Analysis of the Assembly Function of the Human Immunodeficiency Virus Type 1 Gag Protein Nucleocapsid Domain

et al. 2008

Virology

The HIV-1 matrix (MA) protein is similar to nucleocapsid (NC) proteins in its propensity for self-interaction and association with RNA. Here we report on our finding that replacing MA with NC results in the production of wild type (wt)-level RNA and virus-like particles (VLPs). In contrast, constructs containing MA as a substitute for NC are markedly defective in VLP production and form virions with lower densities than wt, even though their RNA content is over 50% that of wt level. We also noted that a DeltaMN mutant lacking both MA and NC produces a relatively higher amount of VLPs than those in which MA was substituted for NC. Although DeltaMN contains approximately 30% the RNA of wt, it still exhibits virion densities equal (or very similar) to those of wt. The data suggest that neither NC nor RNA are major virion density determinants. Furthermore, we noted that NC(ZIP)--a NC replacement with a leucine zipper dimerization motif--produces VLPs as efficiently as wt. However, the markedly reduced assembly efficiency of NC(ZIP) is associated with the formation of VLPs with densities slightly lower than those of wt following MA removal, suggesting that (a) MA is required to help the inserted leucine zipper motif perform efficient Gag multimerization, and (b) MA plays a role in the virus assembly process.

Section: Discussionmentioning

confidence: 70%

Section: Introductionmentioning

confidence: 99%

HIV-1 matrix protein repositioning in nucleocapsid region fails to confer virus-like particle assembly

Chang

et al. 2008

Virology

“…In addition to playing a key role in viral RNA packaging (Berkowitz et al, 1993(Berkowitz et al, , 1995Poon et al, 1996;Zhang and Barklis, 1997), NC contains an I domain that is responsible for Gag-Gag interactions (Bennett et al, 1993;Bowzard et al, 1998). Heterologous polypeptides capable of self-association have been shown to confer the ability to efficiently produce chimeric VLPs when substituted for HIV-1 NC (Accola et al, 2000;Burniston et al, 1999;Johnson et al, 2002;Zhang et al, 1998). However, replacing NC with a leucine-zipper motif that does not encapsidate RNA abolishes hA3G packaging without significantly affecting HIV-1 virion production (Zennou et al, 2004), suggesting RNA involvement in hA3G incorporation.…”

Section: Introductionmentioning

confidence: 99%

APOBEC3G cytidine deaminase association with coronavirus nucleocapsid protein

2009

Virology

We previously reported that replacing HIV-1 nucleocapsid (NC) domain with SARS-CoV nucleocapsid (N) residues 2-213, 215-421, or 234-421 results in efficient virus-like particle (VLP) production at a level comparable to that of wild-type HIV-1. In this study we demonstrate that these chimeras are capable of packaging large amounts of human APOBEC3G (hA3G), and that an HIV-1 Gag chimera containing the carboxyl-terminal half of human coronavirus 229E (HCoV-229E) N as a substitute for NC is capable of directing VLP assembly and efficiently packaging hA3G. When co-expressed with SARS-CoV N and M (membrane) proteins, hA3G was efficiently incorporated into SARS-CoV VLPs. Data from GST pull-down assays suggest that the N sequence involved in N-hA3G interactions is located between residues 86 and 302. Like HIV-1 NC, the SARS-CoV or HCoV-229E N-associated with hA3G depends on the presence of RNA, with the first linker region essential for hA3G packaging into both HIV-1 and SARS-CoV VLPs. The results raise the possibility that hA3G is capable of associating with different species of viral structural proteins through a potentially common, RNA-mediated mechanism.

“…Plasmid DNAs containing wt (wtZiP) and mutant (KZiP) leucine zipper domains of human CREB [24] were kindly provided by E. Barklis [54]. cDNA fragments of the wt and mutant leucine zipper domains were PCR-amplified, digested, and ligated into DNC, yielding the respective constructs DNC(wtZiP) and DNC(KZiP).…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Likewise, recombinant SARS-CoV nucleocapsid proteins can undergo multimerization via self-association [16,30,42,51]. Since heterologous polypeptides that form interprotein contacts permit efficient VLP production when placed in the HIV-1 NC region [1,9,20,54], we initially tested whether SARS-CoV N substitution for HIV-1 NC supports chimeric VLP assembly and release, and found that HIV-1 Gag mutants containing SARS-CoV N coding sequences as NC substitutes are capable of VLP assembly. This suggests that the HIV-1 NC assembly domain can be functionally replaced with the SARS-CoV N.…”

Section: Introductionmentioning

confidence: 99%

Severe acute respiratory syndrome coronavirus nucleocapsid protein confers ability to efficiently produce virus-like particles when substituted for the human immunodeficiency virus nucleocapsid domain

Chang²,

Chen³

et al. 2008

J Biomed Sci

We replaced the HIV-1 nucleocapsid (NC) domain with different N-coding sequences to test SARS-CoV nucleocapsid (N) self-interaction capacity, and determined the capabilities of each chimera to direct virus-like particle (VLP) assembly. Analysis results indicate that the replacement of NC with the carboxyl-terminal half of the SARS-CoV N resulted in the production of wild type (wt)-level virus-like particles (VLPs) with the density of a wt HIV-1 particle. When co-expressed with SARS-CoV N, chimeras containing the N carboxyl-terminal half sequence efficiently packaged N. However, the same was not true for the chimera bearing the N amino-terminal half sequence, despite its production of substantial amounts of VLPs. According to further analysis, HIV-1 NC replacement with N residues 2-213, 215-421, or 234-421 resulted in efficient VLP production at levels comparable to that of wt HIV-1, but replacement with residues 215-359, 302-421, 2-168, or 2-86 failed to restore VLP production to wild-type levels. The results suggest that the domain conferring the ability to direct VLP assembly and release in SARS-CoV N is largely contained between residues 168 and 421.