Aleš Zábranský

The Mason-Pfizer Monkey Virus Internal Scaffold Domain Enables In Vitro Assembly of Human Immunodeficiency Virus Type 1 Gag

ABSTRACT The Mason-Pfizer monkey virus (M-PMV) Gag protein possesses the ability to assemble into an immature capsid when synthesized in a reticulocyte lysate translation system. In contrast, the human immunodeficiency virus (HIV) Gag protein is incapable of assembly in parallel assays. To enable the assembly of HIV Gag, we have combined or inserted regions of M-PMV Gag into HIV Gag. By both biochemical and morphological criteria, several of these chimeric Gag molecules are capable of assembly into immature capsid-like structures in this in vitro system. Chimeric species containing large regions of M-PMV Gag fused to HIV Gag sequences failed to assemble, while species consisting of only the M-PMV p12 region, and its internal scaffold domain (ISD), fused to HIV Gag were capable of assembly, albeit at reduced kinetics compared to M-PMV Gag. The ability of the ISD to induce assembly of HIV Gag, which normally assembles at the plasma membrane, suggests a common requirement for a concentrating factor in retrovirus assembly. Despite the dramatic effect of the ISD on chimera assembly, the function of HIV Gag domains in that process was found to remain essential, since an assembly-defective mutant of HIV CA, M185A, abolished assembly when introduced into the chimera. This continued requirement for HIV Gag domain function in the assembly of chimeric molecules will allow this in vitro system to be used for the analysis of potential inhibitors of HIV immature particle assembly.

Premature processing of mouse mammary tumor virus Gag polyprotein impairs intracellular capsid assembly

Mouse mammary tumor virus (MMTV) is the prototypical member of the Betaretrovirus genus, but the processes of its morphogenesis are poorly characterized. In this report, we describe an unusual intracellular processing of MMTV Gag polyprotein in human 293T cells transiently expressing MMTV from heterologous promoter. The same specific cleavage products of the viral protease were seen for the wild type as well as for nonmyristylated mutant of MMTV Gag polyprotein completely defective in the particle release. Inactivation of the viral protease resulted in more stable Gag polyprotein and in accumulation of intracytoplasmic particles for nonmyristylated Gag. The intracellular processing of nonmyristylated MMTV Gag indicates that protease activation in betaretrovirus can occur independently of budding.

The Noncanonical Gag Domains p8 and n Are Critical for Assembly and Release of Mouse Mammary Tumor Virus

ABSTRACT The mouse mammary tumor virus (MMTV) Gag contains the unique domains pp21, p3, p8, and n. We investigated the contribution of these domains to particle assembly and found that the region spanning the p8 and n domains is critical for shape determination and assembly. Deletion of pp21 and p3 reduced the number of released particles, but deletion of the n domain resulted in frequent formation of aberrant particles, while deletion of p8 severely impaired assembly. Further investigation of p8 revealed that both the basic and the proline-rich motifs within p8 contribute to MMTV assembly.

Subsequent Selfprocessing of Bovine Leukemia Virus Proteinase in Vitro

Bovine leukemia virus (BLV) is a B-cell lymphotropic retrovirus closely related to human T-cell leukemia viruses (HTLV-1 and -2)1 BLV can naturally infect cattle in which it induces a disease complex termed enzootic bovine leukosis. Experimental infection of rabbits, however, causes severe depression in. immune function.2 Although BLV and HTLV are grouped with C-type oncoviruses, during assembly they can be barely distinguished from lentiviruses, forming thus a specific group.3

One-step separation of myristoylated and nonmyristoylated retroviral matrix proteins

N-terminal myristoylation of retroviral matrix proteins is essential for the targeting of the Gag polyproteins to the plasma membrane. To investigate the effect of the myristoylation on the structure and membrane binding ability of the matrix proteins, it is necessary to prepare their myristoylated forms. We present purification of myristoylated matrix proteins of the mouse mammary tumor virus and murine leukemia virus, two morphogenetically distinct retroviruses. The proteins were expressed in Escherichia coli coexpressing a yeast N-myristoyltransferase. This E. coli expression system yielded a mixture of myristoylated and nonmyristoylated matrix proteins. We established efficient one-step metal affinity purification that enabled to obtain pure myristoylated matrix proteins suitable for structural and functional studies.

Analysis of Autoprocessing of Mason-Pfizer Monkey Virus Proteinase in Vitro

Mason-Pfizer Monkey Virus (M-PMV, also called SRV-3) is a primate retrovirus that was first isolated from a spontaneous breast carcinoma of a female rhesus monkey Macaca mulatta.1,2 Infection of macaque species with M-PMV causes an AIDS-like disease.3,6 M-PMV is characterized by the self-assembly of the Gag, Gag-Pro and Gag-Pro-Pol precursors into intracytoplasmic particles (procapsids) within the infected cell cytoplasm. These morphogenetic properties of the virus are typical for D-type retrovirus.7,8