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Journal of Virology, December 2003, p. 12592-12602, Vol. 77, No. 23
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.23.12592-12602.2003

Human Immunodeficiency Virus Type 1 N-Terminal Capsid Mutants Containing Cores with Abnormally High Levels of Capsid Protein and Virtually No Reverse Transcriptase

Shixing Tang,1 Tsutomu Murakami,2,{dagger} Naiqian Cheng,3 Alasdair C. Steven,3 Eric O. Freed ,2,{ddagger} and Judith G. Levin1*

Laboratory of Molecular Genetics, National Institute of Child Health and Human Development,1 Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases,2 Laboratory of Structural Biology Research, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 208923

Received 3 July 2003/ Accepted 27 August 2003

We previously described the phenotype associated with three alanine substitution mutations in conserved residues (Trp23, Phe40, and Asp51) in the N-terminal domain of human immunodeficiency virus type 1 capsid protein (CA). All of the mutants produce noninfectious virions that lack conical cores and, despite having a functional reverse transcriptase (RT), are unable to initiate reverse transcription in vivo. Here, we have focused on elucidating the mechanism by which these CA mutations disrupt virus infectivity. We also report that cyclophilin A packaging is severely reduced in W23A and F40A virions, even though these residues are distant from the cyclophilin A binding loop. To correlate loss of infectivity with a possible defect in an early event preceding reverse transcription, we modeled disassembly by generating viral cores from particles treated with mild nonionic detergent; cores were isolated by sedimentation in sucrose density gradients. In general, fractions containing mutant cores exhibited a normal protein profile. However, there were two striking differences from the wild-type pattern: mutant core fractions displayed a marked deficiency in RT protein and enzymatic activity (<5% of total RT in gradient fractions) and a substantial increase in the retention of CA. The high level of core-associated CA suggests that mutant cores may be unable to undergo proper disassembly. Thus, taken together with the almost complete absence of RT in mutant cores, these findings can account for the failure of the three CA mutants to synthesize viral DNA following virus entry into cells.

* Corresponding author. Mailing address: Laboratory of Molecular Genetics, NICHD, Building 6B, Room 216, NIH, Bethesda, MD 20892-2780. Phone: (301) 496-1970. Fax: (301) 496-0243. E-mail: levinju{at}mail.nih.gov.

{dagger} Present address: Department of Immunology, Graduate School and Faculty of Medicine, University of the Ryukyus, Uehara 207, Nishihara, Okinawa 903-0215, Japan.

{ddagger} Present address: HIV Drug Resistance Program, NCI-Frederick, Frederick, MD 21702-1201.

Journal of Virology, December 2003, p. 12592-12602, Vol. 77, No. 23
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.23.12592-12602.2003




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