Vector Design Strategies

A. Biology of the Viral Lytic Cycle

HSV-1 is a double-stranded DNA virus whose capsid is surrounded by a dense layer of proteins, the tegument, which is contained within a lipid bilayer envelope (Fig. 1A). Glycopro-teins embedded in the viral envelope mediate infection of the host cell, which takes place in 2 identifiable stages: (1) attachment to the cell surface, and (2) fusion with the cell membrane, resulting in virus penetration. The envelope of HSV-1 contains at least 10 glycoproteins (gB, gC, gD, gE, gG, gH, gl, gJ, gL, and gM) and 4 nonglycosylated integral membrane proteins (products of the UL20, UL34, UL45, and UL49.5 genes). Of the 10 glycoproteins, gB, gD, gH, and gL are essential for viral infection (1-4), whereas gC, gE, gG, gl, gJ, and gM are dispensable for infection in vitro (5-7).

Attachment of the viral particle is mediated by several gly-coproteins (5,6,8). The sequential attachment steps in infection result in fusion of the viral envelope with the cell surface membrane and entry of the viral capsid into the cell cytoplasm. Even though the molecular events of penetration are not well understood, it is clear that multiple viral glycoproteins are required (e.g., gB, gD, gH/gL) (2,4,9-11). In addition, follow ing new virion assembly, viral glycoproteins are also involved in a less well-defined process of egress and release of mature particles from the infected cell membrane. Viral particles are also capable of spreading from cell to cell across cell junctions, a process requiring the functional activities of several glycoproteins that are not required for initial infection (e.g., gl/gE) (12,13).

The genome structure of HSV can be divided into viral genes that are essential or accessory for replication in cell culture (Fig. 1B). The accessory functions may be deleted without significantly hampering virus growth in culture. However, removal of essential genes necessitates the use of complementing cell lines that express the essential products in order to propagate these viral recombinants. In human infections, HSV binds to and enters epidermal cells following direct contact with an infected individual that is shedding virus or has an active lesion. Following virus attachment, the viral capsid penetrates the surface membranes of epithelial cells of the skin or mucosa and is transported to the nuclear membrane where viral DNA is injected through a nuclear pore (Fig. 2A). Once inside the nucleus, the viral DNA is circularized and transported to nuclear domain 10 (ND10) structures (14,15), where the immediate early (IE) genes are expressed as part of the sequential cascade of lytic gene synthesis (16) (Fig. 2B). Transcription of the 5 IE genes (ICP0, ICP4, ICP22, ICP27, and ICP47) does not require de novo viral protein synthesis. Expression of the IE genes is controlled by promoters that contain 1 or more copies of an enhancer element responsive to the viral tegument protein VP16 (a.k.a. Vmw 65 or TIF, a transactivator that is transported into the nucleus along with viral DNA) (17-19). The IE genes ICP4 and ICP27 encode products required for expression of the early (E) and late (L) genes (20-23); the former (E) gene class specifying primarily enzyme functions required for viral DNA synthesis and the latter (L) comprising primarily virion structural components. ICP4 regulates viral promoter function (21), whereas ICP27 affects the processing and transport of viral RNA (24,25). The IE gene products ICP0 and ICP22 contribute to viral gene transcription but are not essential to virus replication in cultured cells (26-29). ICP0 is a promiscuous transacti-vator that exerts its effect prior to the transcription initiation event; it is not a DNA-binding protein (30). ICP22 has been found to regulate the level of ICP0 expression (31). ICP47 does not affect transcription but rather has been reported to interfere with a transporter function transporter of antigen presentation (TAP) that is responsible for loading major histo-compatibility (MHC) class I molecules with antigenic peptides (32-35). Expression of late genes is dependent on both viral DNA synthesis and IE gene functions (21,23,36,37). Following translation of the late gene products, which become viral structural components of the capsid, tegument, and envelope, genome-length copies of viral DNA are packaged into the newly assembled capsids. Tegument proteins accumulate around the capsid and the immature particle buds through the inner nuclear membrane where the viral glycoproteins are localized. Double-membrane-enveloped virus containing virus-encoded glycoproteins modified by the Golgi apparatus

Herpes Simplex Viral Vectors (A)

Figure 1 HSV-1 virion structure and genome organization. (A) Electron micrograph of the HSV particle, showing the capsid, tegument, and glycoprotein-containing lipid envelope. (B) Schematic representation of the HSV genome, showing the unique long (UL) and unique short (US) segments, each bounded by inverted repeat (IR) elements. The location of the essential genes, which are required for viral replication in vitro, and the nonessential or accessory genes, which may be deleted without affecting replication in vitro, are indicated. The 5 IE genes, various glycoprotein genes, LAT, and other important loci are highlighted by asterisks.

Figure 1 HSV-1 virion structure and genome organization. (A) Electron micrograph of the HSV particle, showing the capsid, tegument, and glycoprotein-containing lipid envelope. (B) Schematic representation of the HSV genome, showing the unique long (UL) and unique short (US) segments, each bounded by inverted repeat (IR) elements. The location of the essential genes, which are required for viral replication in vitro, and the nonessential or accessory genes, which may be deleted without affecting replication in vitro, are indicated. The 5 IE genes, various glycoprotein genes, LAT, and other important loci are highlighted by asterisks.

enzymes fuse with the cell membrane forming a mature, extracellular virus particle with a single membrane bilayer (38). The infectious particles can infect neighboring cells by cell-to-cell transmission or can be released for infection of distal cells. With the exception of sensory neurons, cell lysis accompanies productive viral infection.

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