Chimeric Human Immunodeficiency Virus Type 1 Containing Murine Leukemia Virus Matrix Assembles in Murine Cells

Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of the precursor is mediated by the amino-terminal region of polyprotein. To compensate for the assembly block, we substituted the murine leukemia virus matrix coding sequences into an infectious HIV-1 clone. Transfection of murine fibroblasts expressing cyclin T1 with the chimeric proviruses resulted in viruses that were efficiently assembled and released. Chimeric viruses, in which the cytoplasmic tail of the transmembrane subunit, gp41, was truncated to prevent potential interference between the envelope glycoprotein and the heterologous matrix, could infect human and murine cells. They failed to further replicate in the murine cells, but replicated with delayed kinetics in human MT-4 cells. These findings may be useful for establishing a murine model for HIV-1 replication.     

Analysis of the Assembly Function of the Human Immunodeficiency Virus Type 1 Gag Protein Nucleocapsid Domain

Previous studies have shown that in addition to its function in specific RNA encapsidation, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is required for efficient virus particle assembly. However, the mechanism by which NC facilitates the assembly process is not clearly established. Formally, NC could act by constraining the Pr55^gag polyprotein into an assembly-competent conformation or by masking residues which block the assembly process. Alternatively, the capacity of NC to bind RNA or make interprotein contacts might affect particle assembly. To examine its role in the assembly process, we replaced the NC domain in Pr55^gag with polypeptide domains of known function, and the chimeric proteins were analyzed for their abilities to direct the release of virus-like particles. Our results indicate that NC does not mask inhibitory domains and does not act passively, by simply providing a stable folded monomeric structure. However, replacement of NC by polypeptides which form interprotein contacts permitted efficient virus particle assembly and release, even when RNA was not detected in the particles. These results suggest that formation of interprotein contacts by NC is essential to the normal HIV-1 assembly process.Human immunodeficiency virus type 1 (HIV-1) encodes three major genes, gag, pol, and env, which are commonly found in all mammalian retroviruses. It also encodes accessory genes whose protein products are important for regulation of its life cycle (6, 30, 35). However, of all the genes encoded by HIV-1, only the protein product of the gag gene has been found to be necessary and sufficient for the assembly of virus-like particles (11, 13, 17, 22, 32, 33). The HIV-1 Gag protein initially is expressed as a 55-kDa polyprotein precursor (Pr55^gag), but during or shortly after particle release, Pr55^gag ordinarily is cleaved by the viral protease (PR). The products of the protease action are the four major viral proteins matrix (MA), capsid (CA), nucleocapsid (NC), and p6, and the two spacer polypeptides p2 and p1, which represent sequences between CA and NC and between NC and p6, respectively (15, 19, 23, 30).The HIV-1 nucleocapsid proteins have two Cys-X₂-Cys-X₄-His-X₄-Cys (Cys-His) motifs, reminiscent of the zinc finger motifs found in many DNA binding proteins, and NC has been shown to facilitate the specific encapsidation of HIV-1 genomic RNAs. In addition to its encapsidation function, NC influences virus particle assembly (7, 10, 17, 21, 40). In particular, Gag proteins lacking the NC domain fail to assemble virus particles efficiently. Nevertheless, some chimeric Gag proteins which carry foreign sequences in place of NC have been shown to assemble and release virus particles at wild-type (wt) levels (2, 37, 40). Thus, it appears that in some circumstances, the role that NC plays in virus particle assembly can be replaced. To date, it is not clear how NC affects particle assembly, although several possibilities might be envisioned. One possibility is that deletion of NC unmasks inhibitory sequences in p2 or the C terminus of CA. Alternatively, NC may simply provide a stable monomeric folded structure which locks CA or other Gag domains into an assembly-competent conformation. Another possibility is that NC facilitates assembly by forming essential protein-protein contacts between neighbor Pr^gag molecules, as suggested in cross-linking studies (21). Finally, the assembly role of NC may stem from its RNA binding capabilities, a hypothesis supported by studies of Campbell and Vogt (5), which have shown that RNA facilitates the in vitro assembly of retroviral Gag proteins into higher-order structures.To distinguish among possible mechanisms by which NC facilitates HIV-1 assembly, we replaced NC with polypeptides having known structural characteristics and examined particle assembly directed by these chimeric proteins. Using this approach, we have found that NC does not play a passive role in HIV-1 assembly as either a mask to assembly inhibitor domains or a nonspecific, stably folded structure. Rather, sequences known to form strong interprotein contacts were observed to enhance assembly, suggesting a similar role for the NC domain itself. With several assembly-competent chimeric proteins, we detected no particle-associated RNAs. These results suggest that while RNA may be essential to virus assembly in the context of the wt Pr55^gag protein, it is dispensable for formation of virus-like particles from chimeric proteins.     

Particle Size Determinants in the Human Immunodeficiency Virus Type 1 Gag Protein

The retroviral Gag protein plays the central role in the assembly process and can form membrane-enclosed, virus-like particles in the absence of any other viral products. These particles are similar to authentic virions in density and size. Three small domains of the human immunodeficiency virus type 1 (HIV-1) Gag protein have been previously identified as being important for budding. Regions that lie outside these domains can be deleted without any effect on particle release or density. However, the regions of Gag that control the size of HIV-1 particles are less well understood. In the case of Rous sarcoma virus (RSV), the size determinant maps to the CA (capsid) and adjacent spacer sequences within Gag, but systematic mapping of the HIV Gag protein has not been reported. To locate the size determinants of HIV-1, we analyzed a large collection of Gag mutants. To our surprise, all mutants with defects in the MA (matrix), CA, and the N-terminal part of NC (nucleocapsid) sequences produced dense particles of normal size, suggesting that oncoviruses (RSV) and lentiviruses (HIV-1) have different size-controlling elements. The most important region found to be critical for determining HIV-1 particle size is the p6 sequence. Particles lacking all or small parts of p6 were uniform in size distribution but very large as measured by rate zonal gradients. Further evidence for this novel function of p6 was obtained by placing this sequence at the C terminus of RSV CA mutants that produce heterogeneously sized particles. We found that the RSV-p6 chimeras produced normally sized particles. Thus, we present evidence that the entire p6 sequence plays a role in determining the size of a retroviral particle.     

In Vitro Assembly Properties of Human Immunodeficiency Virus Type 1 Gag Protein Lacking the p6 Domain

Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Δp6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Δp6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Δp6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Δp6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Δp6 and nucleic acid are not sufficient for the formation of normal-sized particles.     

Ubiquitin Is Covalently Attached to the p6Gag Proteins of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus and to the p12Gag Protein of Moloney Murine Leukemia Virus

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6^Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12^Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6^Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.     

The C-Terminal Half of the Human Immunodeficiency Virus Type 1 Gag Precursor Is Sufficient for Efficient Particle Assembly

Human immunodeficiency virus type 1 particle assembly is directed by the Gag polyprotein Pr55^gag, the precursor for the matrix (MA), capsid (CA), and nucleocapsid proteins of the mature virion. We now show that CA sequences N terminal to the major homology region (MHR), which form a distinct domain, are dispensable for particle formation. However, slightly larger deletions which extend into the MHR severely impair particle production. Remarkably, a deletion which removed essentially all MA and CA sequences between the N-terminal myristyl anchor and the MHR reduced the yield of extracellular particles only moderately. Particle formation even exceeded wild-type levels when additional MA sequences, either from the N or the C terminus of the domain, were retained. We conclude that no distinct region between the myristyl anchor and the MHR is required for efficient particle assembly or release.     

Nucleoporins Nup98 and Nup214 Participate in Nuclear Export of Human Immunodeficiency Virus Type 1 Rev

Human immunodeficiency virus type 1 (HIV-1) Rev contains a leucine-rich nuclear export signal that is essential for its nucleocytoplasmic export mediated by hCRM1. We examined the role of selected nucleoporins, which are located in peripheral structures of the nuclear pore complex and are thought to be involved in export, in Rev function in human cells. First, we found that upon actinomycin D treatment, Nup98, but not Nup214 or Nup153, is able to translocate to the cytoplasm of HeLa cells, demonstrating that Nup98 may act as a soluble factor. We further showed that Rev can recruit Nup98 and Nup214, but not Nup153, to the nucleolus. We also found that the isolated FG-containing repeat domains of Nup98 and Nup214, but not those of Nup153, competitively inhibit the Rev/RRE-mediated expression of HIV. Taken together, the recruitment of Nup98 and Nup214 by Rev and the competitive inhibition exhibited by their NP domains demonstrate direct participation of Nup98 and Nup214 in the Rev-hCRM1-mediated export.     

Comparison of Heterologous Prime-Boost Strategies against Human Immunodeficiency Virus Type 1 Gag Using Negative Stranded RNA Viruses

This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8⁺ T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8⁺ T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8⁺ T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8⁺ T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.     

Binding of the Human Immunodeficiency Virus Type 1 Gag Protein to the Viral RNA Encapsidation Signal in the Yeast Three-Hybrid System

We have used the yeast three-hybrid system (D. J. SenGupta, B. Zhang, B. Kraemer, P. Pochart, S. Fields, and M. Wickens, Proc. Natl. Acad. Sci. USA 93:8496–8501, 1996) to study binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein to the HIV-1 RNA encapsidation signal (HIVΨ). Interaction of these elements results in the activation of a reporter gene in the yeast Saccharomyces cerevisiae. Using this system, we have shown that the HIV-1 Gag protein binds specifically to a 139-nucleotide fragment of the HIVΨ signal containing four stem-loop structures. Mutations in either the Gag protein or the encapsidation signal that have been shown previously to impair this interaction reduced the activation of the reporter gene. Interestingly, the nucleocapsid portion of Gag retained the RNA binding activity but lost its specificity compared to the full-length Gag. These results demonstrate the utility of this system and suggest that a variety of genetic analyses could be performed to study Gag-encapsidation signal interactions.     

Nonreciprocal Packaging of Human Immunodeficiency Virus Type 1 and Type 2 RNA: a Possible Role for the p2 Domain of Gag in RNA Encapsidation

The ability of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) to cross-package each other’s RNA was investigated by cotransfecting helper virus constructs with vectors derived from both viruses from which the gag and pol sequences had been removed. HIV-1 was able to package both HIV-1 and HIV-2 vector RNA. The unspliced HIV-1 vector RNA was packaged preferentially over spliced RNA; however, unspliced and spliced HIV-2 vector RNA were packaged in proportion to their cytoplasmic concentrations. The HIV-2 helper virus was unable to package the HIV-1 vector RNA, indicating a nonreciprocal RNA packaging relationship between these two lentiviruses. Chimeric proviruses based on HIV-2 were constructed to identify the regions of the HIV-1 Gag protein conferring RNA-packaging specificity for the HIV-1 packaging signal. Two chimeric viruses were constructed in which domains within the HIV-2 gag gene were replaced by the corresponding domains in HIV-1, and the ability of the chimeric proviruses to encapsidate an HIV-1-based vector was studied. Wild-type HIV-2 was unable to package the HIV-1-based vector; however, replacement of the HIV-2 nucleocapsid by that of HIV-1 generated a virus with normal protein processing which could package the HIV-1-based vector. The chimeric viruses retained the ability to package HIV-2 genomic RNA, providing further evidence for a lack of reciprocity in RNA-packaging ability between the HIV-1 and HIV-2 nucleocapsid proteins. Inclusion of the p2 domain of HIV-1 Gag in the chimera significantly enhanced packaging.     

Translation Elongation Factor 1-Alpha Interacts Specifically with the Human Immunodeficiency Virus Type 1 Gag Polyprotein

Human immunodeficiency virus type 1 (HIV-1) gag-encoded proteins play key functions at almost all stages of the viral life cycle. Since these functions may require association with cellular factors, the HIV-1 matrix protein (MA) was used as bait in a yeast two-hybrid screen to identify MA-interacting proteins. MA was found to interact with elongation factor 1-alpha (EF1alpha), an essential component of the translation machinery that delivers aminoacyl-tRNA to ribosomes. EF1alpha was then shown to bind the entire HIV-1 Gag polyprotein. This interaction is mediated not only by MA, but also by the nucleocapsid domain, which provides a second, independent EF1alpha-binding site on the Gag polyprotein. EF1alpha is incorporated within HIV-1 virion membranes, where it is cleaved by the viral protease and protected from digestion by exogenously added subtilisin. The specificity of the interaction is demonstrated by the fact that EF1alpha does not bind to nonlentiviral MAs and does not associate with Moloney murine leukemia virus virions. The Gag-EF1alpha interaction appears to be mediated by RNA, in that basic residues in MA and NC are required for binding to EF1alpha, RNase disrupts the interaction, and a Gag mutant with undetectable EF1alpha-binding activity is impaired in its ability to associate with tRNA in cells. Finally, the interaction between MA and EF1alpha impairs translation in vitro, a result consistent with a previously proposed model in which inhibition of translation by the accumulation of Gag serves to release viral RNA from polysomes, permitting the RNA to be packaged into nascent virions.     

Functional Interaction of Human Immunodeficiency Virus Type 1 Vpu and Gag with a Novel Member of the Tetratricopeptide Repeat Protein Family

Viral protein U (Vpu) is a protein encoded by human immunodeficiency virus type 1 (HIV-1) that promotes the degradation of the virus receptor, CD4, and enhances the release of virus particles from cells. We isolated a cDNA that encodes a novel cellular protein that interacts with Vpu in vitro, in vivo, and in yeast cells. This Vpu-binding protein (UBP) has a molecular mass of 41 kDa and is expressed ubiquitously in human tissues at the RNA level. UBP is a novel member of the tetratricopeptide repeat (TPR) protein family containing four copies of the 34-amino-acid TPR motif. Other proteins that contain TPR motifs include members of the immunophilin superfamily, organelle-targeting proteins, and a protein phosphatase. UBP also interacts directly with HIV-1 Gag protein, the principal structural component of the viral capsid. However, when Vpu and Gag are coexpressed, stable interaction between UBP and Gag is diminished. Furthermore, overexpression of UBP in virus-producing cells resulted in a significant reduction in HIV-1 virion release. Taken together, these data indicate that UBP plays a role in Vpu-mediated enhancement of particle release.     

Human Immunodeficiency Virus Type 1 Gag Polyprotein Multimerization Requires the Nucleocapsid Domain and RNA and Is Promoted by the Capsid-Dimer Interface and the Basic Region of Matrix Protein

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein directs the formation of virions from productively infected cells. Many gag mutations disrupt virion assembly, but little is known about the biochemical effects of many of these mutations. Protein-protein interactions among Gag monomers are believed to be necessary for virion assembly, and data suggest that RNA may modify protein-protein interactions or even serve as a bridge linking Gag polyprotein monomers. To evaluate the primary sequence requirements for HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants was expressed in bacteria and evaluated for the ability to associate with full-length Gag in vitro. The nucleocapsid protein, the major RNA-binding domain of Gag, exhibited activity comparable to that of the complete polyprotein. In the absence of the nucleocapsid protein, relatively weak activity was observed that was dependent upon both the capsid-dimer interface and basic residues within the matrix domain. The relevance of the in vitro findings was confirmed with an assay in which nonmyristylated mutant Gags were assessed for the ability to be incorporated into virions produced by wild-type Gag expressed in trans. Evidence of the importance of RNA for Gag-Gag interaction was provided by the demonstration that RNase impairs the Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags encoded by distantly related retroviruses and with structurally unrelated RNA-binding proteins. These results are consistent with models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions.     

Human Immunodeficiency Virus Type 1 Vectors Efficiently Transduce Human Hematopoietic Stem Cells

Lentiviruses are potentially advantageous compared to oncoretroviruses as gene transfer agents because they can infect nondividing cells. We demonstrate here that human immunodeficiency virus type 1 (HIV-1)-based vectors were highly efficient in transducing purified human hematopoietic stem cells. Transduction rates, measured by marker gene expression or by PCR of the integrated provirus, exceeded 50%, and transduction appeared to be independent of mitosis. Derivatives of HIV-1 were constructed to optimize the vector, and a deletion of most of Vif and Vpr was required to ensure the long-term persistence of transduced cells with relatively stable expression of the marker gene product. These results extend the utility of this lentivirus vector system.