Targeting Human Immunodeficiency Virus Type 1 Assembly, Maturation and Budding

Wapling, Johanna; Srivastava, Seema; Shehu-Xhilaga, Miranda; Tachedjian, Gilda

doi:10.1177/117739280700200020

Cited by 9 publications

(6 citation statements)

References 230 publications

(249 reference statements)

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“…Both release and infectivity are decreased for HIV particles containing excess GagPol, and these decreases are at least partially due to premature PR activation (45,46). This activation may result from dimerization of HIV-1 reverse transcriptase (RT), which, when it occurs in the context of Gag-Pol, can activate PR and have negative effects on budding and maturation (50). In contrast, MoMLV RT is monomeric, at least in solution (51), and thus, MoMLV RT likely cannot contribute to Gag-Pol-Gag-Pol dimerization and premature PR activation to the extent that HIV-1 RT can.…”

Section: Discussionmentioning

confidence: 99%

Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions

Johnson

Collins

D'Souza

et al. 2014

J Virol

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The Moloney murine leukemia virus (MoMLV) ribonucleoprotein complex is composed of an approximately 20:1 mixture of Gag and Gag-Pol polyproteins plus a single genomic RNA (gRNA) dimer. The mechanisms that regulate these proportions are unknown. Here, we examined whether virion proportions of Gag, Gag-Pol, and gRNA were determined by sampling (that is, if they reflected expression ratios or intracellular concentrations) or more specific recruitment. To this end, MoMLV Gag, Gag-Pol, and gRNA were expressed separately or together in various ratios. Varying the expression ratios of Gag and Gag-Pol revealed that Gag-Pol incorporation was stochastic and that the conserved 20:1 Gag/Gag-Pol ratio coincided with maximal particle production. When skewed expression ratios resulted in excess Gag-Pol, the released virions maintained the intracellular Gag/GagPol ratios and the infectivity per virion was largely maintained, but virion production decreased sharply with high levels of GagPol. The determinants of gRNA proportions were addressed by manipulating the amounts and contexts of functional nucleocapsid (NC) and the ratios of Gag to gRNA. The results showed that the NC domain of either Gag or Gag-Pol could provide gRNA packaging functions equally well. Unlike Gag-Pol, gRNA incorporation was saturable. An upper limit of gRNA incorporation was observed, and particle production was not disrupted by excess gRNA expression. These results indicate that the determinants of Gag/Gag-Pol proportions differ from those for Gag/gRNA. On the basis of the assumption that MoMLV evolved to produce virion components in optimal proportions, these data provide a means of estimating the proportion of unspliced MoMLV RNA that serves as genomic RNA. IMPORTANCEViruses assemble their progeny from within the cells that they parasitize, where they must sort through a rich milieu of host proteins and nucleic acids to gather together their own building blocks, which are also proteins and nucleic acids. The research described here addresses whether or not the proportions of viral proteins and nucleic acids that are brought together to form a retroviral particle are determined by random sampling from the cell-and thus dictated by the components' availabilities within the cell-or if the amounts of each molecule are specified by the virus replication process. The results indicated that protein components of the murine retrovirus studied here are recruited by chance but that a specific counting mechanism defines the amount of nucleic acid incorporated into each progeny virion.

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Section: Discussionmentioning

confidence: 99%

Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions

Johnson

Collins

D'Souza

et al. 2014

J Virol

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“…The myristoylation of Gag is generally considered as essential for targeting the protein to the host plasma membrane (Morikawa et al, 1996). Dominant-negative mutations that directly interfere with Gag myristoylation, or indel mutations that block translation from the physiological start site or that create a loss-of-function gag transcript will perturb proper viral particle assembly at the plasma membrane, and hence block retroviral particle budding (Morikawa et al, 1996;Wapling, Srivastava, Shehu-Xhilaga, & Tachedjian, 2007). In addition to Myr, the conserved proline-rich PPxY motif also contributes to retrovirus budding, likely by interacting with the ESCRT machinery (Henzy, Gifford, Johnson, & Coffin, 2014), and its mutation strongly inhibits viral particle release (Segura-Morales et al, 2005).…”

Section: Targeting Group 1 Erv Sequences By Crispr-cas9 Genome Editingmentioning

confidence: 99%

Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

Duroy

Bosshard

Schmid‐Siegert

et al. 2019

Biotech & Bioengineering

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The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type‐C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type‐C ERV sequences clustering into three functionally conserved groups. Transcripts from one type‐C ERV group were full‐length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral‐like particles, suggesting that this ERV group may produce functional viruses. CRISPR‐Cas9 genome editing was used to disrupt the gag gene of the expressed type‐C ERV group. Comparison of CRISPR‐derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA‐loaded viral particles. Clones bearing a Gag loss‐of‐function mutation in this ERV showed a reduction of RNA‐containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.

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“…Genetic studies have demonstrated that mutations in the Pol region of Gag-Pol, comprising protease (PR), reverse transcriptase (RT), and integrase (IN), can lead to defects in particle morphology, virion release, uncoating, reverse transcription, or nuclear localization of the preintegration complex ( 6 – 13 ). The mechanism by which the Pol region within Gag-Pol influences these events is poorly understood.…”

Section: Introductionmentioning

confidence: 99%

“…How IN or Pol mutations could influence assembly has not been elucidated. There are several hypotheses, one of which is that mutations in IN or Pol interfere with Gag and Gag-Pol oligomerization, thereby disrupting the assembly process ( 11 , 13 ). Another hypothesis is that defects in IN may lead to premature protease action within the cells, and it has been shown that the inhibition of protease catalytic activity overcomes the assembly defects mediated by at least some of the IN mutants ( 12 ).…”

Section: Introductionmentioning

confidence: 99%

An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability

Porte

Cano

et al. 2016

J Virol

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INI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production. SID-INI1 dramatically reduced the intracellular Gag/Gag-Pol protein levels and, in addition, decreased viral particle production. The SID-INI1-mediated effects were less dramatic in trans complementation assays using IN deletion mutant viruses with Vpr-reverse transcriptase (RT)-IN. SID-INI1 did not inhibit long-terminal-repeat (LTR)-mediated transcription, but it marginally decreased the steady-state gag RNA levels, suggesting a posttranscriptional effect. Pulse-chase analysis indicated that in SID-INI1-expressing cells, the pr55Gag levels decreased rapidly. RNA interference analysis indicated that small hairpin RNA (shRNA)-mediated knockdown of INI1 reduced the intracellular Gag/Gag-Pol levels and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of posttranscriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibits assembly and particle production. Interfering INI1 leads to a decrease in particle production and Gag/Gag-Pol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events. IMPORTANCE Significant gaps exist in our current understanding of the mechanisms and host factors that influence HIV-1 posttranscriptional events, including gag RNA levels, Gag/Gag-Pol protein levels, assembly, and particle production. Our previous studies suggested that the IN-binding host factor INI1 plays a role in HIV-1 assembly. An ectopically expressed minimal IN-binding domain of INI1, S6, potently and selectively inhibited HIV-1 Gag/Gag-Pol trafficking and particle production. However, whether or not endogenous INI1 and its interacting partners, such as SAP18, are required for late events was unknown. Here, we report that endogenous INI1 and its interaction with SAP18 are necessary to maintain intracellular levels of Gag/Gag-Pol and for particle production. Interfering INI1 or the INI1-SAP18 interaction leads to the impairment of these processes, suggesting a novel strategy for inhibiting posttranscriptional events of HIV-1 replication.

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Targeting Human Immunodeficiency Virus Type 1 Assembly, Maturation and Budding

Cited by 9 publications

References 230 publications

Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions

Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions

Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability

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