Viruses viral protein production

Okui N, Kobayashi N, Kitamura Y (1998) Production of uninfectious human immunodeficiency virus type 1 containing viral protein R fused to a single-chain antibody against viral integrase. J Virol 72 6960-6964... 

In general terms, four main stages can be recognized in the multiplication of human viruses, (i) attachment (ii) penetration and uncoating (iii) production of viral proteins and replication of viral nucleic acid, (iv) assembly and release of progeny viruses. 

Once potent ligands for a viral protein are identified, further advancement depends on demonstrating activity in cells. Unfortunately, reproducible in vitro viral replication assays for HCV have not been reported. There are scattered reports that a very low level of genome replication, or even virus production, can be observed under certain circumstances [56]. However, recently specific sequences yielding relatively reproducible replication, at consistently detectable levels have been reported [57]. In the coming years these may allow routine assays suitable for compound evaluation to be developed, but to date drug discovery must rely on other cell culture models. 

Removal of viruses from the product stream can be achieved in a number of ways. The physicochemical properties of viral particles differ greatly from most proteins, ensuring that effective fractionation is automatically achieved by most chromatographic techniques. Gel-filtration chromatography, for example, effectively separates viral particles from most proteins on the basis of differences in size. 

To determine if the high in vitro potents of the anti-HIV compound 30 translates into antiviral efficiency in vivo, Datema et al. investigated the inhibition of HIV-1 production and of depletion of human T cells in HIV-1-infected SCID-hu Thy/Liv mice [37]. Steady levels of 100 ng of 30 or higher per mL in plasma resulted in significant inhibition of HIV p24 protein formation. Daily injection of 30 caused a dose-dependent decrease in viral p24 production, and this inhibition could be potentiated by coadministration of AZT (or DDI). This study suggested that 30 alone or in combination with the licensed anti-HIV agents AZT and DDI may decrease the virus load in HIV-infected patients and, by extension, that the infectious cell entry step is a valid target for antiviral chemotherapy of HIV disease. 

Recently, it has been found that NO donors inhibit HIV-1 replication in acutely infected human peripheral blood mononuclear cells (PBMCs), and have an additive inhibitory effect on HIV-1 replication in combination with 3 -azido-3 -deoxythymisylate (AZT) [139, 140]. S-nitrosothiols (RSNOs) inhibit HIV-1 replication at a step in the viral replicative cycle after reverse transcription, but before or during viral protein expression through a cGMP-independent mechanism. In the latently infected U1 cell line, NO donors and intracellular NO production stimulate HIV-1 reactivation. These studies suggest that NO both inhibits HIV-1 replication in acutely infected cells and stimulates HIV-1 reactivation in chronically infected cells. Thus, NO donors may be useful in the treatment of HIV-1 disease by inhibiting acute infection, or reactivating a latent virus. 

Despite these theoretical concerns, a number of HIV vaccines are under development. Most of these vaccines have been developed by recombinant DNA techniques that have allowed a large-scale production of individual viral proteins. The predominant HIV proteins that make up these potential vaccines are env proteins (e.g., gp 120) and, to a lesser extent, gag proteins. In addition, inactivated whole HIV virus is being tested. 

One experimental tool in this direction is provided by some enveloped animal viruses which mature at the cell surface of infected cells (K Sri inen and Renkonen, 1977 Lenard, 1978). Such viruses include influenza virus, Semliki Forest virus (SFV), Sindbis virus, and vesicular stomatitis virus (VSV). They are extremely simple in makeup and hence are very well characterized. They can be tagged with biochemical probes in many different ways. They infect many animal cells in culture, and after infection turn the cells into factories for the production of virus progeny. The protein-synthesizing machinery of the host cell is programmed by the viral RNA to make viral proteins exclusively and these include the viral surface glycoproteins. These are synthesized with signal peptides and inserted into the ER membrane (Katz et ai, 1977 Garoff et... 

Interferons (IFN) are glycoproteins that, among other products, are released from virus-infected cells. In neighboring cells, interferon stimulates the production of "antiviral proteins." These inhibit the synthesis of viral proteins by (preferential) destruction of viral DNA or by suppressing its translation. Interferons are not directed against a specific virus, but have a broad spectrum of antiviral action that is, however, species-specific. Thus, interferon for use in humans must be obtained from cells of human origin, such as leukocytes (IFN-a), fibroblasts (IFN-P), or lymphocytes (IFN-y). Interferons are also used to treat certain malignancies and autoimmune disorders (e.g., IFN-a for chronic hepatitis C and hairy cell leukemia IFN-p for severe herpes virus infections and multiple sclerosis). 

Figure 6.1 An RNA viral gene therapy vector, engineered to carry the genetic information for a protein product, is incubated with blood or bone marrow cells removed from a patient. The foreign DNA sequence integrates into the cell s genetic material and when the cells are returned to the patient, they direct the production of the protein product. RNA gene therapy vectors are engineered to be unable to direct the production of new virus particles.

The application of mathematical modelling to baculovirus infection and virus-like particle production was also successfully done to Parvovirus B19 viruslike particle production with two different baculovirus at low MOTs [18]. But in this model the same concepts proposed in the Licari and Bailey Model was applied, i. e. baculovirus infection follows Poisson distribution with mean and variance equal to a.MOI, but with co-infection with two single-vectors, each one encoding a specific viral protein. 

Following their production, the viral components are assembled to form a mature virus particle. The viral genome is encapsulated by viral protein in some cases (e.g. adenovirus, poliovirus), it is not encapsulated. In certain viruses, such as the poxviruses, multiple membranes surround the capsid. Release of the virus from the host cell may be rapid and produce cell lysis and death. A slower process resembling budding may allow the host cell to survive. 

Src kinase was discovered during the search for the tumor-causing principle of retroviruses. The viral oncogene product of these viruses, v-Src kinase, was the first tyrosine kinase to be identified. In comparison to its cellular coimterpart, c-Src kinase, v-Src lacks the autoinhibitory structural element that controls protein kinase activity. As a consequence of this loss, v-Src kinase is constitutively active and is a potent transforming protein. 

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