trans-acting inhibition of genomic RNA dimerization by Rous sarcoma virus matrix mutants.

The genomic RNA of retroviruses exists within the virion as a noncovalently linked dimer. Previously, we identified a mutant of the viral matrix (MA) protein of Rous sarcoma virus that disrupts viral RNA dimerization. This mutant, Myr1E, is modified at the N terminus of MA by the addition of 10 amino acids from the Src protein, resulting in the production of particles containing monomeric RNA. Dimerization is reestablished by a single amino acid substitution that abolishes myristylation (Myr1E-). To distinguish between cis and trans effects involving Myr1E, additional mutations were generated. In Myr1E.cc and Myr1E-.cc, different nucleotides were utilized to encode the same protein as Myr1E and Myr1E-, respectively. The alterations in RNA sequence did not change the properties of the viral mutants. Myr1E.ATG- was constructed so that translation began at the gag AUG, resulting in synthesis of the wild-type Gag protein but maintenance of the src RNA sequence. This mutant had normal infectivity and dimeric RNA, indicating that the src sequence did not prevent dimer formation. All of the src-containing RNA sequences formed dimers in vitro. Examination of MA-green fluorescent protein fusion proteins revealed that the wild-type and mutant MA proteins Myr1E.ATG-, Myr1E-, and Myr1E-.cc had distinctly different patterns of subcellular localization compared with Myr1E and Myr1E.cc MA proteins. This finding suggests that proper localization of the MA protein may be required for RNA dimer formation and infectivity. Taken together, these results provide compelling evidence that the genomic RNA dimerization defect is due to a trans-acting effect of the mutant MA proteins.

RNA dimerization defect in a Rous sarcoma virus matrix mutant.

The retrovirus matrix (MA) sequence of the Gag polyprotein has been shown to contain functions required for membrane targeting and binding during particle assembly and budding. Additional functions for MA have been proposed based on the existence of MA mutants in Rous sarcoma virus (RSV), murine leukemia virus, human immunodeficiency virus type 1, and human T-cell leukemia virus type 1 that lack infectivity even though they release particles of normal composition. Here we describe an RSV MA mutant with a surprising and previously unreported phenotype. In the mutant known as Myr1E, the small membrane-binding domain of the Src oncoprotein has been added as an N-terminal extension of Gag. While Myr1E is not infectious, full infectivity can be reestablished by a single amino acid substitution in the Src sequence (G2E), which eliminates the addition of myristic acid and the membrane-binding capacity of this foreign sequence. The presence of myristic acid at the N terminus of the Myr1E Gag protein does not explain its replication defect, because other myristylated derivatives of RSV Gag are fully infectious (e.g., Myr2 [C. R. Erdie and J. W. Wills, J. Virol. 64:5204-5208, 1990]). Biochemical analyses of Myr1E particles reveal that they contain wild-type levels of the Gag cleavage products, Env glycoproteins, and reverse transcriptase activity when measured on an exogenous template. Genomic RNA incorporation appears to be mildly reduced compared to the wild-type level. Unexpectedly, RNA isolated from Myr1E particles is monomeric when analyzed on nondenaturing Northern blots. Importantly, the insertional mutation does not lie within previously identified dimer linkage sites. In spite of the dimerization defect, the genomic RNA from Myr1E particles serves efficiently as a template for reverse transcription as measured by an endogenous reverse transcriptase assay. In marked contrast, after infection of avian cells, the products of reverse transcription are nearly undetectable. These findings might be explained either by the loss of a normal function of MA needed in the formation or stabilization of RNA dimers or by the interference in such events by the mutant MA molecules. It is possible that Myr1E viruses package a single copy of viral RNA.

Identification of retroviral late domains as determinants of particle size.

Retroviral Gag proteins, in the absence of any other viral products, induce budding and release of spherical, virus-like particles from the plasma membrane. Gag-produced particles, like those of authentic retrovirions, are not uniform in diameter but nevertheless fall within a fairly narrow distribution of sizes. For the human immunodeficiency virus type 1 (HIV-1) Gag protein, we recently reported that elements important for controlling particle size are contained within the C-terminal region of Gag, especially within the p6 sequence (L. Garnier, L. Ratner, B. Rovinski, S.-X. Cao, and J. W. Wills, J. Virol. 72:4667-4677, 1998). Deletions and substitutions throughout this sequence result in the release of very large particles. Because the size determinant could not be mapped to any one of the previously defined functions within p6, it seemed likely that its activity requires the overall proper folding of this region of Gag. This left open the possibility of the size determinant residing in a subdomain of p6, and in this study, we examined whether the late domain (the region of Gag that is critical for the virus-cell separation step) is involved in controlling particle size. We found that particles of normal size are produced when p6 is replaced with the totally unrelated late domain sequences from Rous sarcoma virus (contained in its p2b sequence) or equine infectious anemia virus (contained in p9). In addition, we found that the large particles released in the absence of p6 require the entire CA and adjacent spacer peptide sequences, whereas these internal sequences of HIV-1 Gag are not needed for budding (or proper size) when a late domain is present. Thus, it appears the requirements for budding are very different in the presence and absence of p6.

Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein.

We have previously demonstrated that the Gag p9 protein of equine infectious anemia virus (EIAV) is functionally homologous with Rous sarcoma virus (RSV) p2b and human immunodeficiency virus type 1 (HIV-1) p6 in providing a critical late assembly function in RSV Gag-mediated budding from transfected COS-1 cells (L. J. Parent et al., J. Virol. 69:5455-5460, 1995). In light of the absence of amino acid sequence homology between EIAV p9 and the functional homologs of RSV and HIV-1, we have now designed an EIAV Gag-mediated budding assay to define the late assembly (L) domain peptide sequences contained in the EIAV p9 protein. The results of these particle budding assays revealed that expression of EIAV Gag polyprotein in COS-1 cells yielded extracellular Gag particles with a characteristic density of 1.18 g/ml, while expression of EIAV Gag polyprotein lacking p9 resulted in a severe reduction in the release of extracellular Gag particles. The defect in EIAV Gag polyprotein particle assembly could be corrected by substituting either the RSV p2b or HIV-1 p6 protein for EIAV p9. These observations demonstrated that the L domains of EIAV, HIV-1, and RSV were interchangeable in mediating assembly of EIAV Gag particles in the COS-1 cell budding assay. To localize the L domain of EIAV p9, we next assayed the effects of deletions and site-specific mutations in the p9 protein on its ability to mediate budding of EIAV Gag particles. Analyses of EIAV Gag constructs with progressive N-terminal or C-terminal deletions of the p9 protein identified a minimum sequence of 11 amino acids (Q20N21L22Y23P24D25L26S27E28I29K30) capable of providing the late assembly function. Alanine scanning studies of this L-domain sequence demonstrated that mutations of residues Y23, P24, and L26 abrogated the p9 late budding function; mutations of other residues in the p9 L domain did not substantially affect the level of EIAV Gag particle assembly. These data indicate that the L domain in EIAV p9 utilizes a YXXL motif which we hypothesize may interact with cellular proteins to facilitate virus particle budding from infected cells.

Evidence for a second function of the MA sequence in the Rous sarcoma virus Gag protein.

During retrovirus assembly, Gag proteins bind to the inner leaflet of the plasma membrane to initiate the budding process. The molecular basis of this protein-lipid interaction is poorly understood. For the human, immunodeficiency virus type 1 Gag protein, we recently reported that the membrane-binding domain resides within the N-terminal 31 amino acids and consists of two components: myristate and a cluster of basic residues, which together promote membrane binding in vitro and budding in vivo (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). The positively charged residues associate electrostatically with acidic phospholipids to stabilize membrane binding, while myristate provides membrane-binding energy via hydrophobic interactions. Here we demonstrate that the human immunodeficiency virus type 1 Gag membrane-binding domain can fully replace the membrane-targeting function of the N-terminal 100 residues of the non-myristylated Rous sarcoma virus (RSV) Gag protein. To further explore the importance of myristate and basic residues in membrane binding, we developed a gain-of-function assay whereby budding was restored to defective mutants of RSV Gag. Detailed mutational analysis revealed that the position, number, and context of charged residues are crucial to budding. Myristate provides additional membrane-binding energy, which is critical when a Gag protein is near the threshold of stable membrane association. Finally, viruses with altered matrix (MA) proteins that are noninfectious, even though they produce particles with high efficiency, were identified. Thus, we present the first evidence that the RSV MA sequence plays two distinct roles, membrane binding during particle assembly and a second, as yet undefined function required for viral infectivity.

Disseminated Mycobacterium marinum infection and bacteremia in a child with severe combined immunodeficiency.

We report an unusual case of disseminated dermatitis, osteomyelitis, and bacteremia in an immunocompromised host. An infant presented with a pustular skin rash resembling chicken pox, but culture of a skin lesion yielded Mycobacterium marinum. Upon further evaluation, severe combined immunodeficiency was diagnosed. Radiographs of the hands and feet showed evidence of osteomyelitis. M. marinum was isolated from blood and synovial fluid. We recommend a high level of suspicion for this organism in immunocompromised hosts who have been exposed to fresh water or salt water, particularly those of aquaria. Drug susceptibility testing of serial clinical isolates from our patient revealed development of high-level resistance to isoniazid and rifampin during therapy. We believe that treatment of disseminated M. marinum infections should include combinations of antimycobacterial agents chosen on the basis of results of susceptibility testing done by an experienced laboratory, thereby limiting the emergence of drug resistance.

Growth signal transduction by the human interleukin-2 receptor requires cytoplasmic tyrosines of the beta chain and non-tyrosine residues of the gamma c chain.

To evaluate the possible role for receptor-based tyrosine phosphorylation in growth signaling induced by interleukin-2 (IL-2), a series of substitution tyrosine mutants of the IL-2 receptor beta and gamma c chains was prepared and analyzed. Concurrent mutation of all six of the cytoplasmic tyrosines present in the beta chain markedly inhibited IL-2-induced growth signaling in both pro-B and T cell lines. Growth signaling in a pro-B cell line was substantially reconstituted when either of the two distal tyrosines (Tyr-392, Tyr-510) was selectively restored in the tyrosine-negative beta mutant, whereas reconstitution of the proximal tyrosines (Tyr-338, Tyr-355, Tyr-358, Tyr-361) did not restore this signaling function. Furthermore, at least one of the two cytoplasmic tyrosines that is required for beta chain function was found to serve as a phosphate acceptor site upon induction with IL-2. Studies employing a chimeric receptor system revealed that tyrosine residues of the beta chain likewise were important for growth signaling in T cells. In contrast, although the gamma c subunits is a target for tyrosine phosphorylation in vivo, concurrent substitution of all four cytoplasmic tyrosines of this chain produced no significant effect on growth signaling by chimeric IL-2 receptors. However, deletion of either the Box 1, Box 2, or intervening (V-Box) regions of gamma c abrogated receptor function. Therefore, tyrosine residues of beta but not of gamma c appear to play a pivotal role in regulating growth signal transduction through the IL-2 receptor, either by influencing cytoplasmic domain folding or by serving as sites for phosphorylation and subsequent association with signaling intermediates. These findings thus highlight a fundamental difference in the structural requirements for IL-2R beta and gamma c in receptor-mediated signal transduction.

Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins.

The Gag proteins of Rous sarcoma virus and human immunodeficiency virus (HIV) each contain a function involved in a late step in budding, defects in which result in the accumulation of these molecules at the plasma membrane. In the Rous sarcoma virus Gag protein (Pr76gag), this assembly domain is associated with a PPPY motif, which is located at an internal position between the MA and CA sequences. This motif is not contained anywhere within the HIV Gag protein (Pr55gag), and the MA sequence is linked directly to CA. Instead, a late assembly function of HIV has been associated with the p6 sequence situated at the C terminus of Gag. Here we demonstrate the remarkable finding that the late assembly domains from these two unrelated Gag proteins are exchangeable between retroviruses and can function in a positionally independent manner.

Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids.

Retroviral Gag proteins are targeted to the plasma membrane, where they play the central role in virion formation. Several studies have suggested that the membrane-binding signal is contained within the amino-terminal matrix sequence; however, the precise location has never been determined for the Gag protein of any retrovirus. In this report, we show that the first 31 residues of human immunodeficiency virus type 1 Gag protein can function independently as a membrane-targeting domain when fused to heterologous proteins. A bipartite membrane-targeting motif was identified, consisting of the myristylated N-terminal 14 amino acids and a highly basic region that binds acidic phospholipids. Replacement of the N-terminal membrane-targeting domain of pp60v-src with that of human immunodeficiency virus type 1 Gag elicits efficient membrane binding and a transforming phenotype. Removal of myristate or the basic region results in decreased membrane binding of Gag-Src chimeras in vitro and impaired virion formation by Pr55gag in vivo. We propose that the N-terminal Gag sequence functions as a targeting signal to direct interaction with acidic phospholipids on the cytoplasmic leaflet of the plasma membrane.

Ethanol: an enhancer of transplantation antigen expression.

Major histocompatibility antigens (MHC) play a pivotal role in the immune response. Abnormal expression of MHC antigens has been correlated with aberrant regulation of the immune response. Studies on the effect of ethanol on class I MHC antigens demonstrate that ethanol significantly enhances their cell surface expression in a variety of cell lines in vitro. These changes in cell surface levels reflect increased intracellular protein synthesis and increased steady state mRNA levels. The effective ethanol concentrations (0.1-1.0%) are physiologically attainable. Measurement of class I MHC antigens on peripheral blood lymphocytes in a population of acutely ethanol-intoxicated patients showed a highly significant increase relative to controls. The possibility that the elevated levels of MHC antigens induced by ethanol may play a role in the evolution of ethanol-related disease is discussed.

Ethanol: an enhancer of major histocompatibility complex antigen expression.

Ethanol enhances expression of cell surface class I major histocompatibility complex (MHC) antigens in a variety of cell lines; up to an eightfold increase is observed in an embryonic cell line. In ethanol-treated L cells, increased cell surface expression of MHC antigens occurs with a concomitant increase in steady-state RNA levels. This effect is promoter dependent and restricted, because not all gene products are elevated. The effective ethanol concentration (1%) is physiologically attainable, leading to speculations about the role of elevated MHC antigens in alcohol-related diseases.