Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.     

Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation

Retroviral RNA encapsidation is a highly selective process mediated through recognition by the viral Gag proteins of cis-acting RNA packaging signals in genomic RNA. This RNA species is also translated, producing the viral gag gene products. The relationship between these processes is poorly understood. Unlike that of human immunodeficiency virus type 1 (HIV-1), the dominant packaging signal of HIV-2 is upstream of the major splice donor and present in both unspliced and spliced viral RNAs, necessitating additional mechanisms for preferential packaging of unspliced genomic RNA. Encapsidation studies of a series of HIV-2-based vectors showed efficient packaging of viral genomes only if the unspliced, encapsidated RNA expressed full-length Gag protein, including functional nucleocapsid. We propose a novel encapsidation initiation mechanism, providing selectivity, in which unspliced HIV-2 RNA is captured in cis by the Gag protein. This has implications for the use of HIV-2 and other lentiviruses as vectors.     

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.     

Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA.

We previously identified blocks of sequence near the 5' end of the human immunodeficiency virus (HIV-1) genome which conferred on RNA the ability to bind specifically to the HIV-1 Gag polyprotein, Pr55gag (J. Luban and S. P. Goff, J. Virol. 65:3203-3212, 1991; R. Berkowitz, J. Luban, and S. P. Goff, J. Virol. 67:7190-7200, 1993). Here we report the use of an RNase protection assay to quantify the effect of deletion of these sequences on RNA packaging into virions. First, we demonstrated with wild-type HIV-1 sequences that in comparison with spliced viral RNA, full-length viral genomic RNA is enriched 20-fold in virions. A previously described mutation with deletion of sequences between the major splice donor and the first codon of gag (A. Lever, H. Gottlinger, W. Haseltine, and J. Sodroski, J. Virol. 63:4085-4087, 1989) disrupted these ratios such that different HIV-1 RNA forms were packaged in direct proportion to cytoplasmic concentrations. The effect of deletion mutations preceding and within gag coding sequence on packaging was then tested in competition with RNAs containing wild-type packaging sequences. Using this system, we were able to demonstrate significant effects on packaging of RNAs with mutations immediately preceding the first codon of gag. The greatest reduction in packaging was seen with RNAs lacking the first 40 nucleotides of gag coding sequence, although sequences more 3' had slight additional effects.     

The human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site

Several retroviruses have recently been shown to promote translation of their gag gene products by internal ribosome entry. In this report, we show that mRNAs containing the human immunodeficiency virus type 1 (HIV-1) gag open reading frame (ORF) exhibit internal ribosome entry site (IRES) activity that can promote translational initiation of Pr55(gag). Remarkably, this IRES activity is driven by sequences within the gag ORF itself and is not dependent on the native gag 5'-untranslated region (UTR). This cap-independent mechanism for Pr55(gag) translation may help explain the high levels of translation of this protein in the face of major RNA structural barriers to scanning ribosomes found in the gag 5' UTR. The gag IRES activity described here also drives translation of a novel 40-kDa Gag isoform through translational initiation at an internal AUG codon found near the amino terminus of the Pr55(gag) capsid domain. Our findings suggest that this low-abundance Gag isoform may be important for wild-type replication of HIV-1 in cultured cells. The activities of the HIV-1 gag IRES may be an important feature of the HIV-1 life cycle and could serve as a novel target for antiretroviral therapeutic strategies.     

Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes.

Human immunodeficiency virus type 1 (HIV-1) can be used to generate recombinant viral vectors for delivery of heterologous genes to human CD4-positive lymphocytes. To define the cis-acting sequences required for efficient gene transfer, a number of HIV-1 vectors containing a previously identified packaging signal, long terminal repeats, and additional gag, pol, and env viral sequences were designed. By providing the viral proteins in trans, recombinant viruses were generated and analyzed for their abilities to transfer genes into human T lymphocytes. Inclusion of up to 653 nucleotides derived from the 5' end of the gag gene in the vector improved the efficiency of gene transfer, but inclusion of additional gag or pol sequences did not further improve this efficiency. The increased efficiency of gene transfer associated with the inclusion of 5' gag sequences in the vector arose, at least in part, from an increase in the packaging of vector RNA. The presence of the Rev-responsive element (RRE) increased the efficiency of transfer of vectors containing significant lengths of gag sequence, as expected from the Rev requirement for nucleus-to-cytoplasm transport of unspliced vector RNA containing intact packaging signals. However, the presence of a RRE did not affect the transfer efficiency of smaller vectors lacking significant lengths of gag sequences, arguing against a specific role for the RRE in packaging or vector transfer. These results contribute to an understanding of the minimal cis-acting sequences that operate in the context of HIV-1 vectors for delivering genes into human lymphocytes.     

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.     

Stable expression of primary human immunodeficiency virus type 1 structural gene products by use of a noncytopathic sindbis virus vector

Efficient expression of the human immunodeficiency virus type 1 (HIV-1) structural gene products Gag, Pol, and Env involves the regulation by viral Rev and Rev-responsive elements (RRE). Removal of multiple inhibitory sequences (INS) in the coding regions of these structural genes or modification of the codon usage patterns of HIV-1 genes to those used by highly expressed human genes has been found to significantly increase HIV-1 structural protein expression in the absence of Rev and RRE. In this study, we show that efficient and stable expression of the HIV-1 structural gene products Gag and Env could be achieved by transfection with a noncytopathic Sindbis virus expression vector by using HIV-1 sequences from primary isolates without any sequence modification. Stable expression of these Gag and Env proteins was observed for more than 12 months. The fact that the Sindbis virus expression vector replicates its RNA only in the cytoplasm of the transfected cells and the fact that the lack of expression of HIV-1 Gag by the DNA vector containing unmodified HIV-1 gag sequences was associated with a lack of detectable cytoplasmic gag RNA suggest that a major blockage in the expression of HIV-1 structural proteins in the absence of Rev/RRE is caused by inefficient accumulation of mRNA in the cytoplasm. Efficient long-term expression of structural proteins of diverse HIV-1 strains by the noncytopathic Sindbis virus expression system may be a useful tool for functional study of HIV-1 gene products and vaccine research.     

Efficient particle formation can occur if the matrix domain of human immunodeficiency virus type 1 Gag is substituted by a myristylation signal.

Lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), assemble at and bud through the cytoplasmic membrane. Both the matrix (MA) domain of Gag and its amino-terminal myristylation have been implicated in these processes. We have created HIV-1 proviruses lacking the entire matrix domain of gag which either lack or contain an amino-terminal myristate addition sequence at the beginning of the capsid domain. Myristate- and matrix-deficient [myr(-)MA(-)] viruses produced after transient transfection are still able to assemble into particles, although the majority do not form at the plasma membrane or bud efficiently. Myristylation of the amino terminus of the truncated Gag precursor permits a much more efficient release of the mutant virions. While myr(-)MA(-) particles were inefficient in proteolytic processing of the Gag precursor, myristylation enabled efficient proteolysis of the mutant Gag. All matrix-deficient viruses are noninfectious. Particles produced by matrix-deficient mutants contain low levels of glycoproteins, indicating the importance of matrix in either incorporation or stable retention of Env. Since matrix-deficient viruses contain a normal complement of viral genomic RNA, a role for MA in genomic incorporation can be excluded. Contrary to previous reports, the HIV-1 genome does not require sequences between the 5' splice donor site and the gag start codon for efficient packaging.