Functional Analysis of the Transmembrane (TM) Domain of the AcMNPV GP64 Protein: Substitution of Heterologous TM Domains

Boyce Thompson Institute at Cornell University, Ithaca, New York 14853

^* To whom correspondence should be addressed. Email: gwb1{at}cornell.edu.

	Abstract

GP64, the major envelope glycoprotein of the Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV) budded virion, is important for host cell receptor binding and mediates low-pH-triggered membrane fusion during entry by endocytosis. In the current study, we examined the functional role of the AcMNPV GP64 transmembrane (TM) domain by replacing the 23 amino acid GP64 TM domain with corresponding TM domain sequences from a range of viral or cellular type I membrane proteins, including OpMNPV GP64 and F, Thogotovirus GP75, LdMNPV F, HIV-1 GP41, Human CD4 and glycophorin A (GpA), Influenza virus HA, and with a glycosylphosphatidylinositol (GPI) anchor addition sequence. In transient expression experiments in Sf9 cells, chimeric GP64 proteins containing either a GPI anchor or TM domains from LdMNPV F or HIV-1 GP41 failed to localize to the cell surface and thus appear to be incompatible with either GP64 structure or cell transport. All of the mutant constructs detected at the cell surface mediated hemifusion (outer leaflet merger) upon low pH treatment but only those containing TM domains from CD4, GpA, OpMNPV GP64, and Thogotovirus GP75 mediated pore formation and complete membrane fusion activity. This supports a model in which partial fusion (hemifusion) proceeds by a mechanism that is independent of the TM domain, and the TM domain participates in the enlargement or expansion of fusion pores after hemifusion. GP64 proteins containing heterologous TM domains mediated virion budding with dramatically differing levels of efficiency. In addition, chimeric GP64 proteins containing TM domains from CD4, GpA, HA, and OpMNPV F were incorporated into budded virions but were unable to rescue the infectivity of a gp64null virus, whereas those with TM domains from OpMNPV GP64 and Thogoto GP75 rescued infectivity. These results show that in addition to its basic role in membrane anchoring, the GP64 TM domain is critically important for GP64 trafficking, membrane fusion, virion budding, and virus infectivity. These critical functions were substituted only by TM domains from related viral membrane proteins.