cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging.

We have previously described a series of human immunodeficiency virus type 1-based vectors in which efficient RNA encapsidation appeared to correlate with the presence of a 1.1-kb env gene fragment encompassing the Rev-responsive element (RRE). In this report, we explore in detail the role of the RRE and flanking env sequences in vector expression and RNA encapsidation. The analysis of a new series of vectors containing deletions within the env fragment failed to identify a discrete packaging signal, although the loss of certain sequences reduced packaging efficiency three- to fourfold. Complete removal of the env fragment resulted in a 100-fold decrease in the vector transduction titer but did not abolish RNA encapsidation. We conclude that the RRE and 3' env sequences are not essential for human immunodeficiency virus type 1 vector encapsidation but may be important in vectors in which a heterologous gene has been placed adjacent to the 5' packaging signal, potentially disrupting its structure.     

Contribution of the gag and pol sequences to the leukemogenicity of Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV) is a highly leukemogenic replication-competent murine retrovirus. Both the F-MuLV envelope gene and the long terminal repeat (LTR) contribute to its pathogenic phenotype (A. Oliff, K. Signorelli, and L. Collins, J. Virol. 51:788-794, 1984). To determine whether the F-MuLV gag and pol genes also possess sequences that affect leukemogenicity, we generated recombinant viruses between the F-MuLV gag and pol genes and two other murine retroviruses, amphotrophic clone 4070 (Ampho) and Friend mink cell focus-inducing virus (Fr-MCF). The F-MuLV gag and pol genes were molecularly cloned on a 5.8-kilobase-pair DNA fragment. This 5.8-kilobase-pair F-MuLV DNA was joined to the Ampho envelope gene and LTR creating a hybrid viral DNA, F/A E+L. A second hybrid viral DNA, F/Fr ENV, was made by joining the 5.8-kilobase-pair F-MuLV DNA to the Fr-MCF envelope gene plus the F-MuLV LTR. F/A E+L and F/Fr ENV DNAs generated recombinant viruses upon transfection into NIH 3T3 cells. F/A E+L virus (F-MuLV gag and pol, Ampho env and LTR) induced leukemia in 20% of NIH Swiss mice after 6 months. Ampho-infected mice did not develop leukemia. F/Fr ENV virus (F-MuLV gag and pol, Fr-MCV env, F-MuLV LTR) induced leukemia in 46% of mice after 3 months. Recombinant viruses containing the Ampho gag and pol, Fr-MCF env, and F-MuLV LTR caused leukemia in 38% of mice after 6 months. We conclude that the F-MuLV gag and pol genes contain sequences that contribute to the pathogenicity of murine retroviruses. These sequences can convert a nonpathogenic virus into a leukemia-causing virus or increase the pathogenicity of viruses that are already leukemogenic.     

Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus.

To identify RNA and protein sequences involved in packaging of human immunodeficiency virus type 1 (HIV-1), various mutations were introduced into the viral genome. Portions of the human immunodeficiency virus type 1 genome between the first splice donor site and the gag initiation codon were deleted to investigate the RNA packaging site (psi). Point mutations that alter cysteine residues in one or both zinc finger motifs of p7, a cleavage product of the gag precursor, were created to study the role of the gag zinc fingers in packaging. The psi site mutants and the gag mutants exhibited similar phenotypes. Cells transfected with the mutant genomes, while expressing normal levels of human immunodeficiency virus type 1 RNA and proteins, produced viral particles that were normal in protein content but lacked detectable viral RNA. These mutant virions were unable to productively infect cells. The combination of human immunodeficiency virus type 1 packaging mutations should minimize fortuitous assembly of infectious virus and may provide a means to produce noninfectious particles for candidate vaccines.     

Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region.

Replication-competent retroviruses can be modified to carry nonviral genes. Such gene transfer vectors help define regions of the retroviral genome that are required in cis for retroviral replication. Moloney murine leukemia virus has been used extensively in vector construction, and all of the internal protein-encoding regions can be removed and replaced with other genes while still allowing production of virions containing and transmitting the altered retroviral genome. However, inclusion of a portion of the gag region from Moloney murine leukemia virus markedly increases the titer of virus derived from these vectors. We determined that this effect was due to more efficient packaging of the vector RNA into particles and did not depend on protein synthesis from the gag region. We conclude that the retrovirus packaging signal extends into the gag region. We have found that retroviral vectors containing the complete packaging signal allow more efficient gene transfer into a variety of cell types. In addition, these results may help explain why many oncogenic retroviruses have retained gag sequences and often express transforming proteins that are gag-onc hybrids.     

Phosphorylation regulates RNA binding by the human T-cell leukemia virus Rex protein.

The Rex protein of human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) regulates the expression of the viral structural genes and is critical for viral replication. Rex acts by specifically binding to RNAs containing sequences of the R region of the 5' long terminal repeat. Two forms of Rex detected in HTLV-II-infected cells, p26rex and p24rex, differ in the extent of serine phosphorylation. Two-dimensional phosphopeptide analysis indicates that p26rex is extensively phosphorylated at multiple sites. Using a sensitive immunobinding assay, we show that the phosphorylation state of Rex determines the efficiency of binding of Rex to HTLV-II target RNAs. Thus, the phosphorylation state of Rex in the infected cell may be a switch that determines whether virus exists in a latent or productive state. These studies also suggest that phosphorylation of RNA-binding regulatory proteins is a more general mechanism of gene regulation.     

Structure of glycosylated and unglycosylated gag polyproteins of Rauscher murine leukemia virus: carbohydrate attachment sites.

The structural relationships among the gag polyproteins Pr65gag, Pr75gag, and gPr80gag of Rauscher murine leukemia virus were studied by endoglycosidase H digestion and formic acid cleavage. Fragments were identified by precipitation with specific antisera to constituent virion structural proteins followed by one-dimensional mapping. Endoglycosidase H reduced the size of gPr80gag to that of Pr75gag. By comparing fragments of gPr80gag and the apoprotein Pr75gag, the former was shown to contain two mannose-rich oligosaccharide units. By comparing fragments of Pr65gag and Pr75gag, the latter was shown to differ from Pr65gag at the amino terminus by the presence of a leader peptide approximately 7,000 daltons in size. The internal and carboxyl-terminal peptides of the two unglycosylated polyproteins were not detectably different. The location of the two N-linked carbohydrate chains in gPr80gag has been specified. One occurs in the carboxyl-terminal half of the polyprotein at asparagine177 of the p30 sequence and the other is found in a 23,000-dalton fragment located in the amino-terminal region of gPr80gag and containing the additional amino acid sequences not found in Pr65gag plus a substantial portion of p15.     

Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding.

We have mutated amino acids within the receptor-binding domain of Moloney murine leukemia virus envelope in order to identify residues involved in receptor binding. Analysis of mutations in the region of amino acids 81 to 88 indicates that this region is important for specific envelope-receptor interactions. None of the aspartate 84 (D-84) mutants studied bind measurably, although they are efficiently incorporated into particles. D-84 mutants have titers that correspond to the severity of the substitution. This observation suggests that D-84 may provide a direct receptor contact. Mutations in the other charged amino acids in this domain (R-83, E-86, and E-87) yield titers similar to those of wild-type envelope, but the affinity of the mutant envelope in the binding assay is decreased by nonconservative substitutions in parallel to the severity of the change. These other amino acids may either provide secondary receptor contacts or assist in maintaining a structure in the domain that favors efficient binding. We also studied other regions of high hydrophilicity. Our initial characterization indicates that amino acids 106 to 111 and 170 to 188 do not play a major role in receptor binding. Measurements of relative binding affinity and titer indicate that most mutations in the region of amino acids 120 to 131 did not significantly affect receptor binding. However, SU encoded by mutants H123V, R124L, and C131A as well as C81A could not be detected in particles and therefore did not bind measurably. Therefore, the region encompassed by amino acids 81 to 88 appears to be directly involved in receptor binding.     

Evidence that a central domain of nucleocapsid protein is required for RNA packaging in murine leukemia virus.

We have analyzed RNA packaging by a series of mutants altered in the nucleocapsid (NC) protein of Moloney murine leukemia virus (Mo-MuLV). We found that mutants lacking residues 8 through 11 or 44 through 60 of NC package Mo-MuLV RNA with virtually the same efficiency as wild-type Mo-MuLV. In contrast, point mutants altered at the conserved cysteines in the cysteine array (residues 26 and 29) and a mutant lacking residues 16 through 23 packaged Mo-MuLV RNA with approximately 1% of the efficiency of wild-type Mo-MuLV. The deficiency in packaged RNA was observed not only in Northern (RNA) analysis but also in an RNA-PCR assay, which would detect degraded as well as intact RNA. One of the cysteine array mutants was also shown to be defective with respect to encapsidation of hygromycin phosphotransferase mRNA containing a Mo-MuLV packaging signal. We suggest that a central region of NC, consisting of the cysteine array and flanking basic residues, is required for RNA packaging in Mo-MuLV.     

Phylogenetic and physical analysis of the 5'' leader RNA sequences of avian retroviruses.

A study of the secondary structures of the 5'-leader RNA sequences of avian leukosis/sarcoma viruses was conducted using phylogenetic sequence alignment, theoretical structures calculated from base-pairing interactions involving the calculated minimal delta G values, and RNaseT1 sensitivity. The results suggest that all of the avian retroviral RNA leaders may be able to adopt similar conformations. Open reading frames in the leader RNAs may be positioned to facilitate viral activities such as translation and packaging of the genomic RNA into virus particles.     

Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA.

In Moloney murine leukemia virus, the encapsidation Psi element was shown to be necessary and sufficient to promote packaging of viral RNA, and to be required for dimerization. The conformation of the Psi domain (nucleotides 215 to 565) was investigated in solution by chemical probing. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate at cytosine N3 and adenosine N1, and with a carbodiimide derivative at guanosine N1 and uridine N3. Position N7 of adenine residues was probed with diethylpyrocarbonate. The analyses were conducted on in vitro transcribed fragments corresponding either to the isolated Psi domain or to the 5'-terminal 725 nucleotides. The RNA fragments were analyzed in their monomeric and dimeric forms. A secondary structure model was derived from probing data, computer prediction and sequence analysis of related murine retroviruses. One major result is that Psi forms an independent and highly structured domain. Dimerization induces an extensive reduction of reactivity in region 278 to 309 that can be interpreted as the result of intermolecular interactions and/or intramolecular conformational rearrangements. A second region (around position 215) was shown to display discrete reactivity changes upon dimerization. These two regions represent likely elements of dimerization. More unexpectedly, reactivity changes (essentially enhancement of reactivity) were also detected in another part of Psi (around position 480) not believed to contain elements of dimerization. These reactivity changes could be interpreted as dimerization-induced allosteric transitions.     

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.     

Sequence-specific binding of DNA by the Moloney murine leukemia virus integrase protein.

Genetic studies have indicated that integration of retroviral DNA into the host genome depends on the presence of the inverted repeats at the free termini of the long terminal repeats on the unintegrated DNA and on the product of the 3' end of the pol gene (the integrase [IN] protein). While the precise function of the Moloney murine leukemia virus IN protein is uncertain, others have shown that it is a DNA-binding protein and functions in the processing of the inverted repeats prior to integration. By using site-directed mutagenesis, we cloned and expressed the IN protein in Escherichia coli. Crude extracts of total cellular protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, denatured in guanidine, renatured, and incubated with oligonucleotide probes. Single- and double-stranded oligonucleotides corresponding to the termini of unintegrated linear viral DNA were specifically bound by the IN protein in this assay. These data suggest that the role of the Moloney IN protein in the early steps of integration involves sequence-specific recognition of the DNA sequences found at the ends of the long terminal repeats.     

Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein.

The expression of Gag, Pol, Vif, Vpr, Vpu, and Env proteins from unspliced and partially spliced human immunodeficiency virus type 1 (HIV-1) mRNAs depends on the viral protein Rev, while the production of Tat, Rev, and Nef from multiply spliced mRNAs does not require Rev. To investigate the difference between gag and tat mRNAs, we generated plasmids expressing tat-gag hybrid mRNAs. Insertion of the gag gene downstream of the tat open reading frame in the tat cDNA resulted in the inhibition of Tat production. This inhibition was caused, at least in part, by a decrease in the stability of the produced mRNA. Deletions in gag defined a 218-nucleotide inhibitory sequence named INS-1 and located at the 5' end of the gag gene. Further experiments indicated the presence of more than one inhibitory sequence in the gag-protease gene region of the viral genome. The inhibitory effect of INS-1 was counteracted by the positive effect mediated by the Rev-Rev-responsive element interaction, indicating that this sequence is important for Rev-regulated gag expression. The INS-1 sequence did not contain any known HIV-1 splice sites and acted independently of splicing. It was found to have an unusually high AU content (61.5% AU), a common feature among cellular mRNAs with short half-lives. These results suggest that HIV-1 and possibly other lentiviruses have evolved to express unstable mRNAs which require additional regulatory factors for their expression. This strategy may offer the virus several advantages, including the ability to enter a state of low or latent expression in the host.     

An analytical study of the dimerization of in vitro generated RNA of Moloney murine leukemia virus MoMuLV.

The genome of Moloney murine leukemia virus(MoMuLV) is composed of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Recently it was shown that in vitro generated MuLV RNA formed dimeric molecules and that dimerization sequences are located within the Psi encapsidation domain between positions 215 and 420. Conditions for the spontaneous dimerization of a MuLV RNA fragment encompassing the Psi domain have been investigated. The rate of spontaneous MuLV RNA dimer formation is dependent upon RNA, NaCl and MgCl2 concentrations as well as temperature. Thermal denaturation of in vitro generated dimer RNA of 350 nt, from positions 215 to 565, gave a Tm of about 58 degrees C in 100 mM NaCl. This Tm value is very close to that found for RNA corresponding to the 5' 755 nt and to the genomic 70 S RNA isolated from virions. According to thrermodynamic parameters derived from denaturation curves of MuLV dimer RNA generated in vitro, the dimer linkage structure probably involves short sequences.     

Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays.

Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.