Restricted species tropism of maedi-visna virus strain EV-1 is not due to limited receptor distribution.

The distribution of receptors for maedi-visna virus (MVV) was studied using co-cultivation assays for virus fusion and PCR-based assays to detect the formation of virus-specific reverse transcription products after virus entry. Receptors were present on cell lines from human, monkey, mouse, chicken, quail, hamster and ovine sources. Thus, the distribution of the receptor for MVV is more similar to that of the amphotropic type C retroviruses than to that of other lentiviruses. The receptor was sensitive to proteolysis by papain, but was resistant to trypsin. Chinese hamster ovary (CHO) and lung cells (V79 TOR) did not express functional receptors for MVV. The receptor was mapped to either chromosome 2 or 4 of the mouse using somatic cell hybrids. This allowed several candidates (e.g. MHC-II, CXCR4) that have been proposed for the MVV receptor to be excluded.

Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs.

The enzymatic activity of recombinant influenza virus RNA polymerase is strictly dependent on the addition of a template RNA containing 5' and 3' viral sequences. Here we report the analysis of the binding specificity and physical characterization of the complex by using gel shift, modification interference, and density gradient techniques. The 13S complex binds specifically to short synthetic RNAs that mimic the partially double stranded panhandle structures found at the termini of both viral RNA and cRNA. The polymerase will also bind independently to the single-stranded 5' or 3' ends of viral RNA. It binds most strongly to specific sequences within the 5' end but is unable to bind these sequences in the context of a completely double stranded structure. Modification interference analysis identified the short sequence motifs at the 5' ends of the viral RNA and cRNA templates that are critical for binding.

Sequence requirements for Rev multimerization in vivo.

Multimerization of the human immunodeficiency virus type 1 (HIV-1) Rev protein is believed to be critical to its biological activity. However, the precise protein sequence requirements for Rev multimerization in vivo, and whether multimerization is facilitated by specific RNA binding or vice versa, has remained controversial. In this report, we describe a sensitive in vivo assay for the multimerization of HIV-1 Rev on its cognate RRE primary RNA binding site. Using this assay, we demonstrate that an intact Rev arginine-rich domain, while critical to specific RNA binding, is dispensable for multimerization on the RRE. Mutations introduced into Rev sequences that flank this basic domain produce a partial multimerization phenotype in vivo even though these mutations are known to block Rev multimerization in vitro. Similarly, mutations introduced into the leucine-rich activation domain of Rev, which appear to have no effect on in vitro multimerization, also markedly inhibit multimerization of Rev on the RRE in vivo. Overall, these data appear consistent with the hypothesis that in vivo formation of the multimeric Rev:RRE ribonucleoprotein complex is facilitated by both the RRE RNA substrate and, as first proposed by Bogerd and Greene U. Virol. 67, 2496-2502, 1993), by bridging by a cellular cofactor for Rev that likely interacts with multiple Rev activation domains.

Specific binding of the human T-cell leukemia virus type I Rex protein to a short RNA sequence located within the Rex-response element.

Expression of the structural proteins of human T-cell leukemia virus type I is dependent upon the interaction of the viral Rex trans activator with its highly structured cis-acting RNA target sequence, the 254-nucleotide Rex-response element. Nucleotides critical for Rex binding in vitro have been mapped by modification interference analysis to a discrete 12-nucleotide RNA sequence that is predicted to form a stem-bulge-stem structure. This minimal RNA binding site was sufficient to mediate specific Rex binding in vitro when analyzed in the context of a short RNA probe. The critical importance of this short RNA sequence in mediating Rex function in vivo is supported by its complete conservation among all primate T-cell leukemia virus isolates.

The VP16 transcription activation domain is functional when targeted to a promoter-proximal RNA sequence.

Among eukaryotic transcription trans-activators, the human immunodeficiency virus type 1 (HIV-1) Tat protein is exceptional in that its target site TAR is an RNA rather than a DNA sequence. Here, we confirm that fusion of Tat to the RNA-binding domain of the HIV-1 Rev protein permits the efficient activation of an HIV-1 long terminal repeat (LTR) promoter in which critical TAR sequences have been replaced by RNA sequences derived from the HIV-1 Rev response element (RRE). An RRE target sequence as small as 13 nucleotides is shown to form an effective in vivo target for Rev binding. More important, a fusion protein consisting of Rev attached to the VP16 transcription activation domain was also observed to efficiently activate the HIV-1 LTR from this nascent RNA target. These data demonstrate that trans-activation of transcription by acidic activation domains does not require a stable interaction with the promoter DNA and suggest that VP16, like Tat, can act on steps subsequent to the formation of the HIV-1 LTR preinitiation complex. The finding that the activation domains of VP16 and Tat are functionally interchangeable raises the possibility that these apparently disparate viral trans-activators may nevertheless act via similar mechanisms.

Structural and functional analysis of the visna virus Rev-response element.

The distantly related lentiviruses human immunodeficiency virus type 1 (HIV-1) and visna virus each encode a posttranscriptional regulatory protein, termed Rev, that is critical for expression of the viral structural proteins. We genetically mapped the cis-acting target sequence for visna virus Rev, the visna virus Rev-response element or RRE-V, to a complex 176-nucleotide RNA stem-loop structure that coincides with sequences encoding the N terminus of the transmembrane component of envelope. The computer-predicted structure of the RRE-V was validated by in vitro analysis of structure-specific RNase cleavage patterns. The visna virus Rev protein was shown to interact specifically with the genetically defined RRE-V in vitro but was unable to bind the HIV-1 RRE. Similarly, HIV-1 Rev was also unable to bind the RRE-V specifically. We therefore conclude that the HIV-1 and visna virus Rev proteins, while functionally analogous, nevertheless display distinct RNA sequence specificities. These findings provide a biochemical explanation for the observation that these two viral regulatory proteins are functional only in the homologous viral system.

Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein.

Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev response element (RRE). Nucleotides critical for Rev monomer binding have been mapped by chemical interference to a single site flanking the base of an RNA helix (stem IIB) located within the 234-nucleotide RRE. Binding of additional Rev molecules to an RRE probe did not require any RNA primary sequence information detectable by modification interference beyond that required for binding of a single Rev protein molecule. A synthetic 29-nucleotide RNA molecule designed to incorporate nucleotides identified as critical for Rev binding retained the ability to bind Rev specifically and, therefore, represents a minimal Rev-binding site. We propose that Rev binding to the RRE initiates with the direct interaction of a Rev monomer with a high-affinity binding site located at the base of the IIB stem of the RRE. The subsequent formation of Rev multimers on the RRE appears, in contrast, primarily driven by specific protein-protein interactions.

Mutational definition of the human immunodeficiency virus type 1 Rev activation domain.

Replication of human immunodeficiency virus type 1 requires the functional expression of the virally encoded Rev protein. The binding of this nuclear trans activator to its viral target sequence, the Rev-response element, induces the cytoplasmic expression of unspliced viral mRNAs. Mutation of the activation domain of Rev generates inactive proteins with normal RNA binding capabilities that inhibit wild-type Rev function in a trans-dominant manner. Here, we report that the activation domain comprises a minimum of nine amino acids, four of which are critically spaced leucines. The preservation of this essential sequence in other primate and nonprimate lentivirus Rev proteins indicates that this leucine-rich motif has been highly conserved during evolution. This conclusion, taken together with the observed permissiveness of a variety of eukaryotic cell types for Rev function, suggests that the target for the activation domain of Rev is likely to be a highly conserved cellular protein(s) intrinsic to nuclear mRNA transport or splicing.

Effect of RNA secondary structure on polyadenylation site selection.

Functional polyadenylation [poly(A)] sites consist of two sequence elements, the AAUAAA and G/U box signals, that closely flank the site of mRNA 3'-end formation. In agreement with previous results, random sequence insertions between the AAUAAA and G/U box signals were observed to inhibit poly(A) site function. However, sequence insertions of similar size that were predicted to form RNA stem-loop structures were found to have little effect on the efficiency of polyadenylation and instead induced a 3' shift in the site of polyadenylation that was equal to the length of the inserted stem-loop. The in vivo utilization of a poly(A) site bearing an internal RNA stem-loop structure was inhibited by mutations that destabilized the predicted stem but was restored by compensatory mutations. These results strongly support the hypothesis that the appropriate spacing of the AAUAAA and G/U box signals is critical for poly(A) site function. Sequence insertions that are able to form RNA secondary structures that maintain the correct spacing of these two RNA target sequences are well tolerated, whereas sequence insertions that disturb this spacing inhibit poly(A) site recognition. It is proposed that the effect of sequence insertions on poly(A) site function may be sufficiently predictable to allow the development of an assay for in vivo RNA secondary structure that uses poly(A) site selection as a readout.

Conserved functional organization of the human immunodeficiency virus type 1 and visna virus Rev proteins.

Visna virus encodes a posttranscriptional regulatory protein that is functionally analogous to the Rev trans activator of human immunodeficiency virus type 1. Here, we demonstrate that the known functional organization of the human immunodeficiency virus type 1 Rev trans activator is shared by the distantly related visna virus Rev protein. In particular, both Rev proteins contain an N-terminal domain marked by a highly basic core motif that determines RNA sequence specificity, as well as a second C-terminal domain containing an essential leucine-rich motif that functions as an activation domain. Chimeric proteins consisting of the binding domain of one Rev protein fused to the activation domain of the other were fully functional in the viral sequence context cognate for the binding domain. We also describe derivatives of visna virus Rev bearing a defective activation domain that displayed a trans-dominant negative phenotype in transfected cells. These visna virus Rev mutants may prove useful in the derivation of transgenic animals resistant to this agriculturally important retroviral pathogen.

Efficient polyadenylation within the human immunodeficiency virus type 1 long terminal repeat requires flanking U3-specific sequences.

During transcription of the human immunodeficiency virus type 1 provirus, polyadenylation signals present in the 5' long terminal repeat (LTR) are disregarded while the identical polyadenylation signals present in the 3' LTR are utilized efficiently. As both transcribed LTR sequences contain all signals known to be required for efficient polyadenylation, the basis for this differential utilization has been unclear. Here, we describe experiments that suggest that transcribed sequences present within the human immunodeficiency virus type 1 LTR U3 region act in cis to enhance polyadenylation within the 3' LTR.

Rev activates expression of the human immunodeficiency virus type 1 vif and vpr gene products.

The proteins encoded by human immunodeficiency virus type 1 (HIV-1) can be divided into two temporally regulated classes. Early gene products are encoded by multiply spliced mRNA species and are expressed constitutively. In contrast, late proteins are encoded by a class of unspliced or singly spliced viral transcripts whose cytoplasmic expression is induced by the viral Rev trans activator. Here, we demonstrate that the viral Vif and Vpr proteins are encoded by singly spliced viral mRNAs whose expression is activated by Rev. This activation is shown to result from the reduced utilization of splice sites adjacent to or within the vif and vpr coding sequences. Vif and Vpr therefore belong to the class of late HIV-1 gene products.

Does the human immunodeficiency virus Tat trans-activator contain a discrete activation domain?

Human immunodeficiency virus type 1 (HIV-1) encodes a transcriptional trans-activator, termed Tat, that is absolutely required for viral replication in vitro. By analogy to other known transcription factors, it has been suggested that the HIV-1 Tat protein may contain discrete protein domains that determine sequence specificity and transcriptional activation potential. Here, we report the use of site-directed mutagenesis to examine the functional significance of two candidate activation domains within Tat. A 12 amino acid sequence adjacent to the N-terminus of the Tat protein, which includes a proposed acidic amphipathic alpha-helix activation motif, was found to contribute to, but be dispensable for, Tat function in vivo. In contrast, the integrity of a second potential Tat activation motif, centered on a lysine residue at position 41, was found to be essential for Tat function. However, Tat proteins mutated in this area displayed a fully recessive negative phenotype. Therefore, neither of these two regions of the Tat protein appear to be discrete activation domains. We conclude that previous attempts to categorize Tat as a modular transcription factor have not succeeded and suggest that the functional organization of this complex trans-activator remains to be defined.

Visna virus encodes a post-transcriptional regulator of viral structural gene expression.

Visna virus is an ungulate lentivirus that is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1). Replication of HIV-1 and of other complex primate retroviruses, including human T-cell leukemia virus type I (HTLV-I), requires the expression in trans of a virally encoded post-transcriptional activator of viral structural gene expression termed Rev (HIV-1) or Rex (HTLV-I). We demonstrate that the previously defined L open reading frame of visna virus encodes a protein, here termed Rev-V, that is required for the cytoplasmic expression of the incompletely spliced RNA that encodes the viral envelope protein. Transactivation by Rev-V was shown to require a cis-acting target sequence that coincides with a predicted RNA secondary structure located within the visna virus env gene. However, Rev-V was unable to function by using the structurally similar RNA target sequences previously defined for Rev or Rex and, therefore, displays a distinct sequence specificity. Remarkably, substitution of this visna virus target sequence in place of the HIV-1 Rev response element permitted the Rev-V protein to efficiently rescue the expression of HIV-1 structural proteins, including Gag, from a Rev- proviral clone. These results suggest that the post-transcriptional regulation of viral structural gene expression may be a characteristic feature of complex retroviruses.

HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence.

Expression of human immunodeficiency virus type 1 structural proteins requires both the viral Rev trans-activator and its cis-acting RNA target sequence, the Rev response element (RRE). The RRE has been mapped to a conserved region of the HIV-1 env gene and is predicted to form a complex, highly stable RNA stem-loop structure. Site-directed mutagenesis was used to define a small subdomain of the RRE, termed stem-loop II, that is essential for biological activity. Gel retardation assays demonstrated that the Rev trans-activator is a sequence-specific RNA binding protein. The RRE stem-loop II subdomain was found to be both necessary and sufficient for the binding of Rev by the RRE. We propose that the HIV-1 Rev trans-activator belongs to a new class of sequence-specific RNA binding proteins characterized by the presence of an arginine-rich binding motif.