A small element from the Mason-Pfizer monkey virusgenome makes human immunodeficiency virus type 1 expression and replicationRev-independent.

Abstract

A common feature of all replication-competent retroviruses is that the primary transcription product from the proviral DNA contains at least three open reading frames, gag, po0, and env, positioned 5' to 3' in the RNA. This product is always a genome-length RNA that is spliced to generate subgenomic mRNAs. In the case of the "simpler" retroviruses, a single 5' splice site is positioned near the 5' end ofthe primary transcript and splicing involves the use ofone or two 3' acceptor sites positioned downstream in the RNA. Thus the subgenomic molecules are always singly spliced and have had most or all of the gag-pol region removed. However, because splicing is inefficient, enough full-length RNA remains to function both as the mRNA for the gag andpol genes and as the molecule that is packaged into virus particles (1).The situation in human immunodeficiency virus type 1 (HIV-1) is more complex. In this case, the coding regions for several novel genes are positioned near the center of the primary transcript between gag-pol and env and at the 3' end of the genome (2). The central region of the genome also contains several 5' and 3' splice sites, which, in conjunction with the conventionally positioned 5' splice site near the 5' end of the RNA, are used for differential splicing of the primary transcript into over 20 different species of mRNA (3-5). These RNAs are either singly or multiply spliced.In most cases, cellular mRNAs contain introns that are removed by splicing before transport to the cytoplasm occurs. Intron-containing RNAs are usually prevented from exiting the nucleus due to the binding of splicing factors (6, 7), although there are examples of differentially spliced cellular transcripts that are transported with a retained intron (8). Little is known about how these mRNAs are transported.The HIV Rev protein functions to allow nuclear export of unspliced and singly spliced HIV RNA molecules (9-12). These RNAs contain complete introns and are retained in the nucleus in the absence of Rev. The details of how Rev functions are not known, although it is clear it binds to a specific element in the HIV RNA known as the Revresponsive element (RRE) (13,14).Another genus of more complex retroviruses, typified by human T-lymphotropic virus (HTLV) types I and II, seems to have evolved a mechanism similar to that of HIV to facilitate the transport of intron-containing RNA. These viruses utilize a protein called Rex, which, like Rev, must bind to a specific element present in the viral RNA (RxRE) (15). Rex has also been shown to substitute for Rev in promoting the transport of Rev-dependent mRNA (16,17).While the more complex retroviruses have developed Rev and Rex regulation to allow the cytoplasmic expression of their intron-containing RNA, the simpler retroviruses do not seem to have similar trans-acting proteins. Thus, it has been a puzzle how these viruses achieve nuclear export of their full-length RNA that contains the gag-pol intron.Here we report the identification of a 219-nt element from Mason-Pfizer monkey ...

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

“…In contrast, most cellular mRNAs are exported from the nucleus via NXF1/NXT. This pathway is exploited by the simple orthoretrovirus MasonPfizer monkey virus (MMPV), which has a constitutive RNA transport element (CTE) that recruits NXF1 directly (4,19).The trafficking itinerary and assembly competence of HIV-1 Gag can be influenced by the nuclear export history of the mRNA from which it is translated (1, 25, 52). If the HIV-1 RRE is replaced by the MPMV CTE, the normally Rev-dependent unspliced RNA exits the nucleus efficiently, but Gag translation is inefficient (7).…”

Improved safety and titre of murine leukaemia virus (MLV)-based retroviral vectors

“…11-13 It has been reported that such CTEs can substitute for RRE/Rev in HIV and SIV vectors. 7,10,14,15 In this study we demonstrate that the first 420 nucleotides of gag sequence in MLV can increase both cytoplasmic RNA level and packaging efficiency of a vector genome. Its substitution by the MPMV CTE results in MLV vectors with an increased titre (up to 10-fold higher).…”

“…7,8,11 We therefore examined whether the 240 nucleotide MPMV CTE could enhance MLV titre (Figure 1). This sequence shares no homology with the MLV packaging constructs, or human endogenous or infectious retroviruses.…”

HIV-189.6 Gag expressed from a replication competent HSV-1 vector elicits persistent cellular immune responses in mice

“…The gag-pro gene was removed from this vector by digestion with EcoRI and XhoI, and subcloned into identical sites in plasmid pCIM to generate pCMGP89.6. pCIM, originally derived from pSI (Promega, Corp), was constructed as follows: the SV40 late polyadenylation signal was removed from pSI by digestion with EcoRI and ClaI, and replaced with the Mason-Pfizer monkey virus polyadenylation sequence (MPMVpA) and cytoplasmic transport element (CTE) [46]. The MPMVpA/CTE was PCR-amplified from pSR2 (E. Hunter, unpublished) with primers that contained unique restriction sites for EcoR1 (5' end) and ClaI (3' end).…”

“…The M-PMV CTE has previously been shown to mediate transport of unspliced viral transcripts out of the nucleus for protein synthesis in the absence of Rev protein and its cis-acting response element (RRE) [46]. The gag-pro sequence from the primary isolate HIV-1 89.6 [51] was cloned into pCIM, and the entire expression cassette was removed and introduced into a γ 1 34.5 targeting vector to generate pJP200, as described in Materials and Methods.…”