Synthesis and circularization of N- and B-tropic retroviral DNA Fv-1 permissive and restrictive mouse cells.

Abstract

Production of various forms of nonintegrated viral DNA was measured in cultured mouse cells carrying different Fv-1 alleles early after infection with N-tropic or B-tropic retroviruses. Quantitative analyses were performed by agarose gel electrophoresis, transfer to diazobenzyloxymethyl-paper, and molecular hybridization. In permissive infection of Fv-n cells (NIH Swiss and DBA mouse strains) with N-tropic virus and of Fv-1 b cells (BALB/c and C57BL/6 strains) with B-tropic virus, form III (double-stranded linear) DNA first appeared at 3-4 hr and reached a maximum at 8-10 hr; two form I (closed circle) DNAs appeared at 7-8 hr and reached a maximum at or beyond 12 hr. In the two Fv-1 b cells infected with N4ropic virus and in DBA (Fv-1 n) cells infected with B-tropic virus, formation of the two form I DNAs was quantitatively restricted but formation of form III DNA was unaltered. In Fv-1" NIH Swiss mouse embryo cells infected with B-tropic virus, the level of form III DNA was markedly depressed and hence the two form I DNAs were not detectable. In C57BL/6 cells as well as in DBA/2 cells 12 hr after infection, the quantity of form III DNA varied directly with the amount of restricted virus, whereas the quantity of form I DNA varied according to the square of the amount of restricted virus. The significance of these results for understanding the molecular basis of retrovirus replication and its restriction by the Fv-1 gene is discussed. The importance of the mouse Fv-1 gene locus in controlling infection by N-and B-tropic retroviruses has been well recognized in both animal studies and cell culture studies (1-5). This locus, mapped on chromosome 4 of the mouse (6), is presumably responsible for the production of specific inhibitor molecules in the cytoplasm of the cell (7-9). Inactivation of the restricted virus particles appears to be mediated by a virus protein, possibly the p30 core protein (10), which serves as a "target" for the Fv-1 gene product (11,12). However, the precise molecular mechanism of Fv-1 gene restriction is still unknown. Results of various previous investigations (13)(14)(15)(16)(17)(18)(19)(20) indicate that Fv-1 restriction occurs intracellularly at an early step of the virus replication cycle. Of particular significance are the observations (i) that, in Fv-1 restricted infection, the quantity of the cellgenome integrated viral DNA is markedly decreased whereas the formation of nonintegrated viral DNA appears to be unaffected (17-18), and (ii) that transfection by infectious integrated viral DNA is not restricted by the Fv-1 locus of the cell (19,20). These data imply that Fv-1 restriction may occur at or prior to the step of viral DNA integration.In the present study, we quantitatively measured the production of double-stranded linear and circular forms of viral DNA in Fv-1 permissive and restrictive cells early after infection with N-and B-tropic murine retroviruses. The results demonstrate that mouse cells may be assigned to either of two classes with regard to their ability to re...

A conserved mechanism of retrovirus restriction in mammals

“…To define more precisely the stage at which restriction occurs, we examined the appearance of linear (newly synthesized plus integrated) and circular (a dead-end product indicative of nuclear entry) (26) viral DNA in cells 6 h after infection. In murine cells exhibiting Fv1 restriction, as shown previously (27,28), equal levels of freshly synthesized DNA but reduced levels of circular DNA were detected, implying a block in nuclear entry and therefore integration (Fig. 4).…”

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

“…The most striking molecular anomaly, a decrease in the levels of supercoiled viral DNA under nonpermissive conditions, has been seen previously under different experimental conditions. We first described it as the main feature of the Fv-J gene restriction (19), and it was later confirmed by others (3,40). The Fv-1 gene maps on mouse chromosome 4 (30) and has been shown to require the presence of a viral determinant to operate (21,26).…”

The human and African green monkey TRIM5α genes encode Ref1 and Lv1 retroviral restriction factor activities

“…The two alleles allow the division of MLV isolates into N-tropic viruses, able to infect NIH cells but not BALB͞c cells, and B-tropic viruses able to infect BALB͞c cells but not NIH cells. These mice, cell lines derived from these mice, and cell lines exogenously expressing the Fv1 proteins are able to specifically block MLV infection after reverse transcription but before integration of the provirus (14)(15)(16)(17). The viral determinant for Fv1 restriction is at position 110 in the capsid (CA) protein.…”