Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction.

The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10- to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.

Expression and immunogenicity of human immunodeficiency virus type 1 Gag expressed by a replication-competent rhabdovirus-based vaccine vector.

A replication-competent rhabdovirus-based vector expressing human immunodeficiency virus type 1 (HIV-1) Gag protein was characterized on human cell lines and analyzed for the induction of a cellular immune response in mice. We previously described a rabies virus (RV) vaccine strain-based vector expressing HIV-1 gp160. The recombinant RV was able to induce strong humoral and cellular immune responses against the HIV-1 envelope protein in mice (M. J. Schnell et al., Proc. Natl. Acad. Sci. USA 97:3544-3549, 2000; J. P. McGettigan et al., J. Virol. 75:4430-4434, 2001). Recent research suggests that the HIV-1 Gag protein is another important target for cell-mediated host immune defense. Here we show that HIV-1 Gag can efficiently be expressed by RV on both human and nonhuman cell lines. Infection of HeLa cells with recombinant RV expressing HIV-1 Gag resulted in efficient expression of HIV-1 precursor protein p55 as indicated by both immunostaining and Western blotting. Moreover, HIV-1 p24 antigen capture enzyme-linked immunosorbent assay and electron microscopy showed efficient release of HIV-1 virus-like particles in addition to bullet-shaped RV particles in the supernatants of the infected cells. To initially screen the immunogenicity of this new vaccine vector, BALB/c mice received a single vaccination with the recombinant RV expressing HIV-1 Gag. Immunized mice developed a vigorous CD8(+) cytotoxic T-lymphocyte response against HIV-1 Gag. In addition, 26.8% of CD8(+) T cells from mice immunized with RV expressing HIV-1 Gag produced gamma interferon after challenge with a recombinant vaccinia virus expressing HIV-1 Gag. These results further confirm and extend the potency of RV-based vectors as a potential HIV-1 vaccine.

High level expression of a human rabies virus-neutralizing monoclonal antibody by a rhabdovirus-based vector.

Humans exposed to rabies virus must be promptly treated by passive immunization with anti-rabies antibody and active immunization with rabies vaccine. Currently, antibody prepared from pooled human serum or from immunized horses is utilized. However, neither of these reagents are readily available, entirely safe, or consistent in their biological activity. An ideal reagent would consist of a panel of human monoclonal antibodies. Such antibodies are now available, their only drawback being the cost of production. Using recombinant technology, we constructed a rabies virus-based vector which expresses high levels (approximately 60 pg/cell) of rabies virus-neutralizing human monoclonal antibody. The vector is a modified vaccine strain of rabies virus in which the rabies virus glycoprotein has been replaced with a chimeric vesicular stomatitis virus glycoprotein, and both heavy and light chain genes encoding a human monoclonal antibody have been inserted. This recombinant virus can infect a variety of mammalian cell lines and is non-cytolytic, allowing the use of cell culture technology routinely employed to produce rabies vaccines.

Genetic engineering of live rabies vaccines.

Rabies virus is not a single entity but consists of a wide array of variants that are each associated with different host species. These viruses differ greatly in the antigenic makeup of their G proteins, the primary determinant of pathogenicity and major inducer of protective immunity. Due to this diversity, existing rabies vaccines have largely been targeted to individual animal species. In this report, a novel approach to the development of rabies vaccines using genetically modified, reverse-engineered live attenuated rabies viruses is described. This approach entails the engineering of vaccine rabies virus containing G proteins from virulent strains and modification of the G protein to further reduce pathogenicity. Strategies employed included exchange of the arginine at position 333 for glutamine and modification of the cytoplasmic domain. The recombinant viruses obtained were non-neuroinvasive when administered via a peripheral route. The ability to confer protective immunity depended largely upon conservation of the G protein antigenic structure between the vaccine and challenge virus, as well as on the route of immunization.

Rabies virus-based vectors expressing human immunodeficiency virus type 1 (HIV-1) envelope protein induce a strong, cross-reactive cytotoxic T-lymphocyte response against envelope proteins from different HIV-1 isolates.

Novel viral vectors that are able to induce both strong and long-lasting immune responses may be required as effective vaccines for human immunodeficiency virus type 1 (HIV-1) infection. Our previous experiments with a replication-competent vaccine strain-based rabies virus (RV) expressing HIV-1 envelope protein from a laboratory-adapted HIV-1 strain (NL4-3) and a primary HIV-1 isolate (89.6) showed that RV-based vectors are excellent for B-cell priming. Here we report that cytotoxic T-lymphocyte (CTL) responses against HIV-1 gp160 are induced by recombinant RVs. Our results indicated that a single inoculation of mice with an RV expressing HIV-1 gp160 induced a solid and long-lasting memory CTL response specific for HIV-1 envelope protein. Moreover, CTLs from immunized mice were not restricted to the homologous HIV-1 envelope protein and were able to cross-kill target cells expressing HIV-1 gp160 from heterologous HIV-1 strains. These studies further suggest promise for RV-based vectors to elicit a persistent immune response against HIV-1 and their potential utility as efficacious anti-HIV-1 vaccines.

A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection.

To investigate the importance of the rabies virus (RV) glycoprotein (G) in protection against rabies, we constructed a recombinant RV (rRV) in which the RV G ecto- and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein (rRV-VSV-G). We were able to recover rRV-VSV-G and found that particle production was equal to rRV. However, the budding of the chimeric virus was delayed and infectious titers were reduced 10-fold compared with the parental rRV strain containing RV G. Biochemical analysis showed equal replication rates of both viruses, and similar amounts of wild-type and chimeric G were present in the respective viral particles. Additional studies were performed to determine whether the immune response against rRV-VSV-G was sufficient to protect against rabies. Mice were primed with rRV or rRV-VSV-G and challenged with a pathogenic strain of RV 12 days later. Similar immune responses against the internal viral proteins of both viruses indicated successful infection. All mice receiving the rRV vaccine survived the challenge, whereas immunization with rRV-VSV-G did not induce protection. The results confirm the crucial role of RV G in an RV vaccine.

Reinvestigation of the role of the rabies virus glycoprotein in viral pathogenesis using a reverse genetics approach.

The rabies virus glycoprotein (G) gene of the highly neuroinvasive and neurotropic strains SHBRV-18, CVS-N2c, and CVS-B2c was introduced into the non-neuroinvasive and less neurotropic SN-10 strain to provide further insight into the role of G in the pathogenesis of rabies. Phenotypic analyses of the recombinant viruses revealed, as expected, that the neurotropism of a particular rabies virus strain was a function of its G. Nevertheless, the pathogenicity of the recombinant viruses was, in every case, markedly lower than that of the wild-type viruses suggesting that while the G dictates neurotropism, other viral attributes are also important in pathogenesis. The low pathogenicity of the recombinant viruses is at least in part due to a strong increase in transcription activity. On the other hand, the production of infectious virus by the R-SHB18 recombinant virus-infected cells was significantly delayed by comparison with SHBRV-18 wild-type virus infected-cells. Replacement of the R-SHB18 G cytoplasmic domain, transmembrane domain, and stem region with its SN-10 G counterparts neither results in a significant increase in budding efficiency nor an increase in pathogenicity. These results suggest that an optimal match of the cytoplasmic domain of G with the matrix protein may not be sufficient for maximal virus budding efficiency, which is evidently a major factor of virus pathogenicity. Our studies indicate that to maintain pathogenicity, the interactions between various structural elements of rabies virus must be highly conserved and the expression of viral proteins, in particular the G protein, must be strictly controlled.

Recombinant vesicular stomatitis virus expressing respiratory syncytial virus (RSV) glycoproteins: RSV fusion protein can mediate infection and cell fusion.

The genes encoding the respiratory syncytial virus (RSV) attachment (G) and fusion (F) envelope glycoproteins were expressed separately as additional genes in recombinant vesicular stomatitis viruses (VSV). Cells infected with the VSV-RSV F recombinant formed large syncytia illustrating the fusion activity of F in absence of other RSV proteins. Both F and G glycoproteins were expressed at the cell surface and incorporated into virions. Incorporation of these proteins did not require cytoplasmic tail sequences of VSV G. Using a compound, ammonium chloride, that raises the endosomal pH, we showed that presence of the RSV F glycoprotein in the envelope of recombinant VSV allowed for infectivity through a low-pH-independent pathway. Recombinant VSV expressing RSV glycoproteins could be useful as an RSV vaccine.

Requirement for a non-specific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus.

The cytoplasmic domains of viral glycoproteins are often involved in specific interactions with internal viral components. These interactions can concentrate glycoproteins at virus budding sites and drive efficient virus budding, or can determine virion morphology. To investigate the role of the vesicular stomatitis virus (VSV) glycoprotein (G) cytoplasmic and transmembrane domains in budding, we recovered recombinant VSVs expressing chimeric G proteins with the transmembrane and cytoplasmic domains derived from the human CD4 protein. These unrelated foreign sequences were capable of supporting efficient VSV budding. Further analysis of G protein cytoplasmic domain deletion mutants showed that a cytoplasmic domain of only 1 amino acid did not drive efficient budding, whereas 9 amino acids did. Additional studies in agreement with the CD4-chimera experiments indicated the requirement for a short cytoplasmic domain on VSV G without the requirement for a specific sequence in that domain. We propose a model for VSV budding in which a relatively non-specific interaction of a cytoplasmic domain with a pocket or groove in the viral nucleocapsid or matrix proteins generates a glycoprotein array that promotes viral budding.

Construction of a novel virus that targets HIV-1-infected cells and controls HIV-1 infection.

We describe a recombinant vesicular stomatitis virus lacking its glycoprotein gene and expressing instead the HIV-1 receptor CD4 and a coreceptor, CXCR4. This virus was unable to infect normal cells but did infect, propagate on, and kill cells that were first infected with HIV-1 and therefore had the HIV membrane fusion protein on their surface. Killing of HIV-1-infected cells controlled HIV infection in a T cell line and reduced titers of infectious HIV-1 in the culture by as much as 10(4)-fold. Such a targeted virus could have therapeutic value in reducing HIV viral load. Our results also demonstrate a general strategy of targeting one virus to the envelope protein of another virus to control infection.

Specific targeting to CD4+ cells of recombinant vesicular stomatitis viruses encoding human immunodeficiency virus envelope proteins.

We generated replication-competent, recombinant vesicular stomatitis viruses (VSVs) expressing the human immunodeficiency virus (HIV) envelope protein or an HIV-VSV chimeric envelope protein in which the cytoplasmic domain of the HIV envelope protein was replaced with that from the VSV glycoprotein (G). These recombinants were generated with HIV type 1 (HIV-1) envelopes from both laboratory and primary isolates of HIV-1. The replication-competent recombinant viruses were stable and expressed the foreign proteins at high levels from extra transcription units in VSV. The foreign proteins were processed appropriately and transported to the cell surface. The incorporation of HIV gp120 into VSV particles was demonstrated biochemically only for the construct expressing the chimeric envelopes containing the VSV G cytoplasmic domain. The incorporation of the chimeric HIV envelope protein into the membrane of the recombinant VSV was also demonstrated by electron microscopy with gold-conjugated antibodies. To determine whether specific infection of CD4-positive cells could be demonstrated for these recombinants, we neutralized VSV infectivity due to VSV glycoprotein with anti-VSV serum. The neutralized recombinants expressing the chimeric envelope were able to infect only HeLa cells expressing CD4, and this CD4-specific infectivity was neutralized with anti-HIV serum. This assay also detected a 100-fold-lower titer of CD4-specific infectivity for the VSV recombinant expressing the wild-type HIV envelope. Our results illustrate that it is possible to express functional HIV envelopes from the VSV genome and target the recombinant virus to an alternative receptor. The recombinants may also prove useful as HIV vaccines.

Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles.

In a previous study we demonstrated that vesicular stomatitis virus (VSV) can be used as a vector to express a soluble protein in mammalian cells. Here we have generated VSV recombinants that express four different membrane proteins: the cellular CD4 protein, a CD4-G hybrid protein containing the ectodomain of CD4 and the transmembrane and cytoplasmic tail of the VSV glycoprotein (G), the measles virus hemagglutinin, or the measles virus fusion protein. The proteins were expressed at levels ranging from 23-62% that of VSV G protein and all were transported to the cell surface. In addition we found that all four proteins were incorporated into the membrane envelope of VSV along with the VSV G protein. The levels of incorporation of these proteins varied from 6-31% of that observed for VSV G. These results suggest that many different membrane proteins may be co-incorporated quite efficiently with VSV G protein into budding VSV virus particles and that specific signals are not required for this co-incorporation process. In fact, the CD4-G protein was incorporated with the same efficiency as wild type CD4. Electron microscopy of virions containing CD4 revealed that the CD4 molecules were dispersed throughout the virion envelope among the trimeric viral spike glycoproteins. The recombinant VSV-CD4 virus particles were about 18% longer than wild type virions, reflecting the additional length of the helical nucleocapsid containing the extra gene. Recombinant VSVs carrying foreign antigens on the surface of the virus particle may be useful for viral targeting, membrane protein purification, and for generation of immune responses.

Highly stable expression of a foreign gene from rabies virus vectors.

A reverse genetics approach was applied to generate a chimeric nonsegmented negative strand RNA virus, rabies virus (RV) of the Rhabdoviridae family, that expresses a foreign protein. DNA constructs containing the entire open reading frame of the bacterial chloramphenicol acetyltransferase (CAT) gene and an upstream RV cistron border sequence were inserted either into the nontranslated pseudogene region of a full-length cDNA copy of the RV genome or exchanged with the pseudogene region. After intracellular T7 RNA polymerase-driven expression of full-length antigenome RNA transcripts and RV nucleoprotein, phosphoprotein and polymerase from transfected plasmids, RVs transcribing novel monocistronic mRNAs and expressing CAT at high levels, were recovered. The chimeric viruses possessed the growth characteristics of standard RV and were genetically stable upon serial cell culture passages. CAT activity was still observed in cell cultures infected with viruses passaged for more than 25 times. Based on the unprecedented stability of the chimeric RNA genomes, which is most likely due to the structure of the rhabdoviral ribonucleoprotein complex, we predict the successful future use of recombinant rhabdovirus vectors for displaying foreign antigens or delivering therapeutic genes.

The minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus.

A new transcription unit was generated in the 3' noncoding region of the vesicular stomatitis virus (VSV) glycoprotein gene by introducing the smallest conserved sequence found at each VSV gene junction. This sequence was introduced into a DNA copy of the VSV genome from which infectious VSV can be derived. It contained an 11-nucleotide putative transcription stop/polyadenylation signal for the glycoprotein mRNA, an intergenic dinucleotide, and a 10-nucleotide putative transcription start sequence preceding a downstream foreign gene encoding the bacterial enzyme chloramphenicol acetyltransferase. Infectious recombinant VSV was recovered from this construct and was found to express high levels of functional chloramphenicol acetyltransferase mRNA and protein. The recombinant virus grew to wild-type titers of 5 x 10(9)/ml, and expression of the foreign gene was completely stable for at least 15 passages involving 10(6)-fold expansion at each passage. These results define functionally the transcription stop/polyadenylation and start sequences for VSV and also illustrate the utility of VSV as a stable vector that should have wide application in cell biology and vaccine development.

Normal replication of vesicular stomatitis virus without C proteins.

The expression of two small basic proteins (C and C') encoded by a second open reading frame of the New Jersey serotype of vesicular stomatitis virus (VSV) P gene was reported previously (Spiropoulou and Nichol, J. Virol., 67, 3103-3110, 1993). Here we found that the Indiana serotype virus also expressed C and C' proteins from this reading frame. We eliminated C and C' expression by making a single base change that introduced a stop codon in the C and C' coding sequence, but left the P-protein sequence unchanged. This mutated P gene supported normal replication and packaging of VSV minigenomes encoding G and M proteins. The mutated P gene was also recombined into an infectious clone of VSV that was used to recover virus. The mutant virus no longer expressed the C and C' proteins but showed growth kinetics identical to wild-type virus. The amounts of viral mRNAs and proteins synthesized were indistinguishable in mutant and wild-type virus infected cells as were the yields and composition of mutant and wild-type virus particles. The kinetics of host protein-synthesis shut-off were also identical for both viruses. Although the C and C' proteins were dispensable for VSV growth in tissue culture, they are known to be conserved in all vesiculoviruses, and thus perhaps play a role in viral pathogenesis or transmission by insect vectors.

Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles.

A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells.