Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions

Zhu, Ping; Chertova, Elena; Bess, Julian W.; Lifson, Jeffrey D.; Arthur, Larry O.; Li, Jun; Taylor, Kenneth A.; Roux, Kenneth H.

doi:10.1073/pnas.2634931100

Cited by 360 publications

(347 citation statements)

References 42 publications

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“…In the main, HIV is thought to assemble at the plasma membrane of T-cells. The finding that endocytosis signals function to maintain low levels of Env on the cell surface may explain why HIV particles collected from cultured T-cell lines have relatively low Env levels (average 7-10 spikes/virion; Chertova et al, 2002;Zhu et al, 2003). In addition, the levels of Env expression on the cell surface and on virions could be a determinant for host humoral immune responses and viral evasion (Yuste et al, 2004(Yuste et al, , 2005.…”

Section: Discussionmentioning

confidence: 99%

“…For these particles to be infectious, Env must be transported to the cell surface, but the level to which it is incorporated into budding virions is unclear. Recent data have suggested that fewer than 10 Env protein complexes (trimers) are incorporated per virion (Chertova et al, 2002;Zhu et al, 2003), and early studies of HIV infected T-cells showed that much of the newly synthesized Env is transported to lysosomes and degraded (Willey et al, 1988). However, it has recently become evident that in macrophages the assembly of Envcontaining infectious HIV occurs on intracellular membranes, that have some characteristics in common with late endosomes (Raposo et al, 2002;Pelchen-Matthews et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

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A Conserved Dileucine Motif Mediates Clathrin and AP-2–dependent Endocytosis of the HIV-1 Envelope Protein

Byland

Vance

Hoxie

et al. 2007

MBoC

123

133

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During the assembly of enveloped viruses viral and cellular components essential for infectious particles must colocalize at specific membrane locations. For the human and simian immunodeficiency viruses (HIV and SIV), sorting of the viral envelope proteins (Env) to assembly sites is directed by trafficking signals located in the cytoplasmic domain of the transmembrane protein gp41 (TM). A membrane proximal conserved GYxxØ motif mediates endocytosis through interaction with the clathrin adaptor AP-2. However, experiments with SIV mac239 Env indicate the presence of additional signals. Here we show that a conserved C-terminal dileucine in HIV HxB2 also mediates endocytosis. Biochemical and morphological assays demonstrate that the C-terminal dileucine motif mediates internalization as efficiently as the GYxxØ motif and that both must be removed to prevent Env internalization. RNAi experiments show that depletion of the clathrin adaptor AP-2 leads to increased plasma membrane expression of HIV Env and that this adaptor is required for efficient internalization mediated by both signals. The redundancy of conserved endocytosis signals and the role of the SIV mac239 Env GYxxØ motif in SIV pathogenesis, suggest that these motifs have functions in addition to endocytosis, possibly related to Env delivery to the site of viral assembly and/or incorporation into budding virions. INTRODUCTIONThe assembly of enveloped animal viruses requires that the viral and cellular components needed to make infectious particles are brought to the same site within the infected cell at the same time. For the primate lentiviruses (the human immunodeficiency viruses types 1 and 2 [HIV-1 and -2] and the simian immunodeficiency viruses [SIV]) the key viral structural proteins required to generate infectious particles are Gag, Gag-Pol, and Env. Although Gag alone can promote the formation of virus-like particles, Env and Gag-Pol are both essential for the formation of infectious virions. Although a considerable amount is known about these proteins, little is understood of the mechanisms through which they are targeted to the sites of assembly in infected cells.In many cell types, HIV assembles at the plasma membrane and, in the course of Gag assembly, the particles derive their membrane from the plasma membrane. For these particles to be infectious, Env must be transported to the cell surface, but the level to which it is incorporated into budding virions is unclear. Recent data have suggested that fewer than 10 Env protein complexes (trimers) are incorporated per virion (Chertova et al., 2002;Zhu et al., 2003), and early studies of HIV infected T-cells showed that much of the newly synthesized Env is transported to lysosomes and degraded (Willey et al., 1988). However, it has recently become evident that in macrophages the assembly of Envcontaining infectious HIV occurs on intracellular membranes, that have some characteristics in common with late endosomes (Raposo et al., 2002;Pelchen-Matthews et al., 2003). The assembly of virus in distinct...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Conserved Dileucine Motif Mediates Clathrin and AP-2–dependent Endocytosis of the HIV-1 Envelope Protein

Byland

Vance

Hoxie

et al. 2007

MBoC

123

133

View full text Add to dashboard Cite

show abstract

“…To begin the reaction, the RNA genome (50 nM) was annealed to a 24-nt DNA primer (29) and incubated with nucleocapsid (3.0 M), reverse transcriptase (2.4 M), and dNTPs (500 M) in reverse transcriptase buffer in the presence or absence of 200 nM Apo3A or Apo3G. Each of the components of the reverse transcription reactions were at estimated physiological ratios to the HIV genome as found in the HIV-1 literature (24,54,55) or Apo3G literature (56). Analyses used the sequences of 25 mutated clones for each condition tested.…”

Section: Methodsmentioning

confidence: 99%

Biochemical Analysis of Hypermutation by the Deoxycytidine Deaminase APOBEC3A

Love

Chelico

2012

Journal of Biological Chemistry

119

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“…Previous biochemical analyses of mature HIV-1 particles estimated 1,000 to 10,000 copies of viral RNA for 1 pg of capsid (p24) or ϳ25 million capsid molecules (5). The number of Gag molecules in immature HIV-1 particles was estimated to be 1,400 to 2,400 (6,7). Because each Gag polyprotein yields 1 processed, mature capsid protein; taken together, these numbers imply that each virion contains, on average, somewhere between 0.05 and 1 genomes.…”

mentioning

confidence: 99%

High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis

Chen

Nikolaitchik

Singh

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

189

254

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A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging.Bgl ͉ MS2 ͉ dimerization ͉ DIS ͉ fluorescent protein

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Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions

Cited by 360 publications

References 42 publications

A Conserved Dileucine Motif Mediates Clathrin and AP-2–dependent Endocytosis of the HIV-1 Envelope Protein

A Conserved Dileucine Motif Mediates Clathrin and AP-2–dependent Endocytosis of the HIV-1 Envelope Protein

Biochemical Analysis of Hypermutation by the Deoxycytidine Deaminase APOBEC3A

High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis

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