Jpn. J. Infect. Dis., 53 (2), 88-90, 2000

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Laboratory and Epidemiology Communications

Fv-1 Restriction of Murine Leukemia Virus May Not Necessarily Be at Cytoplasmic-Nuclear Transport Phase

Takashi Odawara*

AIDS Research Center, National Institute of Infectious Diseases,
Gakuen 4-7-1, Musashimurayama-shi, Tokyo 208-0011

Communicated by Hiroshi Yoshikura

(Accepted May 29, 2000)


Fv-1 restriction of murine leukemia virus (MuLV) determined by two amino acids in p30 (CA) of the viral protein (1-2) is considered to occur during the post-penetration phase of infection (3-5). Since the formation of circular proviral DNA, which is considered to reflect the entrance of pre-integration complex into the nucleus has been seen to be impaired in the restrictive cells (6-7), it has been speculated that the cytoplasmic-nuclear transport of the provirus was inhibited in restrictive cells. However, whether Fv-1 acts in cytoplasm, in the nucleus, or in both is still unknown.

I report here that an MuLV, which was constructed by replacing the gag region of NB-tropic Moloney MuLV with N-tropic WN1802N-derived gag, N-MoF (8), did not show any inhibition of formation of the circular intermediate, though its titration pattern was that of a typical N-tropic virus.

Figure 1 shows the structures of N-MoF and a similar B-tropic construct, B-MoFdA (9). In XC assay (10), both N-MoF and B-MoFdA showed the expected titration pattern of N-tropic and B-tropic viruses (Fig.2).

In order to examine the formation of linear and circular proviruses in the infected cells, cells plated in an amount of 2 x106 cells/10 cm dish were infected with the viruses in the presence of 0.8 ƒÊg/ml of polybrene for 5 h. At indicated periods after the start of infection, DNAs were extracted according to Hirt's method (11). One third of the DNAs extracted from each plate was electrophoresed in 0.8% agarose gel, and analyzed by Southern blotting using an Xba1-Xba1 (#5766-#8113) fragment of Moloney MuLV env as a probe.

As shown in Figure 3-A, though formation of the circular proviral DNA of WN1802N was inhibited in C57BL/6-derived B-type YH-7 cells, that of N-MoF was not. That of B-MoFdA in N-type NIH3T3 cells was inhibited as expected (Fig. 3-B).

The above observation showed that the Fv-1 restriction could take place without inhibition of the formation of circular DNA. The circular form is not considered a pre-integration molecule itself but rather a by-product (12). Therefore, this discrepancy may not be surprising. However, as the circular form is strictly nuclear (13), the restriction of N-MoF was exerted probably after entrance to the nucleus; i.e., it was inferred that Fv-1 restriction can occur in the nuclear phase. As WN1802N and N-MoF had gag regions with the same sequence, it is speculated that a region other than gag is able to determine the impaired formation of circular DNA.

REFERENCES

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*Corresponding author: E-mail:odawara@cshl.org

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