Capsid is a dominant determinant of retrovirus infectivity in nondividing cells

Abstract

A major difference between lentiviruses such as human immunodeficiency virus (HIV) and most other retroviruses is their ability to productively infect nondividing cells. We present here genetic evidence for involvement of the capsid protein (CA) in the infectious phenotype in nondividing cells. A chimeric HIV type 1 (HIV-1) in which the MA and CA of HIV-1 are replaced with the MA, p12, and CA encoding sequences from murine leukemia virus (MLV) loses the ability to efficiently infect nondividing cells. Analysis of the accumulation of two-long-terminal-repeat circles implies that the impairment of nuclear transport of preintegration complexes is responsible for the restricted infection of this chimeric virus in nondividing cells. Incorporation of MLV MA and MLV p12 into HIV virions alone does not exert any adverse effects on viral infection in interphase cells. These results suggest that CA is the dominant determinant for the difference between HIV and MLV in the ability to transduce nondividing cells.

FIG. 1.

Generation of the infectious chimeric virus MHIV-mMA12CA. (A) Schematic representation of genomic organization of HIV-1, MLV, and MHIV (upper). Protein structure of the gag gene of these viruses is also shown (lower). MHIV-mMAp12CA encodes the MLV MA, p12, and CA and all the HIV-1 genes other than the HIV MA and CA. (B) Western blot analysis of purified virus particles of MHIV-mMA12CA together with HIV-1 and MLV. Polyprotein processing was tested for HIV-1 MA (p17gag) and CA (p24gag) as well as MLV MA (p15gag) and CA (p30gag) in addition to HIV-1 IN. Some unprocessed Gag and Gag-Pol proteins remain in the lane of MHIV-mMA12CA, but the anti-IN profile of MHIV-mMA12CA is the same as that of parental HIV-1 (not shown in this figure). (C) Single-cycle infectivity of MHIV-mMA12CA. Single-cycle infections of MHIV-mMA12CA were tested either alone or with envelope expression of VSV-G or a truncated HIV-1 Env in trans. Infectivity was measured with the MAGI assay by counting β-galactosidase-positive cells 2 days postinfection using viruses harvested after transfection of proviral clones with or without env expression vectors into 293T cells. These data are representative of three independent experiments.

FIG. 2.

Electron microscopy analysis of virions of HIV (left), MHIV-mMA12CA (center), and MLV (right) produced from 293T cells transfected with the respective proviruses. Bar, 100 nm.

FIG. 3.

MHIV-mMA12CA does not establish efficient infection in nondividing cells. (A) Infectivity of MHIV in aphidicolin-treated cells. MAGI cells were treated in the presence or absence of aphidicolin and challenged with virus stocks. Virus titers are expressed as the number of β-galactosidase-expressing blue cells per milliliter. These data are representative of five independent experiments. (B) Infectivity of MHIV in γ-irradiated cells. GHOST cells were treated with 3,500 rads from a ¹³⁷cesium source. HIV and concentrated MHIV, all of which were pseudotyped with the VSV-G envelope protein, were spinoculated into normal (dividing) or γ-irradiated (nondividing) GHOST cells. Two days after infection, the cells were fixed and analyzed with flow cytometry for GFP-expressing GHOST cells. Dotted lines indicate zidovudine-treated control cells. (C) Macrophage infection with MHIV-mMA12CA. Monocyte-derived macrophages were prepared by adherence to the bottom of wells and maintained for 10 to 14 days before infection. Infectivity is shown as the relative percentage of luciferase titer in HeLa cells per that in macrophages. Error bars indicate standard deviations of duplicate cultures. The luciferase titers of these particular data are as follows: 534,677 relative light units (RLU) for HeLa cells infected with HIV; 680,954 RLU for HeLa cells infected with MLV; 137,651 RLU for HeLa cells infected with MHIV; 14,669 RLU for macrophages infected with HIV; 2,281 RLU for macrophages infected with MLV; 178 RLU for macrophages infected with MHIV. These values are averages of duplicate wells. The data presented here are representative of at least four different independent experiments using different blood donors.

FIG. 4.

(A) Dose-independent restriction of MHIV-mMA12CA in nondividing cells. Aphidicolin-treated HeLa cells were infected with increasing amount of luciferase-encoding viruses. Culture supernatants of transfected cells were used as inocula. Virus infectivity was judged by measuring luciferase titers of infected cell lysates 2 days after infection. (B) Transduction of the lacZ gene into dividing and nondividing HeLa cells. MAGI cells were treated in the presence or absence of aphidicolin and challenged with virus stocks by using retroviral vectors encoding the lacZ gene. Virus titers are expressed as the number of β-galactosidase-expressing blue cells per milliliter. These data are representative of two independent experiments.

FIG. 5.

CA determines infectivity in nondividing cells. (A) Schematic illustration of the protein structure of Gag of new chimeric viruses. Note that two new chimeric viruses as well as MHIV-mMA12CA were created based on the HIV-1 infectious clone. (B) Western blot analysis of purified virus particles of new MHIV chimeras. See the legend to Fig. 1B for details. (C) Single-cycle infectivity of MHIV as well as parental MLV and HIV. For details, see the legend to Fig. 1C.

FIG. 6.

Infection of MHIV-mMA12CA is blocked at nuclear transport. Reverse transcription and nuclear transport of MHIV-mMA12CA and parental HIV-1 were monitored by measuring late reverse transcription products (upper) and 2-LTR circles (lower) as markers, respectively. Increasing amounts of virus inocula (x axis) were challenged with either normal or nondividing cells. The y axis indicates the copy number of each DNA analyzed with real-time quantitative PCR.