Incorporation of High Levels of Chimeric Human Immunodeficiency Virus Envelope Glycoproteins into Virus-Like Particles

Abstract

The human immunodeficiency virus (HIV) envelope (Env) protein is incorporated into HIV virions or virus-like particles (VLPs) at very low levels compared to the glycoproteins of most other enveloped viruses. To test factors that influence HIV Env particle incorporation, we generated a series of chimeric gene constructs in which the coding sequences for the signal peptide (SP), transmembrane (TM), and cytoplasmic tail (CT) domains of HIV-1 Env were replaced with those of other viral or cellular proteins individually or in combination. All constructs tested were derived from HIV type 1 (HIV-1) Con-S ⌬CFI gp145, which itself was found to be incorporated into VLPs much more efficiently than full-length Con-S Env. Substitution of the SP from the honeybee protein mellitin resulted in threefold-higher chimeric HIV-1 Env expression levels on insect cell surfaces and an increase of Env incorporation into VLPs. Substitution of the HIV TM-CT with sequences derived from the mouse mammary tumor virus (MMTV) envelope glycoprotein, influenza virus hemagglutinin, or baculovirus (BV) gp64, but not from Lassa fever virus glycoprotein, was found to enhance Env incorporation into VLPs. The highest level of Env incorporation into VLPs was observed in chimeric constructs containing the MMTV and BV gp64 TM-CT domains in which the Gag/Env molar ratios were estimated to be 4:1 and 5:1, respectively, compared to a 56:1 ratio for full-length Con-S gp160. Electron microscopy revealed that VLPs with chimeric HIV Env were similar to HIV-1 virions in morphology and size and contained a prominent layer of Env spikes on their surfaces. HIV Env specific monoclonal antibody binding results showed that chimeric Env-containing VLPs retained conserved epitopes and underwent conformational changes upon CD4 binding.

Incorporation of Membrane-Anchored Flagellin into Influenza Virus-Like Particles Enhances the Breadth of Immune Responses

“…1A, a membrane-anchored flagellin-encoding gene was constructed by merging the coding sequence for the SP from the honeybee protein mellitin at the N terminus and a TM-CT from influenza HA in frame at the C terminus. As a heterologous SP, mellitin SP is known to improve glycoprotein cell surface expression in an insect cell system (60). The TM-CT sequence from HA provides a membrane anchor sequence which would be expected to engage the modified flagellin to assemble into influenza virus matrix protein (M1)-derived VLPs (1).…”

“…The hemagglutination activity of VLPs was determined by the capacity to hemagglutinate chicken red blood cells (42). For electron microscopy, VLP samples (5 to 10 l; 0.1 mg/ml protein) were examined as described previously (60).…”

Dense Array of Spikes on HIV-1 Virion Particles

“…CTT modification also affects the antigenicity of HIV-1 Env (12) and can alter neutralization sensitivity in an Env-dependent manner (13)(14)(15)(16)(17). Stable cell line production of Env (18), and substitutions with a foreign TM domain have been shown to enhance Env on virus-like particles (VLPs); but the effects are either modest, or the reports lack details about the integrity, function, and antigenicity of trimeric Env (19)(20)(21)(22).…”

Inhibition of Envelope-Mediated CD4+-T-Cell Depletion by Human Immunodeficiency Virus Attachment Inhibitors

“…1). The calculated level of Env along with p24 Gag quantification was used to establish a 1-to-10 ratio of Env to p24 Gag associated with purified VLP NL4-3 , revealing that VLP preparations contained approximately 30 copies of gp120/particle, a density that is consistent with that of infectious particles (48,58). Moreover, VLP NL4-3 induces syncitium formation in MOLT4 cell cultures (data not shown), suggesting HIV-1 Env functionality.…”