Structural evidence that MOAP1 and PEG10 are derived from retrovirus/retrotransposon Gag proteins

ABSTRACT Newcastle disease virus (NDV) avirulent strain LaSota was used to coexpress gp160 Env and p55 Gag from a single vector to enhance both Env-specific and Gag-specific immune responses. The optimal transcription position for both Env and Gag genes in the NDV genome was determined by generating recombinant NDV (rNDV)-Env-Gag (gp160 located between the P and M genes and Gag between the HN and L genes), rNDV-Gag-Env (Gag located between the P and M genes and gp160 between the HN and L genes), rNDV-Env/Gag (gp160 followed by Gag located between the P and M genes), and rNDV-Gag/Env (Gag followed by gp160 located between the P and M genes). All the recombinant viruses replicated at levels similar to those seen with parental NDV in embryonated chicken eggs and in chicken fibroblast cells. Both gp160 and Gag proteins were expressed at high levels in cell culture, with gp160 found to be incorporated into the envelope of NDV. The Gag and Env proteins expressed by all the recombinants except rNDV-Env-Gag self-assembled into human immunodeficiency virus type 1 (HIV-1) virus-like particles (VLPs). Immunization of guinea pigs by the intranasal route with these rNDVs produced long-lasting Env- and Gag-specific humoral immune responses. The Env-specific humoral and mucosal immune responses and Gag-specific humoral immune responses were higher in rNDV-Gag/Env and rNDV-Env/Gag than in the other recombinants. rNDV-Gag/Env and rNDV-Env/Gag were also more efficient in inducing cellular as well as protective immune responses to challenge with vaccinia viruses expressing HIV-1 Env and Gag in mice. These results suggest that vaccination with a single rNDV coexpressing Env and Gag represents a promising strategy to enhance immunogenicity and protective efficacy against HIV. IMPORTANCE A safe and effective vaccine that can induce both systemic and mucosal immune responses is needed to control HIV-1. In this study, we showed that coexpression of Env and Gag proteins of HIV-1 performed using a single Newcastle disease virus (NDV) vector led to the formation of HIV-1 virus-like particles (VLPs). Immunization of guinea pigs with recombinant NDVs (rNDVs) elicited potent long-lasting systemic and mucosal immune responses to HIV. Additionally, the rNDVs were efficient in inducing cellular immune responses to HIV and protective immunity to challenge with vaccinia viruses expressing HIV Env and Gag in mice. These results suggest that the use of a single NDV expressing Env and Gag proteins simultaneously is a novel strategy to develop a safe and effective vaccine against HIV.

Download Full-text

The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.79.21.13463-13472.2005 ◽

2005 ◽

Vol 79 (21) ◽

pp. 13463-13472 ◽

Cited By ~ 69

Author(s):

Danso Ako-Adjei ◽

Marc C. Johnson ◽

Volker M. Vogt

Keyword(s):

Nuclear Export ◽

Leukemia Virus ◽

Particle Diameter ◽

Particle Morphology ◽

Structural Protein ◽

Wild Type ◽

Virus Like Particles ◽

Gag Proteins ◽

Hiv 1

ABSTRACT The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.

Download Full-text

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity

Molecular and Cellular Biology ◽

10.1128/mcb.2.4.355-360.1982 ◽

1982 ◽

Vol 2 (4) ◽

pp. 355-360

Author(s):

A Ziemiecki ◽

R R Friis ◽

H Bauer

Keyword(s):

Protein Synthesis ◽

Half Life ◽

Rous Sarcoma Virus ◽

Kinase Activity ◽

Rabbit Serum ◽

Temperature Sensitive ◽

Apparent Contradiction ◽

Gag Proteins ◽

Rous Sarcoma ◽

Sarcoma Virus

The half-life of metabolically labeled pp60src of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp60src with tumor-bearing rabbit serum. These experiments showed that pp60src has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp60src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42 degrees C) or permissive (35 degrees C) temperature. The half-life of the pp60src -associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp60src. The apparent contradiction between the half-lives of the kinase activity and the [35S]methionine-labeled pp60src protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp60src, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.

Download Full-text

Gene Therapy for HIV-1 Using Intracellular Antibodies Against HIV-1 Gag Proteins

Intrabodies ◽

10.1007/978-3-662-12119-1_9 ◽

1998 ◽

pp. 163-181

Author(s):

Isaac J. Rondon ◽

Wayne A. Marasco

Keyword(s):

Gene Therapy ◽

Gag Proteins ◽

Hiv 1

Download Full-text

Assembly of HIV-1 gag-proteins: A new target for antiviral peptide therapy?

Peptides 1992 ◽

10.1007/978-94-011-1470-7_49 ◽

1993 ◽

pp. 133-135 ◽

Cited By ~ 1

Author(s):

Susanne Modrow ◽

A. v. Poblotzki ◽

R. Wagner ◽

H. R. Gelderblom ◽

G. Pauli ◽

...

Keyword(s):

Peptide Therapy ◽

Gag Proteins ◽

Antiviral Peptide ◽

Hiv 1

Download Full-text

Human Immunodeficiency Virus Types 1 and 2 Differ in the Predominant Mechanism Used for Selection of Genomic RNA for Encapsidation

Journal of Virology ◽

10.1128/jvi.73.4.3023-3031.1999 ◽

1999 ◽

Vol 73 (4) ◽

pp. 3023-3031 ◽

Cited By ~ 67

Author(s):

Jane F. Kaye ◽

Andrew M. L. Lever

Keyword(s):

Human Immunodeficiency Virus ◽

Genomic Rna ◽

Rna Packaging ◽

Packaging Signal ◽

Initiation Mechanism ◽

Gag Protein ◽

Gag Proteins ◽

Viral Genomes ◽

Immunodeficiency Virus

ABSTRACT Retroviral RNA encapsidation is a highly selective process mediated through recognition by the viral Gag proteins of cis-acting RNA packaging signals in genomic RNA. This RNA species is also translated, producing the viral gag gene products. The relationship between these processes is poorly understood. Unlike that of human immunodeficiency virus type 1 (HIV-1), the dominant packaging signal of HIV-2 is upstream of the major splice donor and present in both unspliced and spliced viral RNAs, necessitating additional mechanisms for preferential packaging of unspliced genomic RNA. Encapsidation studies of a series of HIV-2-based vectors showed efficient packaging of viral genomes only if the unspliced, encapsidated RNA expressed full-length Gag protein, including functional nucleocapsid. We propose a novel encapsidation initiation mechanism, providing selectivity, in which unspliced HIV-2 RNA is captured in cis by the Gag protein. This has implications for the use of HIV-2 and other lentiviruses as vectors.

Download Full-text