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Measles virus (MV) with attenuated pathogenicity has potential as oncolytic agent. However, the clinical translation of this therapy concept has one major hurdle: the production of sufficient amounts of infectious oncolytic MV particles. The current study describes oncolytic MV production in Vero cells grown on microcarrier using serum-free medium. The impact of the number of harvests, cell concentration at infection (CCI), multiplicity of infection (MOI), and temperature on MV production was determined in different production scales/systems (static T-flasks, dynamic spinner, and bioreactor system) and modes (batch, repeated-batch, and perfusion). Cell growth, metabolic, and production kinetics were analyzed. It was found that the number of harvests had the strongest positive impact on MV yield in each production scale, and that high temperatures affected MV yield adversely. Moderate MV titers were produced in T- and spinner flasks at 37°C (∼107 TCID50 mL−1, where TCID50 is tissue culture infective doses 50%), but stirred tank reactor (STR) MV production at 37°C yielded up to 10 000-fold lower MV titers. In contrast, at lower temperatures (32°C, 27°C), 1.4 × 107 TCID50 mL−1 were achieved in the STR. Variations in MOI and CCI had almost no influence on MV production yield. The current study improves oncolytic MV production process understanding and identifies process bottlenecks for large-scale production.

Differences in fusion activity between measles virus (MV) attenuated, oncolytic strain MVNSe and pathogenic MVwt323 are reflected in amino acid 94 of the fusion (F) proteins. A valine 94 in FNSe (naturally) or Fwt323 (introduced) correlated with enhanced cell–cell fusion activity during transient glycoprotein expression or recombinant MV infections irrespective of the strains’ targeted receptors, whereas the reverse effect was found for methionine 94. Enhanced fusogenicity was determined by weaker glycoprotein interaction and correlated positively with cytotoxicity in both virus strains. Amino acid 94 of F can be used to tailor fusogenicity and cytotoxicity of recombinant MV, while the cellular receptor triggering fusion is not decisive.

Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of human immunodeficiency virus type 1 (HIV-1) Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins and HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C), and African green monkeys (agmA3C), while HIV-2, African green monkey simian immunodeficiency virus (SIVagm), and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif-triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities related to hA3C. The subtype F-1 Vif was identified to be inactive for degradation of hA3C and hA3F. The residues that determined F-1 Vif inactivity in the degradation of A3C/A3F were located in the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 with glycine and R167 with lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F. IMPORTANCE The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.

In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MV vac2 genome, and the respective viruses were rescued (MV vac2 -CoV-S and MV vac2 -CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MV vac2 -CoV-S in Vero cells turned out to be comparable to that of the control virus MV vac2 -GFP (encoding green fluorescent protein), while titers of MV vac2 -CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo , immunization of type I interferon receptor-deficient (IFNAR −/− )-CD46Ge mice with 2 × 10 5 50% tissue culture infective doses of MV vac2 -CoV-S(H) or MV vac2 -CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice. IMPORTANCE Although MERS-CoV has not yet acquired extensive distribution, being mainly confined to the Arabic and Korean peninsulas, it could adapt to spread more readily among humans and thereby become pandemic. Therefore, the development of a vaccine is mandatory. The integration of antigen-coding genes into recombinant MV resulting in coexpression of MV and foreign antigens can efficiently be achieved. Thus, in combination with the excellent safety profile of the MV vaccine, recombinant MV seems to constitute an ideal vaccine platform. The present study shows that a recombinant MV expressing MERS-S is genetically stable and induces strong humoral and cellular immunity against MERS-CoV in vaccinated mice. Subsequent challenge experiments indicated protection of vaccinated animals, illustrating the potential of MV as a vaccine platform with the potential to target emerging infections, such as MERS-CoV.

International audience; Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.

The R peptide in the cytoplasmic tail (C-tail) of γ-retroviral envelope proteins (Env) prevents membrane fusion before budding. To analyse its role in the formation of replication competent, infectious particles, we developed chimeric murine leukaemia viruses (MLV) with unmodified or R-peptide deleted Env proteins of the gibbon ape leukaemia virus (GaLV). While titres of these viruses were unaffected, R-peptide deficiency led to strongly impaired spreading. Most remarkably, we isolated an escape mutant which had restored an open reading frame for a C-terminal extension of the truncated C-tail. A reconstituted virus encoding this escape C-tail replicated in cell culture. In contrast to R-peptide deficient Env, particle incorporation of the escape Env was effective due to an enhanced protein expression and restored intracellular co-localisation with Gag proteins. Our data demonstrate that the R peptide not only regulates membrane fusion but also mediates efficient Env protein particle incorporation in γ-retrovirus infected cells.

Xenotransplantation of porcine cells, tissues, and organs shows promise to surmount the shortage of human donor materials. Among the barriers to pig-to-human xenotransplantation are porcine endogenous retroviruses (PERV) since functional representatives of the two polytropic classes, PERV-A and PERV-B, are able to infect human embryonic kidney cells in vitro , suggesting that a xenozoonosis in vivo could occur. To assess the capacity of human and porcine cells to counteract PERV infections, we analyzed human and porcine APOBEC3 (A3) proteins. This multigene family of cytidine deaminases contributes to the cellular intrinsic immunity and act as potent inhibitors of retroviruses and retrotransposons. Our data show that the porcine A3 gene locus on chromosome 5 consists of the two single-domain genes A3Z2 and A3Z3 . The evolutionary relationships of the A3Z3 genes reflect the evolutionary history of mammals. The two A3 genes encode at least four different mRNAs: A3Z2, A3Z3, A3Z2-Z3, and A3Z2-Z3 splice variant A (SVA). Porcine and human A3s have been tested toward their antiretroviral activity against PERV and murine leukemia virus (MuLV) using novel single-round reporter viruses. The porcine A3Z2, A3Z3 and A3Z2-Z3 were packaged into PERV particles and inhibited PERV replication in a dose-dependent manner. The antiretroviral effect correlated with editing by the porcine A3s with a trinucleotide preference for 5′ TGC for A3Z2 and A3Z2-Z3 and 5′ CAC for A3Z3. These results strongly imply that human and porcine A3s could inhibit PERV replication in vivo , thereby reducing the risk of infection of human cells by PERV in the context of pig-to-human xenotransplantation.

Background Lentiviral vectors allow stable gene transfer into nonreplicating cells and are increasingly used in clinical gene therapy approaches. Vectors derived from different origins can show distinct target cell transduction properties. Therefore, the construction of modern vector systems of different viral origin remains desirable. The generation of safe and efficient lentivirus-derived transfer vectors by gradual enhancing cloning steps is a time-consuming process that depends on the presence of suitable restriction sites. Multiple-step cloning protocols also enhance the risk of acquisition of mutations or other genetic instabilities. Methods We constructed novel HIV-2 and SIVsmmPBj-derived transfer vectors by amplification of three essential segments of the viral genome [5′-long terminal repeat (LTR), rev responsive element, ΔU3-3′-LTR] on the template of the lentiviral full-length genome by a highly flexible three-step fusion polymerase chain reaction approach. Further necessary vector elements, as well as a multiple cloning site, were included into the resulting vector by extension of the primer sequences. The respective vesicular stomatitis virus G pseudotyped lentiviral vector particles were generated and analysed. Results Two novel transfer vectors of different lentiviral origin were successfully generated. Titers for the corresponding SIVsmmPBj- and HIV-2-derived vectors reached up to 9.9 × 107 transforming units (TU)/ml and 1.2 × 108 TU/ml, respectively. The specific capacity to transduce primary human monocytes was maintained in both newly-generated vector systems. Conclusions We anticipate that this novel and fast way of generating any lentiviral transfer vector will improve the generation of such vectors. The HIV-2- and SIVsmmPBj-derived vectors described will prove valuable for future gene therapy strategies. Copyright © 2010 John Wiley & Sons, Ltd.

The accessory protein Vpx is encoded by lentiviruses of the human immunodeficiency virus type 2 (HIV-2) and the simian immunodeficiency SIVsm/SIVmac lineage. It is packaged into virions and is indispensable in early steps of monocyte infection. HIV-1, which does not encode Vpx, is not able to infect human monocytes, but Vpx enables infection with HIV-1. The underlying mechanism is not completely understood. In this work, we focus on Vpx-mediated intracellular postentry events as counteraction of host cell proteins. We found that Vpx binds to apolipoprotein B mRNA-editing catalytic polypeptide 3 family member A (APOBEC3A; A3A), a member of the family of cytidine deaminases, present in monocytes. This interaction led to a reduction of the steady-state protein level of A3A. A single-point mutation in Vpx (H82A) abrogated binding to A3A and single-round infection of monocytes by HIV-1. Taken together, our data indicate that lentiviral Vpx counteracts A3A in human monocytes.

The mammalian APOBEC3 (A3) proteins comprise a multigene family of cytidine deaminases that act as potent inhibitors of retroviruses and retrotransposons. The A3 locus on the chromosome 28 of the horse genome contains multiple A3 genes: two copies of A3Z1, five copies of A3Z2, and a single copy of A3Z3, indicating a complex evolution of multiple gene duplications. We have cloned and analyzed for expression the different equine A3 genes and examined as well the subcellular distribution of the corresponding proteins. Additionally, we have tested the functional antiretroviral activity of the equine and of several of the human and nonprimate A3 proteins against the Equine infectious anemia virus (EIAV), the Simian immunodeficiency virus (SIV), and the Adeno-associated virus type 2 (AAV-2). Hematopoietic cells of horses express at least five different A3s: A3Z1b, A3Z2a-Z2b, A3Z2c-Z2d, A3Z2e, and A3Z3, whereas circulating macrophages, the natural target of EIAV, express only part of the A3 repertoire. The five A3Z2 tandem copies arose after three consecutive, recent duplication events in the horse lineage, after the split between Equidae and Carnivora. The duplicated genes show different antiviral activities against different viruses: equine A3Z3 and A3Z2c-Z2d are potent inhibitors of EIAV while equine A3Z1b, A3Z2a-Z2b, A3Z2e showed only weak anti-EIAV activity. Equine A3Z1b and A3Z3 restricted AAV and all equine A3s, except A3Z1b, inhibited SIV. We hypothesize that the horse A3 genes are undergoing a process of subfunctionalization in their respective viral specificities, which might provide the evolutionary advantage for keeping five copies of the original gene.

Lentiviral vectors are potent gene transfer vehicles frequently applied in research and lately also in clinical applications. Recent improvements have come from combining lentiviral vectors with engineered envelope proteins, which now allow targeting of cell entry to any cell population of interest, as well as the transduction of quiescent cells of the haematopoietic system. We propose that measles virus envelope glycoproteins are especially well suited for this purpose because they can mediate pH-independent cell entry at the cell surface membrane and can induce cytoskeleton rearrangements that facilitate the transport of lentiviral core particles to the cell nucleus. Lentiviral vectors pseudotyped with measles virus glycoproteins are expected to improve the safety and efficacy of gene transfer to human cells.

The APOBEC3 cytidine deaminases are part of the intrinsic defense of cells against retroviruses. Lentiviruses and spumaviruses have evolved essential accessory proteins, Vif and Bet, respectively, which counteract the APOBEC3 proteins. We show here that Bet of the Prototype foamy virus inhibits the antiviral APOBEC3C activity by a mechanism distinct to Vif: Bet forms a complex with APOBEC3C without inducing its degradation. Bet abolished APOBEC3C dimerization as shown by coimmunoprecipitation and cross-linking experiments. These findings implicate a physical interaction between Bet and the APOBEC3C. Subsequently, we identified the Bet interaction domain in human APOBEC3C in the predicted APOBEC3C dimerization site. Taken together, these data support the hypothesis that Bet inhibits incorporation of APOBEC3Cs into retroviral particles. Bet likely achieves this by trapping APOBEC3C protein in complexes rendering them unavailable for newly generated viruses due to direct immobilization.

The coreceptor usage of HIV-1 envelope proteins (Env) is mainly dependent on a defined variable region within the V3-loop of Env. Thus, retroviral vectors derived from murine leukemia virus (MLV), which have been pseudotyped with HIV-1 envelope proteins holding different V3-loops, enable selective gene delivery into either CXCR4 or CCR5 positive cultured cells. Here, we tested the distribution of CD4/CCR5-tropic [MLV(HIV)]-pseudotype vectors in transgenic mice expressing CD4 and either CXCR4 or CCR5 of human origin. The specificity of gene transfer was analyzed by ex vivo transduction of spleen cells as well as after i.v. or i.p. injection of transgenic mice. Expression of the transferred marker gene EGFP and vector sequences could be detected exclusively in lymphocytes expressing (hu)CD4 and (hu)CCR5, whereas MLV vectors pseudotyped with the VSV-G envelope glycoprotein mediated gene transfer in mice of all genotypes investigated. These data demonstrated that cell-specific gene delivery via [MLV(HIV)]-pseudotyped vectors, as previously shown for cultured cells, is also achievable in vivo.

The wild-type (wt) envelope (Env) proteins of spleen necrosis virus (SNV), together with the transmembrane (TM) protein fused to antibody domains (scFv), have been used for the generation of stable packaging cell lines releasing pseudotyped cell targeting vectors derived from SNV andMurine leukemia virus(MLV). As a first step towards assessing whether HIV-1(SNV/TM-scFv) packaging cells could be established for the production of lentiviral cell targeting vectors, it is reported here that infectious HIV-1-derived particles pseudotyped with wt SNV Env proteins could be generated. Using novel chimeric SNV-derived Env proteins encompassing wt and engineered cytoplasmic domains (C-tail) of the Gibbon ape leukemia virus (GaLV) TM protein, it was further shown that the wt C-tail not only excludes the GaLV TM protein from incorporation into HIV-1 particles, but confers this phenotype to other retroviral envelopes upon C-terminal fusion.

Genome hypermutation of different orthoretroviruses by cellular cytidine deaminases of the APOBEC3 family during reverse transcription has recently been observed. Lentiviruses like HIV-1 have acquired proteins preventing genome editing in the newly infected cell. Here we show that feline foamy virus (FFV), a typical member of the foamy retrovirus subfamily Spumaretrovirinae, is also refractory to genome deamination. APOBEC3-like FFV genome editing in APOBEC3-positive feline CRFK cells only occurs when the accessory FFV Bet protein is functionally inactivated. Editing of bet -deficient FFV genomes is paralleled by a strong decrease in FFV titer. In contrast to lentiviruses, cytidine deamination already takes place in APOBEC3-positive FFV-producing cells, because edited proviral DNA genomes are consistently present in released particles. By cloning the feline APOBEC3 orthologue, we found that its homology to the second domain of human APOBEC3F is 48%. Expression of feline APOBEC3 in APOBEC3-negative human 293T cells reproduced the effects seen in homologous CRFK cells: Bet-deficient FFV displayed severely reduced titers, high-level genome editing, reduced particle release, and suppressed Gag processing. Although WT Bet efficiently preserved FFV infectivity and genome integrity, it sustained particle release and Gag processing only when fe3 was moderately expressed. Similar to lentiviral Vif proteins, FFV Bet specifically bound feline APOBEC3. In particles from Bet-deficient FFV, feline APOBEC3 was clearly present, whereas its foamy viral antagonist Bet was undetectable in purified WT particles. This is the first report that, in addition to lentiviruses, the foamy viruses also developed APOBEC3-counter-acting proteins.

To achieve specific gene transfer into human CD4(+) cells, murine leukaemia virus (MLV)-based pseudotype vector particles were generated employing Env variants derived from human or simian immunodeficiency virus (HIV-1 or SIVagm). Here, we describe the generation of full-length onco/lentivirus hybrid genomes comprising components of MLV and HIV-1 or SIVagm, respectively, to assess the possibility of replication-competent hybrid virus formation. The env reading frame of an infectious molecular clone of MLV was replaced with the analogous coding regions of HIV-1 or SIVagm encompassing the env gene and accessory genes. Resulting MLV/HIV-1 or MLV/SIVagm hybrid genomes were transfected into 293T cells. Expression of viral proteins and budding of retroviral particles was shown by specific immunostaining and electron microscopy. The viral particles mediated CD4- and co-receptor-specific infection of human cells as demonstrated by PCR and immunostaining in the respective target cells. However, no productive infection resulting in the generation of infectious virus was detected in these cells. Thus, these onco/lentivirus hybrids, although able to initiate single-round infection, were not replication competent. Thus, MLV-based pseudotype vectors carrying Env variants of HIV-1 or SIVagm are not prone to form replication-competent retroviruses, suggesting a favourable safety profile for MLV-based CD4-specific pseudotype vectors.

Retroviral vectors derived from murine leukemia virus (MLV) have been pseudotyped with a variant of the envelope glycoprotein (Env) of nonpathogenic simian immunodeficiency virus from African green monkeys (SIVagm) to result in [MLV(SIVagm-wt)] vector particles. The variant env gene encodes a full-length surface envelope glycoprotein (SU) and a C-terminally truncated transmembrane protein (TM). To change the coreceptor usage of this vector from CCR5 to CXCR4, which is predominant on human CD4-positive lymphocytes, the putative V3-loop of SIVagm SU was replaced by that of the T cell tropic HIV-1 variant BH10. The resulting [MLV(SIVagm-X4)] vectors were shown to specifically transduce CD4/CXCR4-positive cell lines, demonstrating the equivalent function in cell entry and choice of coreceptor usage of the V3-loops of SIVagm and HIV-1. These modified vectors were able to transduce primary human lymphocytes and were resistant to neutralization by sera from HIV-1-infected individuals. The [MLV(SIVagm-X4)] pseudotype vector generated is thus a promising candidate vector, e.g., for in vivo gene therapy of HIV-1 infection.

The improvement of gene transfer efficiency in growth-arrested cells using human immunodeficiency virus type 1 (HIV-1)-derived vectors led to the development of vectors derived from other members of the lentivirus family. Here we report the generation of a lentiviral vector derived from the apathogenic molecular virus clone SIVagm3mc of the simian immunodeficiency virus from African green monkeys (Cercocebus pygerythrus). Upon pseudotyping with the G-protein of vesicular stomatitis virus (VSV-G), the SIVagm-derived vector was shown to transduce proliferating and growth-arrested mammalian cell lines, including human cells. After in vivo inoculation into the striatum of the adult rat brain, the vector was shown to transduce terminally differentiated neurons and oligodendrocytes as well as quiescent and reactive astrocytes. Moreover, SIVagm transfer vector mRNA was efficiently packaged by HIV-1 vector particles. Homologous [SIV(SIV)] vectors generated by using the SIVagm-derived envelope glycoproteins allowed selective gene transfer into human CD4+/CCR5+ cells. Thus, the SIVagm3mc-derived vector is a useful alternative to HIV-1-derived lentiviral vectors in somatic gene therapy.

In contrast to murine leukaemia virus (MLV)-derived vector systems, vector particles derived from the avian spleen necrosis virus (SNV) have been successfully targeted to subsets of human cells by envelope modification with antibody fragments (scFv). However, an in vivo application of the SNV vector system in gene transfer protocols is hampered by its lack of resistance against human complement. To overcome this limitation we established pseudotyping of MLV vector particles produced in human packaging cell lines with the SNV envelope (Env) protein. Three variants of SNV Env proteins differing in the length of their cytoplasmic domains were all efficiently incorporated into MLV core particles. These pseudotype particles infected the SNV permissive cell line D17 at titers of up to 10(5) IU/ml. A stable packaging cell line (MS4) of human origin released MLV(SNV) pseudotype vectors that were resistant against human complement inactivation. To redirect their tropism to human T cells, MS4 cells were transfected with the expression gene encoding the scFv 7A5 in fusion with the transmembrane domain (TM) of the SNV Env protein, previously shown to retarget SNV vector particles to human lymphocytes. MLV(SNV-7A5)-vector particles released from these cells were selectively infectious for human T cell lines. The data provide a proof of principle for targeting MLV-derived vectors to subpopulations of human cells through pseudotyping with SNV targeting envelopes.

One of the strategies used to alter the tropism of retroviral vectors involves the substitution of their envelope glycoproteins with those of other viruses. This results in the generation of pseudotype vector particles exhibiting properties that may be advantageous for gene transfer into certain human cells. Moreover, when the envelope glycoproteins from simian and human immunodeficiency viruses were used to pseudotype Murine Leukemia Virus (MLV), the resulting vectors mediated selective gene transfer into defined human cell types. Enhanced transduction efficacy and cell targeting via retroviral pseudotype vectors may be important prerequisites for future gene therapy applications.

Retroviral vectors derived from amphotropic murine leukemia viruses (MLV) mediate gene transfer into almost all human cells and are thus not suitable for in vivo applications in gene therapy in which cell-specific gene delivery is required. We and others recently reported the generation of MLV-derived vectors pseudotyped by variants of the envelope glycoproteins (Env) of human immunodeficiency virus type 1 (HIV-1), thus displaying the CD4-dependent tropism of the parental lentivirus (Mammano et al., 1997, J. Virol. 71, 3341–3345; Schnierle et al., 1997, Proc. Natl. Acad. Sci. USA 76, 8640–8645). However, because of their HIV-1-derived envelopes these vectors are neutralized by HIV-specific antibodies present in some infected patients. To circumvent this problem, we pseudotyped MLV capsid particles with variants of Env proteins derived from the apathogenic simian immunodeficiency virus (SIVagm) of African green monkeys (AGM; Chlorocebus pygerythrus ). Truncation of the C-terminal domain of the transmembrane protein was found to be necessary to allow formation of infectious pseudotype vectors. These [MLV(SIVagm)] vectors efficiently transduced various human CD4-expressing cell lines using the coreceptors CCR5 and Bonzo to enter target cells. Moreover, they were resistant to neutralization by antibodies directed against HIV-1. Therefore, [MLV(SIVagm)] vectors will be useful to study the mechanisms of SIVagm cell entry and for the selective gene transfer into CD4+ T-cells of AIDS patients.

Insertional mutagenesis resulting in a leukaemia-like lymphoproliferative disease, as observed in the X-SCID (severe combined immunodeficiency) clinical trial using a gamma-retroviral vector that transferred a functional copy of the defective gene into hematopoietic precursor cells of affected children, sparked a debate about a ban on conventional gamma-retroviral vectors. This commentary summarizes the relevant data on this topic and concludes that there is no preclinical or clinical evidence as yet that SIN vectors, which self-inactivate the retroviral long terminal repeats (LTRs), will indeed show an improved safety profile. Conventional murine leukaemia virus (MLV) vectors can thus be used further in clinical gene therapy trials but require a thorough case-by-case risk-benefit analysis.

CD4-expressing T cells in lymphoid organs are infected by the primary strains of HIV and represent one of the main sources of virus replication. Gene therapy strategies are being developed that allow the transfer of exogenous genes into CD4 + T lymphocytes whose expression might prevent viral infection or replication. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. Major determinants of the tropism of infection are the CD4 molecules on the surface of the target cells and the viral envelope glycoproteins at the viral surface. The best characterized and most widely used gene transfer vectors are derived from Moloney murine leukemia virus (MuLV). To generate MuLV-based retroviral gene transfer vector particles with specificity of infection for CD4-expressing cells, we attempted to produce viral pseudotypes, consisting of MuLV capsid particles and the surface (SU) and transmembrane (TM) envelope glycoproteins gp120-SU and gp41-TM of HIV type 1 (HIV-1). Full-length HIV-1 envelope glycoproteins were expressed in the MuLV env -negative packaging cell line TELCeB6. Formation of infectious pseudotype particles was not observed. However, using a truncated variant of the transmembrane protein, lacking sequences of the carboxyl-terminal cytoplasmic domain, pseudotyped retroviruses were generated. Removal of the carboxyl-terminal domain of the transmembrane envelope protein of HIV-1 was therefore absolutely required for the generation of the viral pseudotypes. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4 + cells during gene therapy of AIDS.

Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancers. Many naturally occurring viruses have a preferential, although nonexclusive, tropism for tumors and tumor cells. In addition, specific targeting of cancer cells can be achieved at the virus entry level. We optimized retargeting of cell entry by elongating the measles virus attachment protein with designed ankyrin repeat proteins (DARPins), while simultaneously ablating entry through the natural receptors. DARPin-targeted viruses were strongly attenuated in off-target tissue, thereby enhancing safety, but completely eliminated tumor xenografts. Taking advantage of the unique properties of DARPins of being fused without generating folding problems, we generated a virus simultaneous targeting two different tumor markers. The bispecific virus retained the original oncolytic efficacy, while providing proof of concept for a strategy to counteract issues of resistance development. Thus, DARPin-targeting opens new prospects for the development of personalized, targeted therapeutics.

Recombinant measles viruses are currently tested in clinical trials as oncolytic agent to be applied in cancer therapy. Contrary to their use as vaccine where 103infectious virus particles per dose are needed, for cancer therapy 109virus particles should be provided per dose. This leads to other challenges for the production process when compared to vaccine production. This study presents measles virus stability with regard to conditions during production and storage of the virus. Relevant process parameters such as temperature (4-37��C), pH (pH 4-11), conductivity (1.5 to 137.5 mS cm-1) and oxygen partial pressure were analyzed. The infectivity of measles virus particles decreased highly at 37 and 32��C, while at 22 and 4��C it remained stable for several hours or even days, respectively. The thermal inactivation reactions followed first order kinetics and the thermodynamic parameters enthalpy and entropy were estimated. Towards changes in pH measles virus particles were very sensitive, while no inactivation could be observed with varying conductivity. Measles virus incubation at an oxygen partial pressure of 100% did not lead to any loss of infectivity. The results show which parameters should be considered and controlled strongly in the production process to further raise measles virus yields for the high amount needed in cancer therapy approaches. �� 2013 Science Publication.

Attenuated measles virus has revealed selective tum or cell killing and is currently tested in clinical trials for the therapy of cancer patients. The amount of i nfectious particles per dose needed for oncolytic therapy can be more than a million times higher compared to vaccination. This requires highly effectiv e production processes which guarantee the measles vi rus quantities needed for its use in cancer therapy . Referring to measles virus production itself, sever al factors are influencing process parameters and subsequent virus yields. These factors are medium o ptimization, feeding of nutrients, an optimal multiplicity of infection, localization of produced virus particles, temperature sensitivity and time of harvest. This review summarizes the available data concerning measles virus production in cell culture and factors influencing the virus yield.

Oncogenic retroviruses are generated by transduction of the coding region of a protooncogene and acquire genetic changes during subsequent replication. Critical genetic events which occurred during and after transduction of rat proto-ras-1Ha into Harvey sarcoma virus were identified by evaluating the transforming activity of plausible synthetic progenitor proviruses encompassing the complete proto-ras genomic region with or without various 5' deletions. All progenitor proviruses induced phenotypic transformation of mouse NIH 3T3 cells, although with a 5- to 10-fold lower frequency than Harvey sarcoma provirus. Although no tumor formation was observed in vivo after inoculation in the absence of helper murine retrovirus, both wild-type and progenitor viruses inoculated in the presence of helper virus induced tumors in newborn BALB/c mice. No critical alterations of the p21ras coding region and no deletion of 5' genomic elements were detected in a progenitor virus encompassing the complete proto-ras genomic region that had been isolated from tumors. However, one progenitor virus that included all proto-ras exons induced tumors with a decreased latency. This virus contained a mutation in codon 12 (glycine to valine), which had apparently been selected during tumorigenesis in vivo. During the genesis of Harvey sarcoma virus, critical steps conferring transforming function are therefore transduction of coding proto-ras exons and enhancement of their transforming function by specific amino acid changes in p21ras.

Primary and secondary cancers of the liver are a significant health problem with limited treatment options. We sought here to develop an oncolytic measles virus (MV) preferentially activated in liver tumor tissue, thus reducing infection and destruction of healthy tissue. We documented that in primary tumor tissue, urokinase-type plasminogen activator and especially matrix metallproteinase-2 (MMP-2) are significantly more active than in adjacent nontumorous tissue. We then generated variants of the MV fusion protein by inserting different MMP substrate motifs at the protease cleavage site and identified the motif PQGLYA as the most efficient cleavage site as determined by syncytia formation on protease-positive tumor cells. The corresponding MMP-activatable oncolytic MV-MMPA1 virus was rescued and shown to be strongly restricted on primary human hepatocytes and healthy human liver tissue, while remaining as effective as the parental MV in the tumor tissue sections. Our findings underline the clinical potency of the MMP activation concept as a strategy to generate safer oncolytic viruses for the treatment of primary and secondary cancers of the liver. Cancer Res; 70(19); 7620–9. ©2010 AACR.

We pseudotyped HIV-1 vectors with cytoplasmic tail-truncated envelope glycoproteins of a wild-type (WT) measles virus (MV). The particles entered the lymphatic cells exclusively through the signaling lymphocyte activation molecule (SLAM, CD150), whereas particles pseudotyped with the MV vaccine strain glycoproteins also recognized the ubiquitous membrane cofactor protein (CD46) as receptor and had less specific cell entry. MV(WT)-HIV vectors reached titers of 10(8) t.u. ml(-1), which were up to 10-fold higher than those of MV(Vac)-HIV vectors, and discriminated between SLAM-positive and SLAM-negative cells, also in mixed cell cultures. As these vectors transduce primary human cells more efficiently than vesicular stomatitis virus-G pseudotyped vectors do, they are promising candidates for gene transfer to human lymphocytes and certain epithelial cells.

A primary safety issue presented by human hematopoietic stem cells/progenitor cells (HS/PC) genetically modified by gammaretroviral or lentiviral vectors is the risk of oncogenesis. This risk is a potential consequence of either of the following events: (a) the possible unintended generation of replication-competent vector-derived viruses (replication-competent retrovirus, RCR; replication-competent lentivirus, RCL) leading to neoplasia due to RCR/RCL infection of target and nontarget cells in vivo, or (b) intended vector integration in the chromosomal DNA of the target somatic cells leading to neoplasia due to insertional mutagenesis. These risks should be addressed in nonclinical and clinical studies. In the US and the EU, a combination of regulations and guidance documents are available to investigators and sponsors of gene therapy clinical trials. Guidance documents provide a facile way to adapt regulatory recommendations, in line with the changing state of the art in medical science. In the field of retroviral vectors, a number of innovations are being tested in nonclinical or clinical investigations, and each of these will raise their own regulatory issues. Some recent examples of these types of innovations include development of novel vector structures to minimize risks associated with vector integration, such as lentiviral vectors currently used in clinical trials for HS/PC modification that have been designed with deletions of the strong retroviral enhancer associated with oncogenesis.

Retargeting of lentiviral vector entry to cell types of interest is a key factor in improving the safety and efficacy of gene transfer. In this study we show that the retargetable envelope glycoproteins of measles virus (MV), namely, the hemagglutinin (H) responsible for receptor recognition and the fusion protein (F), can pseudotype human immunodeficiency virus 1 (HIV-1) vectors when their cytoplasmic tails are truncated. We then pseudotyped HIV-1 vectors with MV glycoproteins displaying on H either the epidermal growth factor or a single-chain antibody directed against CD20, but without the ability to recognize their native receptors. Gene transfer into cells that expressed the targeted receptor was several orders of magnitude more efficient than into cells that did not. High-target versus nontarget cell discrimination was demonstrated in mixed cell populations, where the targeting vector selectively eliminated CD20-positive cells after suicide gene transfer. Remarkably, primary human CD20-positive B lymphocytes were transduced more efficiently by the CD20-targeted vector than by a vector pseudo-typed with the vesicular stomatitis virus G (VSV-G) protein. In addition, the CD20-targeted vector was able to transduce even unstimulated primary B cells, whereas VSV-G pseudotyped vectors were unable to do so. Because MV enters cells through direct fusion at the cell membrane, this novel targeting system should be widely applicable.

Virotherapy is currently being developed for many different types of viruses including replication-competent murine leukaemia virus (MLV) as a novel tool in cancer therapy. However, there is the risk of insertional mutagenesis associated with this virus, making careful preclinical studies necessary before its first application in man. We have previously generated conditionally replication-competent MLV variants that require activation by tumour-associated proteases to become infectious. Here we analysed in a comparative study the spreading of non-targeted and of such tumour-targeted MLV variants to tumour and extratumoural organs in immunodeficient mice. Both virus types were able to efficiently infect tumour cells after systemic administration. The non-targeted virus, however, also infected extratumoural organs like bone marrow, spleen and liver efficiently. In contrast, the targeted viruses revealed in a quantitative analysis of virus spreading an up to 500-fold more selective infection of tumour tissue than the non-targeted virus. The data raise serious doubts about a safe clinical use of non-targeted MLV. Engineering the virus to become activatable by tumour-associated proteases can significantly improve the safety of MLV.

Retroviral vectors have yet not been tested for their potential as vaccines despite their frequent utilization in gene therapy allowing for highly efficient gene transfer into a number of cell types and their suitability for large-scale production in biotechnology. To investigate MLV-based vectors suitability for inducing immune response against HIV-1-antigens, we generated a MLV(HIV-1) pseudotype vector enabling CD4-specific transduction of HIV-1 genes env, vpu , tat and rev originating from the pathogenic SHIV-89.6P . Functional expression of the lentiviral genes in packaging cells, human and rhesus CD4+ target cells was demonstrated by various assays. Following highly efficient ex vivo transduction, up to 3.4 × 10 7 autologous, transfer vector-positive rhesus peripheral blood mononuclear cells (rhPBMCs) were re-inoculated into a rhesus macaque. Five weeks after the initial inoculation HIV-1 Env-specific antibodies were detected using ELISA. ELIspot-assay revealed the induction of a HIV-1 Rev and Env-specific CTL-response 7.5 weeks after immunization. Thus, these novel MLV(HIV-1) vectors facilitate efficient transduction and subsequent expression of HIV-1-genes in CD4-positive host cells. Induction of both humoral and cellular HIV-1-specific immune responses in vivo confirmed their potential as an effective HIV-1 vaccine to be further studied in SHIV/rhesus macaque model of lentivirus infection.

Viruses conditionally replicating in cancer cells form an attractive novel class of antitumoral agents. To engineer such viruses infectivity can be coupled with proteolytic activity of the target cell by modifying the envelope (Env) protein of murine leukaemia virus (MLV) with blocking domains that prevent cell entry unless they are cleaved off by tumour-associated proteases like the matrix metalloproteases (MMP). Here we show that MLV variants selectively spreading through MMP-positive cells can be evolved from virus libraries, in which a standard MMP-2 substrate peptide connecting the blocking domain CD40L with the Env protein was diversified. Passaging the virus library on human fibrosarcoma or glioma cell lines resulted in the selection of about 10 virus clones, of which the three most frequent ones were shown to become activated by MMPs and to be replication competent on MMP-positive cells only. On these cells, the selected linker peptides improved the spreading by several orders of magnitude in vitro, as well as in tumour xenografts in vivo, approaching the kinetic of the unmodified wild-type virus. The data suggest that retroviral protease substrate libraries form a potent tool for the engineering of viruses conditionally replicating in a given cancer cell type of interest.

The successful application of cancer gene therapy has been hampered by the low efficiency of in vivo gene delivery by currently used replication-defective vectors. Accordingly, considerable efforts are now being directed toward development and use of vectors capable of replicating in cancer cells. However, for replicating retroviruses, insertion of additional reading frames into the viral genome often resulted in the generation of unstable viruses. Here, we report a novel concept for the generation of replication-competent murine leukemia virus (MLV) vectors capable of mediating the secretion of soluble therapeutic proteins from infected cells. As a proof of principle, we inserted transgene regions encoding either a single-chain variable region fragment (scFv), here, the laminin-specific L36-scFv, or the T-cell-specific 7A5-scFv, or the cytokine GM-CSF into the MLV envelope (env) gene after +1 codon of the envelope (Env) protein, followed by a sequence specifying a furin protease cleavage site. The resulting viruses, termed L36-furin-A, 7A5-furin-A and GMCSF-furin-Mo, respectively, infected a variety of human cell lines, including HMEC-1 (endothelial), A301 (lymphoid), MDA-MB231 and MDA-MB468 (breast cancer) and HT1080 (fibrosarcoma) cells. Western blot analysis of conditioned culture medium from HT1080 cells infected by replicating L36-furin A, as an example, revealed that more than 90% of the Env fusion protein molecules were indeed intracellularly cleaved. After 5 days of infection, up to 3-4 mug/ml of soluble L36-scFv accumulated in the supernatant of HT1080 cells. The eukaryotically produced L36-scFv and 7A5-scFv were able to recognize their native antigens with high avidity, as assessed by ELISA and flow cytometry. Furthermore, the replicating viruses were genetically stable for more than 12 cell passages. In conclusion, a new generation of replication-competent retroviral vectors capable of mediating long-term and efficient secretion of therapeutic proteins suitable for cancer therapy was generated.

In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.

Top of pageAbstract Retroviral vectors derived from murine leukemia virus (MLV) and pseudotyped with the HIV-1 envelope (Env) protein enable specific gene transfer into CD4+ cells and may therefore be suitable for in vivo gene transfer for the treatment of various inherited or acquired disorders involving T cells including severe combined immunodeficiency disease (SCID), AIDS or T cell lymphoma. In cell culture, MLV(HIV) vectors are not only specific for CD4, but also for HIV coreceptors CCR5 or CXCR4, depending on the particular HIV Env protein used for pseudotyping. Here we tested the in vivo distribution of a CD4/CCR5-specific MLV(HIV) pseudotype vector transferring the EGFP gene in a transgenic mouse strain expressing human CD4 and CCR5 under control of the murine CD4 promoter. At first, gene transfer specificity was demonstrated by ex vivo transduction of a mixed population of spleen lymphocytes. Only cells from double transgenic CD4+/CCR5+ mice were transduced by CD4/CCR5-tropic vectors, whereas gene transfer into lymphocytes from single transgenic CD4+or CCR5+ mice or double transgenic CD4+/CXCR4+ mice was not detectable. Subsequently, MLV(HIV) vectors were administered intravenously or intraperitoneally, and various tissues were analyzed for successful vector transfer. EGFP expression and vector sequences could be detected exclusively in lymphocytes from CD4+/CCR5+ mice, but neither in CD4+/CXCR4+mice nor in single transgenic CD4+, CXCR4, or CCR5+mice. In contrast, when using MLV control vectors pseudotyped with VSV-G, EGFP-expressing lymphocytes were readily detected in mice of all genotypes described.In summary, the suitability of MLV(HIV) vector particles for in vivo gene transfer and their specific tropism for CD4+/CCR5+ lymphocytes was demonstrated.

The chemokine interferon-inducible protein 10 (IP-10) is proposed to have antitumor activity in vivo on the basis of its immunomodulatory and antiangiogenic effects. However, the utility of this angiostatic molecule in cancer therapy is hampered by difficulties in manufacturing large quantities of recombinant proteins and the cumbersome administration schedules. This study is aimed to explore IP-10 gene-based anticancer therapy using replicating murine leukemia virus (MLV) vectors, the latter used in order to achieve sustained and efficient delivery of this protein in tumors.

Viruses conditionally replicating in cancer cells form an attractive novel class of anti-tumoural agents. To engineer such viruses we couple infectivity with proteolytic activity of the target cell by modifying the envelope (Env) protein of murine leukemia virus (MLV) with blocking domains that prevent cell entry unless they are cleaved off by tumor-associated proteases like the matrix metalloproteases (MMP).

replication in the murine cells of these mice in vivo. We are measuring the quality of the cellular and humoral immune responses to different forms and amounts of the SIV-smmPBj1.9 Env proteins presented by MV in mice. Conclusion These studies will be used to select optimal candidate bivalent MV-SIV vaccines for the analysis of the immune response and levels of protection in monkeys.

APOBEC3 (A3, Apolipoprotein B mRNA-editing catalytic polypeptide 3) genes in the genome of domestic cat (Felis catus) were identified and characterized, Background Over the past years a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly, and/or cross-species transmission. Among these host-encoded restriction factors, the APOBEC3 (A3; apolipoprotein B mRNA-editing catalytic polypeptide 3) proteins are potent inhibitors of retroviruses and retrotransposons. While primates encode seven of these genes (A3A to A3H), rodents carry only a single A3 gene. Results Here we identified and characterized several A3 genes in the genome of domestic cat (Felis catus) by analyzing the genomic A3 locus. The cat genome presents one A3H gene and three very similar A3C genes (a-c), probably generated after two consecutive gene duplications. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by read-through alternative splicing. Specific feline A3 proteins selectively inactivated only defined genera of feline retroviruses: Bet-deficient feline foamy virus was mainly inactivated by feA3Ca, feA3Cb, and feA3Cc, while feA3H and feA3CH were only weakly active. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. Within Felidae, A3C sequences show significant adaptive selection, but unexpectedly, the A3H sequences present more sites that are under purifying selection. Conclusion Our data support a complex evolutionary history of expansion, divergence, selection and individual extinction of antiviral A3 genes that parallels the early evolution of Placentalia, becoming more intricate in taxa in which the arms race between host and retroviruses is harsher.

We have determined the entire nucleotide sequence of a full-length molecular clone, termed SIVagm3, which is infectious in vitro and in vivo. The genomic organization was found to be similar to other immunodeficiency viruses of human and simian origin. Comparison of SIVagm3 with SIVagmTYO-1, the only other completely sequenced molecular SIVagm clone, revealed a novel type of intragroup divergence, which is characterized by (1) an unusually high degree of variability in pol in relation to gag and env and (2) a high degree of divergence in the rev and tat genes. Thus, since SIVagm3 and SIVagmTYO-1 evolved from their common ancestor, they diverged in a different manner than human immunodeficiency viruses. Hypervariable regions in env were defined and shown to be relatively restricted in comparison to HIV-1 and HIV-2.

Top of pageAbstract Viruses conditionally replicating in cancer cells form an attractive novel class of anti-tumoural agents. To engineer such viruses we couple infectivity with proteolytic activity of the target cell by modifying the envelope (Env) protein of murine leukemia virus (MLV) with blocking domains that prevent cell entry unless they are cleaved off by tumor-associated proteases like the matrix metalloproteases (MMP). Here we show that MLV variants selectively spreading through MMP-positive cells can be evolved from virus libraries, in which a standard MMP-2 substrate peptide connecting the blocking domain CD40L with the Env protein was diversified. Passaging the virus library on a human fibrosarcoma cell line resulted in the selection of about 10 virus clones of which the two most frequent ones were reconstituted as molecular virus clones and characterized. While the origin of the activating protease for the AKGLYK linker peptide encompassing virus remained elusive, the PSGLYQ linker peptide encompassing virus was cleaved by MMP-2 in vitro and could be inhibited by a broad-range MMP inhibitor. Yet, both linker peptides conferred a remarkable advantage in the spreading through MMP-positive tumor cell lines. The in vivo spreading behavior of these viruses was monitored in SCID mice transplanted with HT1080 tumor cells. In a first experimental setting we applied mixtures of HT1080 cells infected with the selected viruses or the parental virus, respectively, and uninfected HT1080 cells. The AKGLYK linker peptide encompassing virus was able to spread through the complete tumor mass within two weeks when 0.2% infected cells were injected. The parental virus, in contrast, reverted to wildtype virus within the same period by deletion of the blocking domain, which is most likely enhanced by its low spreading efficiency. Similar results were obtained when the selected viruses were injected intratumorally into established HT1080 tumor tissue. Moreover, initial biodistribution data indicate, that the selected viruses, in contrast to the wildtype virus, show a clear preference for tumour tissue. Thus, the growth advantage of MMP-activatable viruses engineered by directed evolution observed in vitro was confirmed in vivo in mouse tumor models. The data suggest that retroviral protease substrate libraries form a potent tool for the engineering of viruses conditionally replicating in a given cancer cell type.

Preclinical evaluation in a small animal model would help the development of gene therapies and vaccines based on foamy virus vectors. The establishment of persistent, non-pathogenic infection with the prototype foamy virus in mice and rabbits has been described previously. To extend this spectrum of available animal models, hamsters were inoculated with infectious cell supernatant or bioballistically with a foamy virus plasmid. In addition, a novel foamy virus from a rhesus macaque was isolated and characterised genetically. Hamsters and mice were infected with this new SFVmac isolate to evaluate whether hamsters are also susceptible to infection. Both hamsters and mice developed humoral responses to either virus subtype. Virus integration and replication in different animal tissues were analysed by PCR and co-cultivation. The results strongly indicate establishment of a persistent infection in hamsters. These studies provide a further small animal model for studying FV-based vectors in addition to the established models.

Lentiviral vectors pseudotyped with the envelope glycoproteins (Env) of amphotropic murine leukemia virus (MLV) and the G protein of vesicular stomatitis virus (VSV-G) have been successfully used in recent preclinical gene therapy studies. We report here the generation of infectious HIV-1-derived vector particles pseudotyped with the Env of the molecular clone 10A1 of MLV and with chimeric envelope glycoprotein variants derived from gibbon ape leukemia virus (GaLV) and MLV. Formation of infectious HIV-1 (GaLV) pseudotype vectors was only possible with the substitution of the cytoplasmic tail of GaLV Env with that of MLV. The lentiviral vectors exhibited a host cell range identical with that of MLV(GaLV) and MLV(10A1) vectors, which are known to enter cells either via the GaLV-receptor Glvr-1 (Pit-1) or via the amphotropic receptor Ram-1 (Pit-2) in addition to Glvr-1, respectively. Thus, HIV-1(GaLV) and HIV-1(10A1) pseudotype vectors may be useful for efficient gene transfer into a variety of human tissues like primary human hematopoietic cells.

SIVagm does not induce disease in its African green monkey (AGM) host. In comparison, the hybrid simian-human immunodeficiency virus SHIV89.6P that carries the HIV env gene induces disease in rhesus macaques more rapidly than the SIVmac parent virus. To address the possibility that this enhancement of disease by HIV env would also occur when present in SIVagm, a full-length SIVagm/89.6Penv chimeric lentivirus genome (termed SHIV-MP) was constructed. SHIV-MP replicated similarly to SIVagm in simian peripheral blood mononuclear cells (PBMCs). In inoculated AGMs, rhesus macaques and pig-tailed (PT) macaques the absolute number of CD4+ T lymphocytes remained at normal levels. The peak levels of productively infected cells in SHIV-MP-infected monkeys ranged from 101 to 102 per 106 PBMCs, while in SIVagm infected macaques the levels were 10–100-fold higher. The env gene of SHIV89.6P therefore appears insufficient to confer acute pathogenicity to a non-pathogenic primate lentivirus due to poor in vivo replication.

Cellular cytidine deaminases from the APOBEC3 family are potent restriction factors that are able to block the replication of retroviruses. Consequently, retroviruses have evolved a variety of different mechanisms to counteract inhibition by APOBEC3 proteins. Lentiviruses such as human immunodeficiency virus (HIV) express Vif, which interferes with APOBEC3 proteins by targeting these restriction factors for proteasomal degradation, hence blocking their ability to access the reverse transcriptase complex in the virions. Other retroviruses use less-well-characterized mechanisms to escape the APOBEC3-mediated cellular defense. Here we show that the prototype foamy virus Bet protein can protect foamy viruses and an unrelated simian immunodeficiency virus against human APOBEC3G (A3G). In our system, Bet binds to A3G and prevents its encapsidation without inducing its degradation. Bet failed to coimmunoprecipitate with A3G mutants unable to form homodimers and dramatically reduced the recovery of A3G proteins from soluble cytoplasmic cell fractions. The Bet-A3G interaction is probably a direct binding interaction and seems to be independent of RNA. Together, these data suggest a novel model whereby Bet uses two possibly complementary mechanisms to counteract A3G: (i) Bet prevents encapsidation of A3G by blocking A3G dimerization, and (ii) Bet sequesters A3G in immobile complexes, impairing its ability to interact with nascent virions.

Background The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). Results Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. Conclusions In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.