 100-nt deletion (⌬5MSD) upstream of the MSD resulted in 15-fold lower cellular genomic RNA levels than the wild-type levels in 293T cells. The ⌬5MSD mutant and a double mutant (DM) (⌬5MSD and ⌬3MSD) did not express detectable levels of virion proteins in 293T cells. In contrast, the region deleted in ⌬5MSD was dispensable in FOS cells, and the DM had the same REE as the ⌬3MSD virus. Thus, the region upstream of the MSD contains sequences critical for RNA and protein expression in a cell type-specific fashion. Our results indicate that MVV encapsidation determinants are located downstream of the MSD. These results provide comparative insight into lentiviral encapsidation and can be utilized in the design of MVV-based gene transfer vectors.The 5Ј-untranslated region (5Ј-UTR) in retrovirus genomes contains functional cis-acting stem-loop structures that are involved in various critical steps of the retrovirus life cycle. These steps are regulation of RNA transcription, mRNA splicing, initiation of translation, dimerization of genomic RNA, encapsidation of the viral genomic RNA into virions, and initiation of reverse transcription (6,13,44). While transcription, splicing, and initiation of reverse transcription are associated with short and well-defined regions, translation and encapsidation determinants are less well defined in length and structure.Retroviral genomic RNA is the predominant RNA in virions, yet it constitutes only 1% of the total RNA in the cytoplasm of the infected cell. Thus, the process of encapsidation of two copies of retroviral genomic RNA in the assembling virion is specific. Encapsidation requires interaction of the nucleocapsid (NC) region of the Gag polyprotein with conserved RNA motifs in the 5Ј-UTR, collectively referred to as encapsidation determinants (⌿). In mammalian retroviruses, these RNA determinants are often located between the major splice donor (MSD) and the gag initiation codon (8). Thus, splicing to generate various retroviral mRNAs removes cis encapsidation determinants. This arrangement promotes encapsidation of genomic RNA but excludes spliced subgenomic RNAs.Maedi-visna virus (MVV) is a retrovirus belonging to the lenti...

Small Ruminant Lentiviruses and Human Immunodeficiency Virus: Cousins that Take a Long View

“…[120][121][122]. Whether there is cotranslational packaging of genome as GAG is translated, as is seen in HIV-2, is not known but is unlikely given the position of the encapsidation motif [123,124]. In HIV-1 infected cells up to 40 viral RNA species may be detected which may be functional or aberrant due to inefficient splice donor and acceptor sites [125].…”

“…Genome dimers may stabilise after budding as the virion matures [163] in a similar way to HIV-1 and other retroviruses [118]. Encapsidation motifs to allow packaging of the genome into the virion core are known to be present in the SRLV RNA between the major splice donor site and the gag initiation codon although other regions of the viral RNA are also important [124]. This is more similar to the position of encapsidation motifs of HIV-1 than to HIV-2 [164].…”

Detection of small ruminant Lentivirus proviral DNA in red deer from Poland

“…In addition, primers that bind to sequences located between the major splicing donor (MSD) and the gag initiation codon were used in the second round of amplification. These sequences are highly conserved in sheep and goat lentiviruses, implying a critical functional role for encapsidation [ 39 , 40 ]. The high similarity of the sequences obtained from the two deer in this work supports this thesis.…”

Evaluation of Serological Methods and a New Real-Time Nested PCR for Small Ruminant Lentiviruses

“…M33677), respectively (outer primer-R). The forward primers for the genotype-specific real-time PCRs are located in a previously described conserved region encompassing a stem-loop structure that contains the dimer initiation site (DIS), just upstream of the major splice donor (MSD) of the small ruminant lentiviruses [63,64]. The reverse primers and probes are located immediately downstream of the predicted gag start codon.…”