Characterization of Vif domains that mediate Feline Immunodeficiency Virus antagonism of APOBEC3-H and APOBEC3-CH restriction

Mapping Intimacies ◽

10.1101/144113 ◽

2017 ◽

Author(s):

Olivia L. Sims ◽

Ernest L. Maynard ◽

Eric M. Poeschla

Keyword(s):

Zinc Finger ◽

Feline Immunodeficiency Virus ◽

E3 Ligase ◽

Terminal Region ◽

Restriction Factor ◽

C Terminus ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Human Apobec3g

AbstractFeline immunodeficiency virus (FIV) Vif mediates degradation of two anti-lentiviral feline APOBEC3 (fA3) proteins, fA3Z3 and fA3Z2bZ3. HIV-1 Vif targets the restriction factor human APOBEC3G (A3G, hA3Z2g-Z1c) for proteasome degradation to mediate viral evasion. Despite this similarity, FIV and HIV-1 Vif share limited homology. Vif binds hA3Z2g-Z1c through its N-terminal region, while its C-terminal region binds to an E3-ligase complex containing Cullin5 and Elongin B/C. Further, HIV-1 Vif contains critical domains in its C-terminus, including an adjacent BC box, the only shared domain between FIV and HIV-1 Vif, and a non-classical zinc finger (HCCH) domain. Felid lentivirus Vif, however, contains a highly conserved KCCC motif. While both Vifs have evolved to counteract select A3 antiretroviral proteins, the FIV Vif domains necessary to target fA3s for degradation are incompletely understood. To identify these domains, we used the well-characterized HIV-1 Vif domains to show that distinct mutations within the BC box of FIV Vif prevent fA3Z3 and fA3Z2bZ3 degradation and reduce virion infectivity. We also found that mutating any single residue in the KCCC motif blocked fA3 targeting and impaired FIV infectivity and replication. These mutations also failed to disrupt the FIV Vif and Cullin5 interaction. Further, we showed that, in contrast to the HCCH domain in HIV-1 Vif, the KCCC domain of FIV Vif does not bind zinc. However, unlike HIV-1 Vif, FIV Vif (C36 isolate) reduces intracellular levels of co-expressed Cullin5 proteins, a novel finding. Our results reveal important C-terminal residues in FIV Vif and show that the BC box and KCCC regions are critical for fA3 degradation, infectivity, and spreading replication.

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Molecular Characterization of Feline Immunodeficiency Virus Budding

Journal of Virology ◽

10.1128/jvi.02337-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2106-2119 ◽

Cited By ~ 33

Author(s):

Benjamin G. Luttge ◽

Miranda Shehu-Xhilaga ◽

Dimiter G. Demirov ◽

Catherine S. Adamson ◽

Ferri Soheilian ◽

...

Keyword(s):

Feline Immunodeficiency Virus ◽

Equine Infectious Anemia Virus ◽

Binding Motif ◽

Virus Release ◽

Related Sequence ◽

Endosomal Sorting ◽

Peptide Motifs ◽

C Terminus ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Infection of domestic cats with feline immunodeficiency virus (FIV) is an important model system for studying human immunodeficiency virus type 1 (HIV-1) infection due to numerous similarities in pathogenesis induced by these two lentiviruses. However, many molecular aspects of FIV replication remain poorly understood. It is well established that retroviruses use short peptide motifs in Gag, known as late domains, to usurp cellular endosomal sorting machinery and promote virus release from infected cells. For example, the Pro-Thr/Ser-Ala-Pro [P(T/S)AP] motif of HIV-1 Gag interacts directly with Tsg101, a component of the endosomal sorting complex required for transport I (ESCRT-I). A Tyr-Pro-Asp-Leu (YPDL) motif in equine infectious anemia virus (EIAV), and a related sequence in HIV-1, bind the endosomal sorting factor Alix. In this study we sought to identify and characterize FIV late domain(s) and elucidate cellular machinery involved in FIV release. We determined that mutagenesis of a PSAP motif in FIV Gag, small interfering RNA-mediated knockdown of Tsg101 expression, and overexpression of a P(T/S)AP-binding fragment of Tsg101 (TSG-5′) each inhibited FIV release. We also observed direct binding of FIV Gag peptides to Tsg101. In contrast, mutagenesis of a potential Alix-binding motif in FIV Gag did not affect FIV release. Similarly, expression of the HIV-1/EIAV Gag-binding domain of Alix (Alix-V) did not disrupt FIV budding, and FIV Gag peptides showed no affinity for Alix-V. Our data demonstrate that FIV relies predominantly on a Tsg101-binding PSAP motif in the C terminus of Gag to promote virus release in HeLa cells, and this budding mechanism is highly conserved in feline cells.

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Amino-Terminal Region of the Human Immunodeficiency Virus Type 1 Nucleocapsid Is Required for Human APOBEC3G Packaging

Journal of Virology ◽

10.1128/jvi.78.21.11841-11852.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 11841-11852 ◽

Cited By ~ 149

Author(s):

Kun Luo ◽

Bindong Liu ◽

Zuoxiang Xiao ◽

Yunkai Yu ◽

Xianghui Yu ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Terminal Region ◽

Genomic Rna ◽

Immunodeficiency Virus ◽

Viral Genomic Rna ◽

Hiv 1 ◽

Human Apobec3g

ABSTRACT APOBEC3G exerts its antiviral activity by targeting to retroviral particles and inducing viral DNA hypermutations in the absence of Vif. However, the mechanism by which APOBEC3G is packaged into virions remains unclear. We now report that viral genomic RNA enhances but is not essential for human APOBEC3G packaging into human immunodeficiency virus type 1 (HIV-1) virions. Packaging of APOBEC3G was also detected in HIV-1 Gag virus-like particles (VLP) that lacked all the viral genomic RNA packaging signals. Human APOBEC3G could be packaged efficiently into a divergent subtype HIV-1, as well as simian immunodeficiency virus, strain mac, and murine leukemia virus Gag VLP. Cosedimentation of human APOBEC3G and intracellular Gag complexes was detected by equilibrium density and velocity sucrose gradient analysis. Interaction between human APOBEC3G and HIV-1 Gag was also detected by coimmunoprecipitation experiments. This interaction did not require p6, p1, or the C-terminal region of NCp7. However, the N-terminal region, especially the first 11 amino acids, of HIV-1 NCp7 was critical for HIV-1 Gag and APOBEC3G interaction and virion packaging. The linker region flanked by the two active sites of human APOBEC3G was also important for efficient packaging into HIV-1 Gag VLP. Association of human APOBEC3G with RNA-containing intracellular complexes was observed. These results suggest that the N-terminal region of HIV-1 NC, which is critical for binding to RNA and mediating Gag-Gag oligomerization, plays an important role in APOBEC3G binding and virion packaging.

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Development of a User-Friendly Pipeline for Mutational Analyses of HIV Using Ultra-Accurate Maximum-Depth Sequencing

Viruses ◽

10.3390/v13071338 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1338

Author(s):

Morgan E. Meissner ◽

Emily J. Julik ◽

Jonathan P. Badalamenti ◽

William G. Arndt ◽

Lauren J. Mills ◽

...

Keyword(s):

Error Rates ◽

Maximum Depth ◽

Sequencing Data ◽

Background Error ◽

High Background ◽

Immunodeficiency Virus ◽

User Friendly ◽

Viral Mutagenesis ◽

Hiv 1

Human immunodeficiency virus type 2 (HIV-2) accumulates fewer mutations during replication than HIV type 1 (HIV-1). Advanced studies of HIV-2 mutagenesis, however, have historically been confounded by high background error rates in traditional next-generation sequencing techniques. In this study, we describe the adaptation of the previously described maximum-depth sequencing (MDS) technique to studies of both HIV-1 and HIV-2 for the ultra-accurate characterization of viral mutagenesis. We also present the development of a user-friendly Galaxy workflow for the bioinformatic analyses of sequencing data generated using the MDS technique, designed to improve replicability and accessibility to molecular virologists. This adapted MDS technique and analysis pipeline were validated by comparisons with previously published analyses of the frequency and spectra of mutations in HIV-1 and HIV-2 and is readily expandable to studies of viral mutation across the genomes of both viruses. Using this novel sequencing pipeline, we observed that the background error rate was reduced 100-fold over standard Illumina error rates, and 10-fold over traditional unique molecular identifier (UMI)-based sequencing. This technical advancement will allow for the exploration of novel and previously unrecognized sources of viral mutagenesis in both HIV-1 and HIV-2, which will expand our understanding of retroviral diversity and evolution.

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Human TRIM5α: Autophagy Connects Cell-Intrinsic HIV-1 Restriction and Innate Immune Sensor Functioning

Viruses ◽

10.3390/v13020320 ◽

2021 ◽

Vol 13 (2) ◽

pp. 320 ◽

Cited By ~ 1

Author(s):

Alexandra P. M. Cloherty ◽

Anusca G. Rader ◽

Brandon Compeer ◽

Carla M. S. Ribeiro

Keyword(s):

Therapeutic Potential ◽

Innate Immune ◽

Health Concern ◽

Restriction Factor ◽

Viral Reservoirs ◽

Minimal Restriction ◽

Immunodeficiency Virus ◽

A Cell ◽

Species Specific ◽

Hiv 1

Human immunodeficiency virus-1 (HIV-1) persists as a global health concern, with an incidence rate of approximately 2 million, and estimated global prevalence of over 35 million. Combination antiretroviral treatment is highly effective, but HIV-1 patients that have been treated still suffer from chronic inflammation and residual viral replication. It is therefore paramount to identify therapeutically efficacious strategies to eradicate viral reservoirs and ultimately develop a cure for HIV-1. It has been long accepted that the restriction factor tripartite motif protein 5 isoform alpha (TRIM5α) restricts HIV-1 infection in a species-specific manner, with rhesus macaque TRIM5α strongly restricting HIV-1, and human TRIM5α having a minimal restriction capacity. However, several recent studies underscore human TRIM5α as a cell-dependent HIV-1 restriction factor. Here, we present an overview of the latest research on human TRIM5α and propose a novel conceptualization of TRIM5α as a restriction factor with a varied portfolio of antiviral functions, including mediating HIV-1 degradation through autophagy- and proteasome-mediated mechanisms, and acting as a viral sensor and effector of antiviral signaling. We have also expanded on the protective antiviral roles of autophagy and outline the therapeutic potential of autophagy modulation to intervene in chronic HIV-1 infection.

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KCTD proteins as Cullin3 E3 Ligase adapters

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314096946 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C305-C305

Author(s):

Alan Ji ◽

Gilbert Privé

Keyword(s):

Crystal Structure ◽

Structural Characterization ◽

Dissociation Constants ◽

E3 Ligase ◽

Terminal Region ◽

Btb Domain ◽

Substrate Protein ◽

Ubiquitin E3 Ligase ◽

Ubiquitin Moiety

Cullin3 (Cul3) is an ubiquitin E3 ligase responsible for catalyzing the transfer of an ubiquitin moiety from an E2 enzyme to a target substrate protein. The C-terminal region of Cul3 binds RBX1/E2-ubiquitin, while, the N-terminal region interacts with various BTB domain proteins which serve as substrate adaptors. Previously, our group determined the crystal structures of the homodimeric BTB proteins SPOP and KLHL3 in complex with the N-terminal domain of Cul3, revealing the determinants responsible for the BTB/Cul3 interaction [1, 2]. A second class of BTB-domain containing proteins, the KCTD proteins, are also Cul3 substrate adaptors but these do not share many of the previously determined features for Cul3 binding. Furthermore, KCTD proteins form homotetramers and homopentamers via BTB oligomerization rather than the previously described homodimers. Despite these differences, many KCTD proteins interact with Cul3 with dissociation constants of approximately 50 nM. While the target substrates for many of the KCTD/Cul3 E3 ligase complexes are unknown, recent studies have implicated the GABAβ2 receptor as an interactor of KCTD 8, 12, 12b and 16. Here, we report the pentameric crystal structure of the KCTD9 BTB domain and our progress on the structural characterization of Cul3/KCTD/substrate complexes.

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