Baculovirus Biology

Baculoviruses are a diverse group of rod-shaped viruses having a restricted host range, which is often limited to specific invertebrate species, especially insects (21). Baculoviruses constitute a family of viruses, the Baculoviridae, including more than 500 known members (22), which are believed to infect permissively only arthropod hosts. The double-stranded circular DNA genome (80-200 kbp) of baculoviruses (23,24) is condensed into a nucleoprotein structure known as a core (25). The core is located within a flexible rod-shaped capsid, which is 25-50 nm in diameter and 200-320 nm in length (26-28) and can expand relatively freely to accommodate even very large recombinant molecules (29). The core and the capsid are known collectively as a nucleocapsid. The nu-cleocapsids are made in the nucleus of infected cells, and they acquire membrane envelopes either by budding through the plasma membrane of the cell or by a nuclear envelopment process. Membrane-enveloped nucleocapsids are referred to as virus particles or virions (4).

Baculoviruses are divided into 2 morphologically distinct genera: nuclear polyhedrosis viruses (NPVs), and granulosis viruses (GVs). In the NPV group, virions that obtain an envelope from nuclear membrane are occluded within a paracrys-talline protein matrix [occluded viruses, (OVs)], forming large (1-5 ^m) polyhedral inclusion bodies (PIBs) containing multiple virions. NPVs are further distinguished on the basis of whether they contain a single nucleocapsid or multiple nucleo-capsids per envelope in the polyhedrin matrix (30). In contrast to NPVs, GVs have only a single virion embedded in a very small inclusion body (4).

Baculoviruses have 2 distinct forms involved in their life cycle that play different roles during the natural life cycle of the virus (Fig. 1). OVs are responsible for horizontal transmission between insect hosts, whereas systemic spread within the insect and propagation in tissue culture is dependent on budded viruses (BVs) (8,31,32). Structurally, BVs and OVs differ by the origin and composition of their envelopes (33). They also differ in the mechanisms by which they enter the host cells. BVs enter the cells by adsorptive endocytosis (34), but OVs enter the midgut epithelial cells via direct membrane fusion at the cell surface (35,36). PIBs are formed during late phase of natural infection by embedding the virions in the crystalline protein matrix, which is composed mostly of polyhedrin protein (37). PIBs enable the horizontal infection of larva by contaminating the plant on which the larva feeds (38). The viruses are protected from environmental factors within the PIB, but in the alkaline midgut of the larva the crystalline polyhedrin matrix is solubilized (39) and the released OVs enter the midgut cells by fusion with the membrane of the microvilli (40). During the lytic cycle of infection, the cells release BVs from the basolateral area of the midgut cells (41). The spread of infection within the insect occurs from the midgut to most tissues by hemolymph (42). Eventually the larva dies and the PIBs that are produced in the very late phase of infection are released into the environment, and the cycle begins again. Most of the naturally occurring bacu-loviruses kill their target host within 4 to 7 days (9).

Baculovirus infection can be divided into early, late, and very late phases. Biologically these phases correspond to reprogramming the cell for virus replication (BV and PIB production). In the early phase (the first 6 h), the virus prepares infected cell for viral DNA replication. This phase is also known as viral synthesis phase. Virus-specific RNAs can be detected in the cells by 30 min postinfection (pi) (43). The late phase extends from 6 h pi to approximately 20 to 24 h pi. During this viral structural phase, late genes are expressed and the production of BVs starts around 12 h pi. Progeny nucleocapsids leave the nucleus and are transported onto the plasma membrane where they acquire their envelope. The occlusion-specific phase begins around 20 h pi. Production of infectious BVs decreases and packaging of virus particles into polyhedrin matrix as OVs begin followed by the cell lysis (4). The packaging into polyhedrin matrix (i.e., production of PIBs) does not take place with BEVS because the polyhedrin gene has been deleted from most baculovirus genomes used in biotechnology procedures.

A. Autographs californica Multiple Nuclear Polyhedrosis Virus

The prototype of the family Baculoviridea and the most extensively studied NPV-type baculovirus is the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Its genome (~ 134 kbp) has been sequenced and predicted to contain 154 open reading frames (44). Although the 3-dimen-sional structure remains to be determined, the components of its cigar-shaped loosely enveloped virion have been extensively studied by protein chemistry (45). Vp39, p80, and p24 represent the major capsid proteins of the BV form of AcMNPV (Fig. 2). Among them, the vp39 is the most studied and is shown to be randomly distributed over the capsid surface. The major glycoprotein of AcMNPV is gp64, which is the only virus-encoded protein found to be associated with the BV envelope. It is believed to be responsible for the forma-

Figure 1 The life cycle of nuclear polyhedrosis virus (AcMNPV). Larva ingests occluded viruses (OVs) containing polyhedral inclusion bodies (PIBs) with their nourishment. In the alkaline environment of the midgut, the PIB break down and the nucleocapsids are released. They infect primarily epithelia of the gut wall. Viruses are fused directly to the plasmalemma of epithelium and the capsids are transported into the nucleus. A minor proportion of viruses may travel through the cells without infecting them in a process called transcytosis. The infection spreads through hemolymph inside the larva via budded viruses (BVs) that contain an essential protein gp64 in their envelopes. Later in the infection, occluded viruses are packed into PIBs inside the nucleus of the infected cells where these particles are released to the environment ready to infect the next hosts (horizontal infection) when cells are lysed and larva dies. , Polyhedral inclusion body;^, occluded virus; -J-, budded virus;';» , gp64 adhesion point in the plasma membrane of infected cells; | nucleocapsid. See the color insert for a color version of this figure.

Figure 1 The life cycle of nuclear polyhedrosis virus (AcMNPV). Larva ingests occluded viruses (OVs) containing polyhedral inclusion bodies (PIBs) with their nourishment. In the alkaline environment of the midgut, the PIB break down and the nucleocapsids are released. They infect primarily epithelia of the gut wall. Viruses are fused directly to the plasmalemma of epithelium and the capsids are transported into the nucleus. A minor proportion of viruses may travel through the cells without infecting them in a process called transcytosis. The infection spreads through hemolymph inside the larva via budded viruses (BVs) that contain an essential protein gp64 in their envelopes. Later in the infection, occluded viruses are packed into PIBs inside the nucleus of the infected cells where these particles are released to the environment ready to infect the next hosts (horizontal infection) when cells are lysed and larva dies. , Polyhedral inclusion body;^, occluded virus; -J-, budded virus;';» , gp64 adhesion point in the plasma membrane of infected cells; | nucleocapsid. See the color insert for a color version of this figure.

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