High titers of retrovirus (vesicular stomatitis virus) pseudotypes, at last.

In a recent issue of the Proceedings, Burns et al. (1) presented evidence that they can make very high titer stocks of a pseudotype virus whose genome is derived from the Moloney retrovirus and whose envelope is the G protein of vesicular stomatitis virus (VSV). As expected, the virus has the broad host range characteristic of VSV. For example, unlike Moloney virus, it is able to infect hamster cell lines. Less predictable, but strikingly, the pseudotype also infects fish cell lines including a zebrafish line. The titers of virus reported are a spectacular 109 colony-forming units/ml. This stunning technical leap-a leap of six to seven orders of magnitude in titer over previous reports for this virus-may have important applications in human gene therapy. It also has important implications for basic research. The report by Burns et al. (1) comes more than 20 years after the discovery that retroviruses and VSV can swap envelope proteins (2), and it appears after years of unsuccessful efforts by various laboratories and biotech companies to produce high titers ofretroviral pseudotypes bearing VSV G protein on their surface. It has long been known that when two viruses infect the same cell, the proteins coded by one viral genome can sometimes package the genome of the other. This process, called phenotypic mixing, is hardly surprising when the viruses are closely related. However, phenotypic mixing can also occur between viruses that belong to completely different taxonomic groups. In 1972 Zavada (2) discovered that phenotypic mixing occurs between retroviruses and VSV, the prototype rhabdovirus. VSV is a lytic virus that replicates in the cytoplasm, has a single negativestranded RNA genome of 11,000 nt, a bullet-shaped virion surrounded by a membrane, and the ability to shut offhost transcription and translation on its way to killing its host cell (see ref. 3). Retroviruses have a positive-strand RNA genome of 8-10,000 bases, replicate via a DNA intermediate that must integrate into the host genome to be efficiently transcribed, and in most cases, do not kill the cells they infect (see ref. 4). Both viruses release progeny virions by budding. Retroviruses use specific cell surface proteins as receptors to get into cells and attach to the receptors via their envelope glycoproteins. This interaction is very often species specific, and in some cases even tissue specific. As a result, retroviruses usually have a quite limited host range. In contrast, VSV has a notoriously broad host range, infecting everything from human to insect cells. It is thought that the virus must use a universal membrane component to get into cells, possibly phospholipid (5). VSV G glycoprotein is responsible for the viral host range. When VSV and a typical mouse or chicken retrovirus infect the same cell, the progeny are a mixture of viruses that include parental types and pseudotypes. The latter, the result of phenotypic mixing, (i) contain a retroviral genome but the G protein and, hence, have the expanded host range of VSV, (ii) contain a VSV genome and the envelope protein and, hence, have the restricted host range ofthe coinfecting retrovirus, or (iil) have a mixture of envelope proteins derived from both viruses (2, 6, 7). Before molecular cloning was possible, the only tools available for separating the various types of particles emerging from a mixed infection were neutralizing antisera and temperature-sensitive mutants of VSV that could prevent replication ofparticles with a VSV genome (2, 6-9). Both methods were messy and it was difficult to obtain either pure or high titer stocks of pseudotypes. Molecular cloning offered new ways to solve the problems. Molecular cloning of viral genomes and the development of retroviral packaging lines lead to the recent report by Burns et al. (1). Packaging cell lines are engineered to synthesize all the proteins needed to make and release infectious retroviral virions (proteins encoded by the viral gag, pol, and env genes) (10, 11). The cells are then provided with a viral genome, designed to be the only RNA molecule in the cell with the capability of being packaged efficiently into virus particles. To meet this requirement, anRNA molecule needs a packaging sequence, termed psi (4i), which in the case of Moloney virus is contained within a few hundred nucleotides near the 5' end of the RNA (10, 12). The other requirement to be a successful viral genome are sequences needed for reverse transcription and sequences required for integration of the resulting proviral DNA. These include a primer binding site and sequences at and near the termini of the RNA that become the long terminal repeats in the double-stranded DNA copy of the viral RNA (see ref. 4). The rest ofthe RNA, up to about 10,000 bases worth, can encode anything at all, including, for example, a gene to correct cystic fibrosis, sickle cell anemia, etc. The particles released from packaging cell lines are infectious but defective, since they lack gag, pol, and env genes. They can infect cells just once, integrating a copy of their genes into the host cell genome. It is these particles that are potentially so useful for gene therapy. Knowledge of how to make retroviral packaging cell lines suggested that one might be able to construct a line that would produce retroviral pseudotypes with VSV host range. In 1991, Emi, Friedmann, and Yee (13) took an important step in this direction. They showed conclusively that G protein was the only VSVcoded protein needed to make mouse retroviral pseudotypes with the VSV host range (13, 14). This result, and a similar finding by Hunter and coworkers (15) that influenza hemagglutinin protein can be used in place of the Rous sarcoma virus env glycoprotein, is interesting. How is G protein, or hemagglutinin protein, recruited into budding retroviral virions? In VSV particles, it has been thought that a matrix protein (M) binds the nucleocapsid and also the tail of the G protein to pull it into the budding particle. But G protein has no sequence similarity to retroviral envelope proteins, so it is unlikely that it interacts specifically with retroviral capsid proteins (14). In fact, how G or hemagglutinin, or even env glycoproteins, are recruited into retroviral virions remains unclear. The current hypothesis is that they may simply get in because they are not attached to anything else (15, 16). In any case, if G or hemagglutinin can be incorporated into retroviruses and determine their host range, who knows what other interesting molecules with interesting host ranges might also work? Emi et al. (13) achieved Moloney (VSV-G) pseudotype titers no higher than 102 to 103 infectious units/ml. Thus, a remaining technical problem to make the