Hantaviruses are enveloped, negative-strand RNA viruses which can be lethal to humans, causing either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome. The viral genomes consist of three RNA segments: the L segment encodes the viral polymerase, the M segment encodes the viral surface glycoproteins G1 and G2, and the S segment encodes the nucleocapsid (N) protein. The N protein is a 420-to 430-residue, 50-kDa protein which appears to direct hantavirus assembly, although mechanisms of N protein oligomerization, RNA encapsidation, budding, and release are poorly understood. We have undertaken a biochemical and genetic analysis of N protein oligomerization. Bacterially expressed N proteins were found by gradient fractionation to associate not only as large multimers or aggregates but also as dimers or trimers. Chemical cross-linking of hantavirus particles yielded N protein cross-link products with molecular masses of 140 to 150 kDa, consistent with the size of an N trimer. We also employed a genetic, yeast two-hybrid method for monitoring N protein interactions. Analyses showed that the C-terminal half of the N protein plus the N-terminal 40 residues permitted association with a full-length N protein fusion. These N-terminal 40 residues of seven different hantavirus strains were predicted to form trimeric coiled coils. Our results suggest that coiled-coil motifs contribute to N protein trimerization and that nucleocapsid protein trimers are hantavirus particle assembly intermediates. Hantaviruses, such as the Sin Nombre hantavirus (SNV) andProspect Hill virus (PHV), are members of the bunyavirus class of viruses (3,20,24). They are enveloped, negative-strand RNA viruses and carry three genomic RNA segments: the L segment, which encodes an RNA-dependent RNA polymerase; the M segment, which encodes envelope glycoproteins G1 and G2; and the S segment, which encodes the viral nucleocapsid (N) protein (24). Hantaviruses are of medical importance, because many of them cause either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome, which is characterized by lung damage and cardiac dysfunction (25).Models for hantavirus replication at the cellular level have been based on direct experiments and by inference from work on other bunyaviruses. The general replication cycle starts with G1 and G2 binding to B3 integrins (7,8) or other cell surface receptors, followed by virus entry and uncoating. After entry, L protein-mediated primary transcription of mRNAs occurs in the cytoplasm, apparently using an orthomyxovirus-like capsnatching mechanism (4, 14). Following mRNA translation, transcription shifts from mRNA to cRNA and viral RNA synthesis, and ribonucleoprotein (RNP) structures are formed (4, 14, 24). The RNPs appear to be composed of viral RNAs, N proteins, and presumably L proteins and accumulate on the cytoplasmic sides of cellular membranes, possibly through interactions with the G1 and G2 proteins (9, 21). Evidence suggests that RNPs use microfilaments for transport to ...

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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...