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

Abstract

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...

Analysis of HIV-1 Gag Protein Interactions via Biotin Ligase Tagging

“…In particular, viruses with MA deletions that retain only the N-terminal myristylation signal and MA-CA juncture sequences were shown to be conditionally infectious, as long the viruses employed either pseudotyped envelope proteins or HIV-1 Env proteins bearing cytoplasmic tail (CT) truncations (30,32,48). These observations support a model in which an Env CT-MA interaction is necessary for proper Env assembly into virions but do not explain how deletion MA (⌬MA) PrGag proteins are transported to the PM and or how they manage to perform the essential functions of wild-type (WT) PrGag proteins (27,28,30,32,48,49). Taking advantage of the relative insensitivity of HIV-1 MA to certain genetic perturbations, researchers also have generated PrGag proteins that carry protein insertions near the C terminus of MA (14,(50)(51)(52).…”

“…For virus protein analysis, virus samples were pelleted through 20% sucrose cushions in phosphate-buffered saline (PBS; 9.5 mM sodium potassium phosphate [pH 7.4], 137 mM NaCl, 2.7 mM KCl) for 45 min at 273,000 ϫ g at 4°C (49,52,57,60,61). Pelleted viruses were carefully resuspended in cold PBS and stored at Ϫ80°C.…”

“…Protein levels were quantified densitometrically using NIH ImageJ software (62), and molecular masses were estimated based on migrations compared to log (molecular mass)-versus-mobility plots of size standards run in parallel. Virus particle release levels were calculated as done previously (49,57,60,61) from at least two independent transfections by quantification of viral versus cellular Gag levels, and normalization of these levels relative to WT HIV-1 transfection data was performed in parallel. In experiments that involved ⌬MA-BirA* or MA-BirA* cotransfection with WT or PR Ϫ HIV-1 vectors, to distinguish between the HIV-1 variants, only the Gag precursor proteins for the two BirA* constructs were included in calculations.…”

“…MT-4 T cells (48,57,60) were maintained in RPMI medium with the same supplements and under the same conditions. Analysis of cellular protein expression and virus particle assembly and release was performed at 72 h after calcium phosphate transfections of HEK 293T cells (5 million cells per 10-cm-diameter plate) by using a total of 24 g plasmid DNA constructs as described previously (49,52,57,60,61). In cotransfection experiments, 16 g MA-BirA* or ⌬MA-BirA* was transfected with 8 g WT NL4-3, 2498T, or pVSV-G. To generate virus for infections, 18 g NL4-3-based plasmids was cotransfected with 6 g pVSV-G into HEK293T cells, and at 3 days posttransfection, viral supernatants were filtered through 0.45-m-pore-size filters.…”

A Mutation in Alpha Helix 3 of CA Renders Human Immunodeficiency Virus Type 1 Cyclosporin A Resistant and Dependent: Rescue by a Second-Site Substitution in a Distal Region of CA

“…During particle maturation, the CA protein condenses to form a conical core around the ribonucleoprotein complex. Mutations that alter HIV-1 core morphology also reduce infectivity (14,28,31,33,38). These observations suggest that proper formation of the conical HIV-1 core is essential for the early postentry events in HIV infection.…”