Basic residues in human immunodeficiency virus type 1 nucleocapsid promote virion assembly via interaction with RNA

Retroviral Gag polyproteins drive virion assembly by polymerizing to form a spherical shell that lines the inner membrane of nascent virions. Deletion of the nucleocapsid (NC) domain of the Gag polyprotein disrupts assembly, presumably because NC is required for polymerization. Human immunodeficiency virus type 1 NC possesses two zinc finger motifs that are required for specific recognition and packaging of viral genomic RNA. Though essential, zinc fingers and genomic RNA are not required for virion assembly. NC promiscuously associates with cellular RNAs, many of which are incorporated into virions. It has been hypothesized that Gag polymerization and virion assembly are promoted by nonspecific interaction of NC with RNA. Consistent with this model, we found an inverse relationship between the number of NC basic residues replaced with alanine and NC's nonspecific RNA-binding activity, Gag's ability to polymerize in vitro and in vivo, and Gag's capacity to assemble virions. In contrast, mutation of NC's zinc fingers had only minor effects on these properties.     

Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference

Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.     

Reciprocal regulatory interaction between human herpesvirus 8 and human immunodeficiency virus type 1

Human herpesvirus 8 (HHV8) is the primary viral etiologic agent in Kaposi's sarcoma (KS). However, individuals dually infected with both HHV8 and human immunodeficiency virus type 1 (HIV-1) show an enhanced prevalence of KS when compared with those singularly infected with HHV8. Host immune suppression conferred by HIV infection cannot wholly explain this increased presentation of KS. To better understand how HHV8 and HIV-1 might interact directly in the pathogenesis of KS, we queried for potential regulatory interactions between the two viruses. Here, we report that HHV8 and HIV-1 reciprocally up-regulate the gene expression of each other. We found that the KIE2 immediate-early gene product of HHV8 interacted synergistically with Tat in activating expression from the HIV-1 long terminal repeat. On the other hand, HIV-1 encoded Tat and Vpr proteins increased intracellular HHV8-specific expression. These results provide molecular insights correlating coinfection with HHV8 and HIV-1 with an unusually high incidence of KS.     

Interactions between human immunodeficiency virus type 1 and vaccinia virus in human lymphoid tissue ex vivo

Vaccinia virus (VACV) has been attracting attention recently not only as a vector for various vaccines but also as an immunization tool against smallpox because of its potential use as a bioterrorism agent. It has become evident that in spite of a long history of studies of VACV, its tissue pathogenesis remains to be fully understood. Here, we investigated the pathogenesis of VACV and its interactions with human immunodeficiency virus type 1 (HIV-1) in the context of human lymphoid tissues. We found that ex vivo-cultured tonsillar tissue supports productive infection by the New York City Board of Health strain, the VACV strain of the Dryvax vaccine. VACV readily infected both T and non-T (B) lymphocytes and depleted cells of both of these subsets equally over a 12-day period postinfection. Among T lymphocytes, CD8(+) cells are preferentially depleted in accordance with their preferential infection: the probability that a CD8(+) T cell will be productively infected is almost six times higher than for a CD4(+) T cell. T cells expressing CCR5 and the activation markers CD25, CD38, and HLA-DR are other major targets for infection by VACV in lymphoid tissue. As a consequence, VACV predominantly inhibits the replication of the R5(SF162) phenotype of HIV-1 in coinfected tissues, as R5-tropic HIV-1 requires activated CCR5(+) CD4(+) cells for productive infection. Human lymphoid tissue infected ex vivo by VACV can be used to investigate interactions of VACV with other viruses, in particular HIV-1, and to evaluate various VACV vectors for the purpose of recombinant vaccine development.     

RNA trafficking signals in human immunodeficiency virus type 1

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.     

Binding of human immunodeficiency virus type 1 nucleocapsid protein to psi-RNA-SL3

The interaction of the nucleocapsid protein NCp7, from the pNL4-3 isolate of HIV-1, with psi-RNA-SL3, with the sequence 5'-GGACUAGCGGAGGCUAGUCC, was studied using non-denaturing gel electrophoresis. Two kinds of experiments were performed, using buffered solutions of radiolabeled RNA and unlabeled protein. In the 'dilution' experiments, the total RNA concentration, RT, was varied for a series of solutions, but kept equal to the total protein concentration, PT, In the 'titration' experiments, solutions having RT constant but with varying PT were analyzed. The solutions were electrophoresed and the autoradiographic spot intensities, proportional to the amounts of the different species present, were measured. The intensities were fit to a number of equilibrium models, differing in species stoichiometries, by finding the best values of the binding constants. It was shown that NCp7 protein and SL3 RNA combine to form at least two complexes. When PT is below approximately 10 microM, a complex that contains two RNAs and one protein forms. Increasing PT to approximately 100 microM causes the 2:1 complex to oligomerize, forming a species having eight RNAs and four proteins. For the dilution experiments, run at 5 degrees C at an ionic strength of 31 mM, we found K1 for the 2:1 complex is approximately 10(11) M(-2) and K2 for the 8:4 complex is approximately 10(16) M(-3). The titration experiments returned K1 approximately 10(7) M(-2) (poorly determined) and K2 approximately 10(19) M(-3). The analysis was complicated by the loss of RNA at higher protein concentrations, due to formation of an insoluble species containing both RNA and protein, which does not enter the gel. Correcting for this changes the calculated values of equilibrium constants, but not the molecularities determined by our analysis. The observation that a small complex can oligomerize to form a larger species is consistent with the fact that NCp7 organizes and condenses the genome in the virus particle.     

Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein

The nucleocapsid protein NCp7, which is the major genomic RNA binding protein of human immunodeficiency virus type 1, plays an important role in several key steps of the viral life cycle. Many of the NCp7 activities, notably the nucleic acid annealing and the genomic RNA wrapping ones, are thought to be linked to a nonspecific binding of NCp7 to its nucleic acid targets. The mechanism of these activities is still debated but several clues are in favor of an intermediate aggregation of nucleic acids by NCp7. To check and characterize the nucleic acid aggregating properties of NCp7, we investigated the interaction of NCp7 with the model RNA homopolymer, polyA, by quasielastic light scattering and optical density measurements. The ordered growth of monodisperse large particles independently of the nucleic acid size and the almost complete covering of polyA by NCp7 strongly suggested an ordered aggregation mechanism. The aggregate kinetics of growth in the optimum protein concentration range (≥2 μM) were governed by a so-called Ostwald ripening mechanism limited by transfer of NCp7-covered polyA complexes from small to large aggregates. The aggregation process was strongly dependent on both Na⁺ and Mg²⁺ concentrations, the optimum concentrations being in the physiological range. Similar conclusions held true when polyA was replaced by 16S + 23S ribosomal RNA, suggesting that the NCp7 aggregating properties were only poorly dependent on the nucleic acid sequence and structure. Finally, as in the NCp7 annealing activities, the basic regions of NCp7, but not the zinc fingers, were found critical in nucleic acid aggregation. Taken together, our data indicate that NCp7 is a highly efficient nucleic acid aggregating agent and strengthen the hypothesis that aggregation may constitute a transient step in various NCp7 functions. © 1997 John Wiley & Sons, Inc.     

Selection and stabilization of the RNA aptamers against the human immunodeficiency virus type-1 nucleocapsid protein

The nucleocapsid (NC) protein of the human immunodeficiency virus-1 (HIV-1) plays an important role in the encapsidation of viral RNA and assembly of viral particle. Since the NC protein is resistant for mutation, it might be an excellent target for the anti-viral therapy. RNA aptamers that bind to the mature form of the NC protein were isolated from a RNA library. Surface plasmon resonance measurement and gel shift assay showed that the RNA aptamers specifically bind to the NC protein with high affinity and compete for the psi RNA binding to the NC protein. Mapping of the RNA aptamer showed at least two sites for the protein binding, suggesting a multiple and cooperative binding by the NC to RNA. In addition, the circular form of RNA avidly binds to the NC protein as a linear counter does. Stabilized RNA aptamer is expected to act as an inhibitor for the viral packaging.     

A dual infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression

This study was designed to examine the impact of human immunodeficiency virus type 1 (HIV-1) fitness on disease progression through the use of a dual competition/heteroduplex tracking assay (HTA). Despite numerous studies on the impact of HIV-1 diversity and HIV-specific immune response on disease progression, we still do not have a firm understanding of the long-term pathogenesis of this virus. Strong and early CD8-positive cytotoxic T-cell and CD4-positive T-helper cell responses directed toward HIV-infected cells appear to curb HIV pathogenesis. However, the rate at which the virus infects the CD4(+) T-cell population and possibly destroys the HIV-specific immune response may also alter the rate of disease progression. For HIV-1 fitness studies, we established conditions for dual HIV-1 infections of peripheral blood mononuclear cells (PBMC) and a sensitive HTA to measure relative virus production. A pairwise comparison was then performed to estimate the relative fitness of various non-syncytium-inducing/CCR5-tropic (NSI/R5) and syncytium-inducing/CXCR4-tropic (SI/X4) HIV-1 isolates. Four HIV-1 strains (two NSI/R5 and two SI/X4) with moderate ex vivo fitness were then selected as controls and competed against primary HIV-1 isolates from an HIV-infected Belgian cohort. HIV-1 isolates from long-term survivors (LTS) were outcompeted by control strains and were significantly less fit than HIV-1 isolates from patients with accelerated progression to AIDS (PRO). In addition, NSI/R5 HIV-1 isolates from PRO overgrew control SI/X4 strains, suggesting that not all SI/X4 HIV-1 isolates replicate more efficiently than all NSI/R5 isolates. Finally, there were strong, independent correlations between viral load and the total relative fitness values of HIV-1 isolates from PRO (r = 0.84, P = 0.033) and LTS (r = 0.86, P = 0.028). Separation of the PRO and LTS plots suggest that HIV-1 fitness together with viral load may be a strong predictor for the rate of disease progression.     

Redundant roles for nucleocapsid and matrix RNA-binding sequences in human immunodeficiency virus type 1 assembly

RNA appears to be required for the assembly of retroviruses. This is likely due to binding of RNA by multiple Gags, which in turn organizes and stabilizes the Gag-Gag interactions that form the virion. While the nucleocapsid (NC) domain is the most conspicuous RNA-binding region of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, we have previously shown that NC is not strictly required for efficient particle production. To determine if an RNA requirement for HIV-1 assembly exists, we analyzed virions produced by an NC deletion mutant for the presence of RNA. The results revealed that virions without NC still contained significant amounts of RNA. Since these packaged RNAs are probably incorporated by other RNA-binding sequences in Gag, an RNA-binding site in the matrix protein (MA) of Gag was mutated. While this mutation did not interfere with HIV-1 replication, a construct with both MA and NC mutations (MX/NX) failed to produce particles. The MX/NX mutant was rescued in trans by coassembly with several forms of Gag: wild-type Gag, either of the single-mutant Gags, or Gag truncations that contain MA or NC sequences. Addition of basic sequences to the MX/NX mutant partially restored particle production, consistent with a requirement for Gag-RNA binding in addition to Gag-Gag interactions. Together, these results support an RNA-binding requirement for Gag assembly, which relies on binding of RNA by MA or NC sequences to condense, organize, and stabilize the HIV-1 Gag-Gag interactions that form the virion.     

Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity.

Interaction of the human immunodeficiency virus type 1 (HIV-1) Gag precursor polyprotein (Pr55Gag) with the viral genomic RNA is required for retroviral replication. Mutations that reduce RNA packaging efficiency have been localized to the highly basic nucleocapsid (NC) p7 domain of Pr55Gag, but the importance of the basic amino acid residues in specific viral RNA encapsidation and infectivity has not been thoroughly investigated in vivo. We have systematically substituted the positively charged residues of the NC domain of Pr55Gag in an HIV-1 viral clone by using alanine scanning mutagenesis and have assayed the effects of these mutations on virus replication, particle formation, and RNA packaging in vivo. Analysis of viral clones with single substitutions revealed that certain charged amino acid residues are more critical for RNA packaging efficiency and infectivity than others. Analysis of viral clones with multiple substitutions indicates that the presence of positive charge in each of three independent domains--the zinc-binding domains, the basic region that links them, and the residues that Hank the two zinc-binding domains--is necessary for efficient HIV-1 RNA packaging. Finally, we note that some mutations affect virus replication more drastically than RNA incorporation, providing in vivo evidence for the hypothesis that NC p7 may be involved in aspects of the HIV life cycle in addition to RNA packaging.     

Distinct functions and requirements for the Cys-His boxes of the human immunodeficiency virus type 1 nucleocapsid protein during RNA encapsidation and replication.

The process of retroviral RNA encapsidation involves interaction between trans-acting viral proteins and cis-acting RNA elements. The encapsidation signal on human immunodeficiency virus type 1 (HIV-1) RNA is a multipartite structure composed of functional stem-loop structures. The nucleocapsid (NC) domain of the Gag polyprotein precursor contains two copies of a Cys-His box motif that have been demonstrated to be important in RNA encapsidation. To further characterize the role of the Cys-His boxes of the HIV-1 NC protein in RNA encapsidation, the relative efficiency of RNA encapsidation for virus particles that contained mutations within the Cys-His boxes was measured. Mutations that disrupted the first Cys-His box of the NC protein resulted in virus particles that encapsidated genomic RNA less efficiently and subgenomic RNA more efficiently than did wild-type virus. Mutations within the second Cys-His box did not significantly affect RNA encapsidation. In addition, a full complement of wild-type NC protein in virus particles is not required for efficient RNA encapsidation or virus replication. Finally, both Cys-His boxes of the NC protein play additional roles in virus replication.     

Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles.

Packaging of retroviral RNA is attained through the specific recognition of a cis-acting encapsidation site (located near the 5' end of the viral RNA) by components of the Gag precursor protein. Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) are two lentiviruses that lack apparent sequence similarity in their putative encapsidation regions. We used SIV vectors to determine whether HIV-1 particles can recognize the SIV encapsidation site and functionally propagate SIV nucleic acid. SIV nucleic acid was replicated by HIV-1 proteins. Thus, efficient lentivirus pseudotyping can take place at the RNA level. Direct examination of the RNA contents of virus particles indicated that encapsidation of this heterologous RNA is efficient. Characterization of deletion mutants in the untranslated leader region of SIV RNA indicates that only a very short region at the 5' end of the SIV RNA is needed for packaging. Comparison of this region with the corresponding region of HIV-1 reveals that both are marked by secondary structures that are likely to be similar. Thus, it is likely that a similar higher-order RNA structure is required for encapsidation.     

Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo.

At least two hairpins in the 5' untranslated leader region, stem-loops 1 and 3 (SL1 and SL3), contribute to human immunodeficiency virus type 1 RNA encapsidation in vivo. We used a competitive assay, which measures the relative encapsidation efficiency of mutant viral RNA in the presence of competing wild-type RNA, to compare the contributions of SL1, SL3, and two adjacent secondary structures, SL2 and SL4, to encapsidation. SL2 is not required for RNA encapsidation, while SL1, SL3, and SL4 all contribute approximately equally to encapsidation. To determine whether these hairpins function in a position-dependent manner, we interchanged the positions of two of these stem-loop structures. This resulted in substantial diminution of encapsidation, indicating that the secondary structures that comprise E, the encapsidation signal, function only in their correct contexts. Mutation of nucleotides flanking SL1 and SL3 had little effect on encapsidation. We also showed that SL1, while present on both genomic and subgenomic viral RNAs, nonetheless contributes to selective encapsidation of genomic RNA. Taken together, these data are consistent with the formation of a higher-order RNA structure, partially composed of SL1, SL3, and SL4, that functions to effect concurrent encapsidation of full-length RNA and exclusion of subgenomic RNA. Finally, it has been reported that E is required for efficient translation of Gag mRNA in vivo. However, we have found that a variety of mutants, including a mutant lacking the entire region encompassing SL1, SL2, and SL3, still produce RNAs that are efficiently translated. These data indicate that E is unlikely to contribute to efficient Gag mRNA translation in vivo.