Mapping and characterization of the N-terminal I domain of human immunodeficiency virus type 1 Pr55(Gag).

Human immunodeficiency virus (HIV) type 1 particles assemble at the plasma membrane of cells in a manner similar to that of the type C oncoretroviruses. The Pr55(Gag) molecule directs the assembly process and is sufficient for particle assembly in the absence of all other viral gene products. The I domain is an assembly domain that has been previously localized to the nucleocapsid (NC) region of Gag. In this study we utilized a series of Gag-green fluorescent protein (GFP) fusion proteins to precisely identify sequences that constitute the N-terminal I domain of Pr55(Gag). The minimal sequence required for the I domain was localized to the extreme N terminus of NC. Two basic residues (arginine 380 and arginine 384) within the initial seven residues of NC were found to be critical for the function of the N-terminal I domain. The presence of positive charge alone in these two positions, however, was not sufficient to mediate the formation of dense Gag particles. The I domain was required for the formation of detergent-resistant complexes of Gag protein, and confocal microscopy demonstrated that the I domain was also required for the formation of punctate foci of Gag proteins at the plasma membrane. Electron microscopic analysis of cells expressing Gag-GFP fusion constructs with an intact I domain revealed numerous retrovirus-like particles (RVLPs) budding from the plasma membrane, while I domain-deficient constructs failed to generate visible RVLPs. These results provide evidence that Gag-Gag interactions mediated by the I domain play a central role in the assembly of HIV particles.

Human immunodeficiency virus replication in a primary effusion lymphoma cell line stimulates lytic-phase replication of Kaposi's sarcoma-associated herpesvirus.

Human immunodeficiency virus (HIV) and Kaposi's sarcoma-associated herpesvirus (KSHV) coinfect many individuals in North America and in parts of Africa. Infection with HIV is a leading risk factor for the development of Kaposi's sarcoma (KS). In this study, we tested the hypothesis that HIV infection of common or adjacent cells would stimulate replication and spread of KSHV. Infection of a primary effusion lymphoma cell line by vesicular stomatitis virus type G-pseudotyped HIV type 1 led to a rapid induction of lytic-phase KSHV replication. Induction of lytic KSHV replication by HIV required active replication of HIV. The addition of the nucleoside reverse transcriptase inhibitor azidothymidine or the protease inhibitor indinavir to the culture prevented HIV spread and inhibited the associated induction of KSHV lytic replication. Lytic replication occurred in both HIV-infected and HIV-uninfected cells within the culture, and could be induced in uninfected cells via a soluble factor released from the HIV-infected cells. Transmission of infectious KSHV to an uninfected target cell was enhanced by HIV replication and was inhibited by antiretroviral drugs. These results may have implications for the pathogenesis and treatment of KS in individuals coinfected with KSHV and HIV.

Semiliki forest virus vector carrying the bovine viral diarrhea virus NS3 (p80) cDNA induced immune responses in mice and expressed BVDV protein in mammalian cells.

Bovine viral diarrhea virus (BVDV) is a primary pathogen responsible for bovine enteric, respiratory and reproductive failure. A genetic region is encoding the p80 (NS3) of BVDV as the most conserved protein among Pestiviruses. BVDV infection in cattle induces NS3 specific lymphocyte proliferation and humoral responses. To generate a DNA vaccine against BVDV, the gene for BVDV-NADL NS3 was cloned into an eukaryotic expression vector of Semiliki Forest virus (pSFV-1). Quadriceps muscles of BALB/c mice were injected with recombinant DNA generated statistically significant cytotoxic T-lymphocyte activity (CTL) and cell mediated immune (CMI) responses against cytopathic and noncytopathic BVDV. Whereas, the BVDV-NS3 did not generate neutralizing antibodies against BVDVin mice. pSFV-1-NS3 DNA was subjected to in vitro transcription into mRNA. The mRNA was transfected into baby hamster kidney cells (BHK-21) and Madin-Darby bovine kidney cells (MDBK). The recombinant cells were used in the detection of DNA antigen responses by immunological assays. This report establishes the ability of BVDV-NS3 DNA inoculation to induce a strong cellular immune responses in mice.

The I domain is required for efficient plasma membrane binding of human immunodeficiency virus type 1 Pr55Gag.

The interaction of the human immunodeficiency virus type 1 (HIV-1) Pr55Gag molecule with the plasma membrane of an infected cell is an essential step of the viral life cycle. Myristic acid and positively charged residues within the N-terminal portion of MA constitute the membrane-binding domain of Pr55Gag. A separate assembly domain, termed the interaction (I) domain, is located nearer the C-terminal end of the molecule. The I domain is required for production of dense retroviral particles, but has not previously been described to influence the efficiency of membrane binding or the subcellular distribution of Gag. This study used a series of Gag-green fluorescent protein fusion constructs to define a region outside of MA which determines efficient plasma membrane interaction. This function was mapped to the nucleocapsid (NC) region of Gag. The minimal region in a series of C-terminally truncated Gag proteins conferring plasma membrane fluorescence was identified as the N-terminal 14 amino acids of NC. This same region was sufficient to create a density shift in released retrovirus-like particles from 1.13 to 1.17 g/ml. The functional assembly domain previously termed the I domain is thus required for the efficient plasma membrane binding of Gag, in addition to its role in determining the density of released particles. We propose a model in which the I domain facilitates the interaction of the N-terminal membrane-binding domain of Pr55Gag with the plasma membrane.

Identification of a 56 kDa putative bovine herpesvirus 1 cellular receptor by anti-idiotype antibodies.

Polyclonal anti-idiotype antibodies (anti-ids) to anti-bovine herpesvirus 1 (BHV-1) MAbs blocked virus infection in cell cultures, indicating that they contain internal images of a viral attachment protein(s). In the present study anti-id (anti-83) of BHV-1 gB was used as a probe to detect the cellular receptor. Anti-id specifically identified a 56 kDa protein in radioimmunoprecipitation analysis (RIPA) of Madin-Darby bovine kidney (MDBK) cell membranes suggesting the involvement of this cell surface component in BHV-1 binding. Anti-83 pretreated with MAb 83 failed to identify the 56 kDa cellular component proving its specificity for the reacting epitope of MAb 83. The recognition of 56 kDa protein by anti-id was inhibited by prior incubation of radiolabelled membrane proteins with BHV-1 suggesting that the ligands competed for the same binding sites on the cells. 35S-Radiolabelled BHV-1 virions also bound a 56 kDa protein from purified MDBK cell membrane proteins in a virus overlay protein blot assay. RIPA using anti-id as probe detected the 56 kDa protein in permissive MDBK cells but not in non-permissive bat lung cells. The protein nature of the 56 kDa component was confirmed by protease treatment of membranes which resulted in abolition of the 56 kDa signal in RIPA. In addition, purified 56 kDa protein inhibited biotinylated BHV-1 attachment in flow cytometry. These findings indicate that the 56 kDa protein identified by anti-id is a putative receptor for BHV-1.