To express the function encoded in its genome, the herpes simplex virus 1 capsid-tegument structure released by deenvelopment during entry into cells must be transported retrograde to the nuclear pore where viral DNA is released into the nucleus. This path is essential in the case of virus entering axons of dorsal root ganglia. The objective of the study was to identify the viral proteins that may be involved in the transport. We report the following findings. The herpes simplex virion contains four structural elements arranged in a concentric fashion. These are the DNA core, the capsid, a set of proteins surrounding the capsid known as the tegument, and an envelope. To initiate infection, herpes simplex virus 1 (HSV-1) and HSV-2 must attach to and penetrate the infected cell. In the process, the viral envelope fuses with the plasma membrane and the capsid-tegument structure is transported to the nuclear pore where it remains docked for at least several hours (3, 32). Viral gene expression necessary to initiate viral replication takes place after viral DNA is released from the docked capsids into the nucleus. The conclusion that at least some of the tegument protein docks with the capsid is based on studies of HSV-1(HFEM)tsB7. At the nonpermissive temperature, the capsids docked at the nuclear pore retain their DNA, whereas at the permissive temperature, the DNA is released in the nucleus and viral gene expression ensues (3, 17). The mutation which leads to the retention of the DNA in the docked capsid maps in the infected cell protein 1-2 (ICP1-2), a tegument protein encoded by the U L 36 open reading frame (ORF) (3).Among the many unresolved issues regarding initiation of infection is the mechanism by which the virus is transported from the site of entry-the junction between the plasma membrane rendered contiguous to the envelope and cytoplasm-to the nuclear pore. Recent studies have focused on the microtubular network and path and dynein as the motor that transports the capsid-tegument structure to the nuclear pore (2,18,28).To define the mechanism of attachment of cytoplasmic dynein to herpesvirus, we tested the ability of viral proteins to interact with the neuronal isoform of the intermediate chain (IC) of cytoplasmic dynein. We report that the amino-terminal domain of the IC (IC-1a) of cytoplasmic dynein interacted in pulldown experiments with three viral proteins, the major capsid protein ICP5 and the products of the U L 34 and U L 31 genes. Since U L 34 interacts with IC-1a and also independently with U L 31 and the major capsid protein, it is likely that the primary interaction is between IC-1a and U L 34 and that the latter pulled down the other viral proteins. Relevant to this report are the following observations.(i) Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport and is the only known retrograde motor in interphase cells (23). It is the largest and most complex of the motor proteins, consisting of four subunit classes: heavy chains responsible for force pro...

Recombinant adeno-associated virus (rAAV) production systems capable of meeting clinical or anticipated commercial-scale manufacturing needs have received relatively little scrutiny compared with the intense research activity afforded the in vivo and in vitro evaluation of rAAV for gene transfer. Previously we have reported a highly efficient recombinant herpes simplex virus type 1 (rHSV) complementation system for rAAV production in multiple adherent cell lines; however, production in a scalable format was not demonstrated. Here we report rAAV production by rHSV coinfection of baby hamster kidney (BHK) cells grown in suspension (sBHK cells), using two ICP27-deficient rHSV vectors, one harboring a transgene flanked by the AAV2 inverted terminal repeats and a second bearing the AAV rep2 and capX genes (where X is any rAAV serotype). The rHSV coinfection of sBHK cells produced similar rAAV1/AAT-specific yields (85,400 DNase-resistant particles [DRP]/cell) compared with coinfection of adherent HEK-293 cells (74,600 DRP/cell); however, sBHK cells permitted a 3-fold reduction in the rHSV-rep2/capX vector multiplicity of infection, grew faster than HEK-293 cells, retained specific yields (DRP/cell) at higher cell densities, and had a decreased virus production cycle. Furthermore, sBHK cells were able to produce AAV serotypes 1, 2, 5, and 8 at similar specific yields, using multiple therapeutic genes. rAAV1/AAT production in sBHK cells was scaled to 10-liter disposable bioreactors, using optimized spinner flask infection conditions, and resulted in average volumetric productivities as high as 2.4 x 10(14) DRP/liter.

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