Biochemical analysis of retroviral structural proteins to identify and quantify retrovirus expressed by an NS0 murine myeloma cell line.

A subclone of the NS0 murine myeloma cell line, frequently used to produce recombinant monoclonal antibodies, was found by a transmission electron microscopy method to express a surprisingly high titer of 10(11) retroviral particles per ml of culture supernatant. Infectivity assays showed a very low infectious titer with the restricted host range expected for a murine amphotropic retrovirus. A Western blot assay for the viral capsid protein was developed to confirm the high titer values and provide a means for monitoring batch consistency and virus removal during the purification process. Mass spectrometry of several of the viral Gag proteins demonstrated that the cell line appeared to produce at least two closely related retroviruses. N-terminal sequencing of three of the Gag proteins demonstrated that these retroviruses were members of the murine leukemia retroviral family. Western blot detection with an antibody for the capsid protein gave a linear standard curve over the range of 0.1-3 ng per lane. This allows the detection of viral titers as low as 6x10(7) virions per ml without the need to concentrate the sample. The Western blot method has higher throughput and less variability than transmission electron microscopy methods and has potential for monitoring viral titer and clearance during development of manufacturing processes.

Quantitative competitive reverse transcription-PCR as a method to evaluate retrovirus removal during chromatography procedures.

Chinese hamster ovary cells used for pharmaceutical protein production express non-infectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing process to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for validation studies. Some chromatography procedures used for pharmaceutical protein purification utilize low pH (< pH 4.0) elution buffers which readily inactivate X-MuLV. Therefore, cell-based infectivity assays are unable to evaluate the physical removal of X-MuLV by these chromatography procedures. To distinguish viral inactivation by low pH treatment from viral removal by chromatography, a quantitative competitive reverse transcription PCR method capable of quantifying both infectious and non-infectious X-MuLV has been developed. This method quantifies X-MuLV particles in chromatography pools by quantifying the X-MuLV particle RNA (pRNA). The difference between the amount of X-MuLV pRNA in the load pool and the product-containing elution pool represents the extent of X-MuLV removal. This method is an extremely powerful complement to cell based-infectivity assays as it allows physical removal of X-MuLV by chromatography and filtration procedures to be distinguished from X-MuLV inactivation when buffers with the ability to inactivate retrovirus are used.

Recent studies on retrovirus-like particles in Chinese hamster ovary cells.

Highly concentrated (4000-7000-fold) culture fluids from CHO cells were analysed for the presence of retrovirus-like activity. Concentrates containing reverse transcriptase activity were detected and further purified by sucrose density gradient centrifugation. Particles banding at 1.13-1.16 g/ml were found to contain nucleic acid sequences and structural proteins related to those found in murine and other retroviruses. Analysis of the endonuclease gene has shown no intact open reading frames. Approximately 100-300 copies of the C-type sequences were present in the genome of CHO cell lines as well as in the DNA extracted from Chinese hamster liver, indicating that these sequences are present in the germ line of the species. Concentrates were analysed for infectivity by direct inoculation and co-cultivation with a series of detector cells. No evidence of infectivity was detected by reverse transcriptase, mink cell S+L- focus assay or by electron microscopic analysis of the inoculated detector cells after at least four passages in culture.

Replication of HIV-1 in a wide variety of animal cells following phenotypic mixing with murine retroviruses.

Human T cells co-infected with the human immunodeficiency virus type 1 (HIV-1) and the xenotropic or dual-tropic mouse type C virus (MuLV) give rise, by phenotypic mixing, to progeny virus that can transfer HIV-1 into a wide variety of mammalian and avian cells. Differences in the extent of HIV-1 replication in these animal cells can be observed. Replication is best in human cells, but occurs substantially in cells from many animal species including mink, horse, and bush wallaby. Virus production in murine and avian cells is very limited. These results confirm that the major block to HIV-1 infection of animal cells is at the cellular surface but that intracellular regulation of viral replication is also involved. Moreover, an enhancement of HIV-1 cytopathic effects can be seen in human cells co-infected by MuLV. All these data suggest phenotypically mixed viruses might be useful for developing an animal model system for studying AIDS, and that the pathological expression of HIV-1 could be modified by the presence in cells of other retroviruses. They also indicate a potential mechanism by which HIV strains can be generated with an increased ability to spread in nature.

Neutralization of mouse xenotropic virus by lipoproteins involves binding to the virions.

We examined the properties of the mouse serum lipoprotein responsible for specific neutralization of the endogenous mouse xenotropic (X-tropic) type C retrovirus. The anti-X-tropic virus activity of the mouse lipoprotein neutralizing factor (NF) could not be blocked by sugars or lectins. Moreover, neutralization did not involve rupture of the virion core. Sucrose density gradient analysis of X-tropic virus mixed with various lipoprotein preparations demonstrated that the binding of complete virions to NF is associated with the neutralization.

The AIDS-associated retrovirus is not sensitive to lysis or inactivation by human serum.

Unheated human serum does not lyse the AIDS-associated retrovirus (ARV), change the density of the virus, or suppress its ability to infect human peripheral mononuclear cells. The results indicate that ARV and the human oncovirus HTLV-I, unlike other animal retroviruses, are resistant to the effect of human serum. These two human viruses coming from different retrovirus subfamilies may be pathogenic because of this lack of sensitivity to human complement components.