Enhanced gene expression of the murine ecotropic retroviral receptor and its human homolog in proliferating cells

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR).Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo r ), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo r alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo r transduction of primary human CD34 ؉ hematopoietic progenitor cells from normal bone marrow was alsodocumented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34 ؉ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo r alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV-and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.

Section: Aav-mediated Transduction Leads To Stable Integration and Losupporting

confidence: 90%

“…The ecoR has been cloned and characterized in the mouse and rat (1,15,22,42). It has been demonstrated to function as the basic amino acid and phosphate transporter, and it has a wide but variable tissue distribution (15,18,40,42,43).…”

mentioning

confidence: 99%

Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells

Qing

Bachelot

Mukherjee

et al. 1997

“…The expression of the rec-1 gene has been shown to vary significantly depending on the differentiation and proliferation states of hepatocytes (8,49) and lymphocytes (45,50). It is likely that cell differentiation, and presumably cell proliferation, also influences the expression of the amphotropic receptor, as suggested by the absence of detectable receptors on mouse fetal liver cells whereas adult hematopoietic tissues express significant levels of the molecule (37).…”

Section: Discussionmentioning

confidence: 99%

Receptor-binding properties of a purified fragment of the 4070A amphotropic murine leukemia virus envelope glycoprotein

Battini

Rodrigues

Müller

et al. 1996

A 208-amino-acid amino-terminal fragment of the 4070A amphotropic murine leukemia virus envelope glycoprotein contains all of the determinants required to recognize cell surface amphotropic receptors. This fragment was fused with a streptavidin-binding tag, expressed in Sf9 insect cells by using a baculovirus vector, and purified to homogeneity. The 125 I-labeled purified fragment (AS208) specifically bound various cell lines susceptible to amphotropic murine leukemia virus infection. The number of AS208-binding sites was in the range of 7 ؋ 10 4 to 17 ؋ 10 4 per cell. Quantitative analysis of binding revealed that AS208-binding sites are heterogeneous with regard to ligand binding affinity or that cooperativity exists between receptors. Competition experiments showed that the concentration of AS208 required to inhibit virus entry was lower than that required to inhibit the binding of virus particles at the cell surface. Taken together, these data suggested that amphotropic envelope-binding sites present at the cell surface do not act independently and do not participate equally in virus infection.

“…They demonstrated that the gene coding for the Ecor is not expressed in the livers of adult mice (31). However, the expression of this gene is enhanced in rapidly proliferating cells of hematopoietic origin (54), which suggests that Ecor plays a role in cellular proliferation. Since retroviruses infect only dividing cells, it is possible that cell cycle-dependent expression of the genes that code for viral receptors might be a significant factor for infectibility of host cells.…”

mentioning

confidence: 99%

Hormonal regulation of the gene for the type C ecotropic retrovirus receptor in rat liver cells

Robinson

Kung

et al. 1994

The infectibility of the regenerating rat liver by ecotropic retroviruses was studied relative to the expression of the gene coding for the ecotropic retrovirus receptor (Ecor) that functions as a cationic amino acid transporter. It is known that the gene for the receptor is expressed in primary hepatocytes and hepatoma cells but is absent in adult liver cells. Isolation of a 2.85-kb cDNA for the rat Ecor suggested that the rat viral receptor is 97% homologous to the mouse viral receptor and that it contains the envelope-binding domain that determines the host range of ecotropic murine retroviruses. This explains the efficient infection of rat cells by ecotropic retroviruses. Since cell division is required for liver cells to be infected, we determined the susceptibility of the regenerating rat liver to infection at different time points after partial hepatectomy (0 to 24 h) in relation to the presence of receptor mRNA. Infection of the liver occurred only when the liver was exposed to virus 4 h after partial hepatectomy. This time course of infection paralleled expression of the gene for the Ecor, which was rapidly induced between 2 and 6 h during liver regeneration. However, expression of the dormant receptor gene in quiescent liver cells can be induced by insulin, dexamethasone, and arginine, indicating that cell division is not required for expression of the receptor gene in liver cells. A diet high in carbohydrate (low in protein) significantly increased the concentration of receptor mRNA in liver cells, indicating that hormones play a role in the regulation of expression of this gene in vivo. We conclude that the gene for the viral receptor is expressed in the regenerating and quiescent liver when the urea cycle enzymes are down regulated. The infection of the regenerating rat liver by ecotropic retroviruses at the time point of expression of the receptor gene supports the requirement of expression of this transporter for infection.