Crosslink Analysis of N-Terminal, C-Terminal, and N/B Determining Regions of the Moloney Murine Leukemia Virus Capsid Protein

We have identified a region near the C terminus of capsid (CA) of murine leukemia virus (MLV) that contains many charged residues. This motif is conserved in various lengths in most MLV-like viruses. One exception is that spleen necrosis virus (SNV) does not contain a well-defined domain of charged residues. When 33 amino acids of the MLV motif were deleted to mimic SNV CA, the resulting mutant produced drastically reduced amounts of virions and the virions were noninfectious. Furthermore, these viruses had abnormal sizes, often contained punctate structures resembling those in the cell cytoplasm, and packaged both ribosomal and viral RNA. When 11 or 15 amino acids were deleted to modify the MLV CA to resemble those from other gammaretroviruses, the deletion mutants produced virions at levels comparable to those of the wild-type virus and were able to complete one round of virus replication without detectable defects. We generated 10 more mutants that displayed either the wild-type or mutant phenotype. The distribution of the wild-type or mutant phenotype did not directly correlate with the number of amino acids deleted, suggesting that the function of the motif is determined not simply by its length but also by its structure. Structural modeling of the wild-type and mutant proteins suggested that this region forms ␣-helices; thus, we termed this motif the "charged assembly helix." This is the first description of the charged assembly helix motif in MLV CA and demonstration of its role in virus budding and assembly.

Section: Discussionsupporting

confidence: 81%

Charged Assembly Helix Motif in Murine Leukemia Virus Capsid: an Important Region for Virus Assembly and Particle Size Determination

Cheslock

Poon

et al. 2003

“…293T and HiL cell lines were passaged at 37°C in 5% CO 2 in culture medium containing Dulbecco's modified Eagle's medium supplemented with 10 mM HEPES (pH 7.4), penicillin, and streptomycin plus 10% fetal calf serum. For transfections, 10-cm plates of 293T cells were transfected by the calcium phosphate method (4,23,29,30,44,45) with either 24 g of HIVLuc DNA, 16 g of HIVLuc plus 8 g of pVSV-G DNA, or 12 g of HIVLuc plus 6 g of pVSV-G plus 6 g of BlaM-vpr DNA. Briefly, confluent 10-cm dishes of 293T cells were split 1:4 the day prior to transfection.…”

Section: Methodsmentioning

confidence: 99%

Virus Particle Core Defects Caused by Mutations in the Human Immunodeficiency Virus Capsid N-Terminal Domain

Scholz

Arvidson

Huseby

et al. 2005

Self Cite

The N-terminal domains (NTDs) of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein have been modeled to form hexamer rings in the mature cores of virions. In vitro, hexamer ring units organize into either tubes or spheres, in a pH-dependent fashion. To probe factors which might govern hexamer assembly preferences in vivo, we examined the effects of mutations at CA histidine residue 84 (H84), modeled at the outer edges of NTD hexamers, as well as a nearby histidine (H87) in the cyclophilin A (CypA) binding loop. Although mutations at H87 yielded infectious virions, mutations at H84 produced assembly-competent but poorly infectious virions. The H84 mutant viruses incorporated wild-type levels of CypA and viral RNAs and showed nearly normal signals in virus entry assays. However, mutant CA proteins assembled aberrant virus cores, and mutant core fractions retained abnormally high levels of CA but reduced reverse transcriptase activities. Our results suggest that HIV-1 CA residue 84 contributes to a structure which helps control either NTD hexamer assembly or the organization of hexamers into higher-order structures.A number of functions have been attributed to the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein.As a portion of the HIV Gag precursor (PrGag) protein, the CA N-terminal domains (NTDs) and C-terminal domains (CTDs) collaborate with each other and with other Gag domains to facilitate virus assembly and budding (4, 9, 10, 13, 15, 17-22, 27, 29, 31, 35, 38-44, 46). Appropriate CA-CA contacts are necessary not only for assembly and release, but also for proper maturation and postmaturation replication steps (9,13,15,20,22,27,29,38,40,41,43,44). Indeed, a variety of HIV-1 capsid mutations have manifested defects in early replication events, such as uncoating and reverse transcription (15,20,27,38,40,41,44). Some of these defects may be attributable to altered interactions with cellular factors, such as cyclophilin A (CypA) (1, 2, 7, 9, 41), and host susceptibility factors, such as Ref1, Trim5␣, and Lv1 (6,12,16,17,24,32,37).In terms of a structural role within virions, CA NTDs appear to assemble hexamer rings that are linked via CTD connections (5,19,20,26,28,33,34,42); evidence suggests that the NTD rings are more tightly packed in immature than in mature virions (28), implying that more CA is assembled into particles than is necessary to build a mature virus core. In vitro experiments have demonstrated unusual pH-dependent characteristics in terms of the structures assembled by HIV-1 Gag proteins. At pH 6.0, PrGag-like proteins have been shown to assemble long tubes, whereas at pH 8.0, spheres are formed (22). The behavior of mature CA is even more complex in that CA dimers predominate at pHs below 6.6, spheres predominate at pH 6.8, and tubes are the major form at pH 7.0, while tubes and spheres may coexist at higher pHs (14). The assembly activities of CA in the pH 6.5 to 7.0 range have led to the speculation that capsid assembly or disassembly may involve a histidine s...

“…Purified fractions were desalted by buffer exchange in Sephadex G25 spin columns with 10 mM sodium phosphate (pH 7.8) and stored under nitrogen at Ϫ80°C. Purified (Ͼ95%) proteins at 0.8 to 3 mg/ml were evaluated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (17,29,30,41,42), staining with 0.4% Coomassie blue R250 (Sigma) in 50% methanol-10% acetic acid, destaining in 50% methanol-10% acetic acid, and gel storage in 10% acetic acid. Proteins also were evaluated by immunoblotting.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Human Immunodeficiency Virus Type 1 Gag Dimerization-Induced Assembly

Alfadhli

Dhenub

Still

et al. 2005

Self Cite

The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.When expressed in cells, the precursor Gag (PrGag) proteins of retroviruses such as human immunodeficiency virus (HIV), Rous sarcoma virus (RSV), and murine leukemia viruses are sufficient for the production of virus-like particles (VLP) (36). These proteins encode N-terminal matrix (MA) domains involved in membrane-binding (M) functions; variably located late (L) domains, which are important for VLP budding; and protein-protein interaction (I) regions, composed of the capsid (CA) and nucleocapsid (NC) domains (36). The NC domains bind viral RNA, facilitating its encapsidation, and also have an assembly function (3,5,6,7,10,11,25,26,32,33,35,37,41,42). Results from investigations on NC mutants are consistent with the notion that NC-RNA binding is essential for efficient VLP assembly (3,5,6,10,11,32,33,35,41,42).Previously, we showed that the assembly function of HIV type 1 (HIV-1) NC could be replaced by heterologous dimerization domains (42), an observation that was confirmed by others (1, 19), and suggested that the assembly role of the NC-RNA interaction is to foster dimerization of PrGag proteins. In vitro investigations on RSV Gag proteins carrying the CA N-terminal domains (NTDs), C-terminal domains (CTDs), and NC domains have supported this view (7,25,26) and led to a dimerization model for VLP assembly. As shown in Fig. 1A, Gag proteins composed of the CA NTD, CA CTD, and NC domains concentrate on RNA by virtue of the NC-RNA interaction. Close juxtaposition of the proteins leads to dimerization, which then triggers the assembly of higher-order oligomers.Assuming that the assembly role of the NC-RNA interaction for all retroviruses is to usher the formation of assembly-competent Gag dimers, then other mechanisms for pairing...