A conserved mechanism of retrovirus restriction in mammals

Abstract

The murine Fv1 gene restricts infection by N- or B-tropic murine leukemia viruses at a postentry, preintegration stage. The Fv1-sensitive viruses previously used for the study of Fv1 encode an ecotropic envelope gene and thus only infect rodent cells. Consequently, the study of Fv1 restriction has been carried out solely in mice and murine cell lines. By infection with retroviral vectors containing N- or B-tropic core and pantropic vesicular stomatitis virus-G envelope protein, we now demonstrate that cell lines derived from various mammalian species, including humans, have an Fv1-like retrovirus restriction function, preventing N-tropic vector infection. Like Fv1, restriction is directed at amino acid 110 of the viral capsid protein. In contrast to Fv1, the novel restriction is characterized by the absence of reverse-transcribed viral DNA. We speculate that these activities have been selected for by retroviral epidemics in the distant past.

Figure 1

Fv1-like restriction to N-tropic virus infection in nonmurine cells. Cells were infected with equal titers of N- and B-tropic virus. Twenty-four hours later, the numbers of infected cells were determined by FACS analysis. Log B/N values were obtained by dividing the percentage of cells infected with N-tropic virus by the percentage cells infected with B-tropic virus and calculating the log value. Cells with Fv1ⁿ-like phenotype therefore have negative values and those with Fv1^b-like phenotype have positive values. Values close to zero represent Fv1-null lines. Errors are standard error. Mu, mouse; Rt, rat; SH, Syrian hamster; CH, Chinese hamster; M, mink; F, ferret; R, rabbit; Ca, cat; D, dog, P, pig; C, cow; AGM, African green monkey.

Figure 2

Viral determinants of the Fv1-like restriction. Human HT1080 cells were infected with equal titers of viruses carrying reciprocal mutations at the four positions distinguishing pCIG3 N and B. FACS profiles (side scatter versus eGFP fluorescent intensity) were measured 1 day later.

Figure 3

Dominant restriction of virus particles containing both N- and B-tropic gag-pol. NIH 3T3 cells (A) and HT1080 cells (B) were infected with equal amounts of chimeric vectors containing different ratios of N- and B-tropic CA molecules within the same virions (squares) or mixtures of N- and B-tropic virus (circles). Twenty-four hours later, the number of eGFP-positive cells was determined by FACS analysis. y axis values were calculated by setting the % infected cells at 100% for neat nonrestricted virus. x axis values were calculated by expressing the amount of N-tropic (gag or virus) as a percentage of the total. Errors are standard error.

Figure 4

Inhibition of viral DNA synthesis in newly infected cells. Mus dunni, BALB 3T3, NIH 3T3, HT1080, and Vero cells were infected with equal amounts of N- and B-tropic virus. Six hours later, total cellular DNA was extracted and tested by PCR for linear (Upper) and circular (Lower) forms of viral DNA. Products of PCR were run out on 1% (Upper) or 1.5% (Lower) agarose gels and visualized by ethidium bromide staining. Lanes labeled −− contain products of PCR from uninfected cells. The position of the 564-bp λ/HindIII marker is indicated with an arrow. The presence of a 1.6-kb band in HT1080 extracts that do not contain viral product was consistently observed. The nature of this product is unknown.