Poeschla, Eric M.

ABSTRACT Encapsidation of retroviral RNA involves specific interactions between viral proteins and cis -acting genomic RNA sequences. Human immunodeficiency virus type 1 (HIV-1) RNA encapsidation determinants appear to be more complex and dispersed than those of murine retroviruses. Feline lentiviral (feline immunodeficiency virus [FIV]) encapsidation has not been studied. To gain comparative insight into lentiviral encapsidation and to optimize FIV-based vectors, we used RNase protection assays of cellular and virion RNAs to determine packaging efficiencies of FIV deletion mutants, and we studied replicative phenotypes of mutant viruses. Unlike the case for other mammalian retroviruses, the sequences between the major splice donor (MSD) and the start codon of gag contribute negligibly to FIV encapsidation. Moreover, molecular clones having deletions in this region were replication competent. In contrast, sequences upstream of the MSD were important for encapsidation, and deletion of the U5 element markedly reduced genomic RNA packaging. The contribution of gag sequences to packaging was systematically investigated with subgenomic FIV vectors containing variable portions of the gag open reading frame, with all virion proteins supplied in trans . When no gag sequence was present, packaging was abolished and marker gene transduction was absent. Inclusion of the first 144 nucleotides (nt) of gag increased vector encapsidation to detectable levels, while inclusion of the first 311 nt increased it to nearly wild-type levels and resulted in high-titer FIV vectors. However, the identified proximal gag sequence is necessary but not sufficient, since viral mRNAs that contain all coding regions, with or without as much as 119 nt of adjacent upstream 5′ leader, were excluded from encapsidation. The results identify a mechanism whereby FIV can encapsidate its genomic mRNA in preference to subgenomic mRNAs.

ABSTRACT Caveolin-1 (Cav-1) is a major protein constituent of caveolae, a type of plasma membrane raft. We observed that coexpression of human Cav-1 with human immunodeficiency virus type 1 (HIV-1) blocked virion production from cells that are ordinarily highly permissive. Further investigation showed that this effect is specific, occurs at low ratios of Cav-1 to HIV-1 DNA, depends on expression of Cav-1 protein, and involves severely impaired expression of HIV-1 proteins. Cav-1 also blocked HIV-2 expression. In contrast, Cav-1 did not inhibit protein expression by a paramyxovirus and did not induce apoptosis or affect cellular morphology, cell viability, or cell cycle progression. Although only small amounts of HIV-1 virions were released from Cav-1-transfected cells, these were fully infectious. Deletion mutagenesis showed that the C-terminal 78 residues were as active as the full-length (178-amino-acid) protein in producing the block. In contrast, the 100 most N-terminal amino acids of Cav-1, which include the previously identified oligomerization and scaffolding domains, were shown to be dispensable. Study of single-amino-acid-exchange mutants of Cav-1 established that palmitoylation was not required. Additional deletion mutants then identified the hydrophobic, membrane-associated domain (residues 101 to 135) as the main determinant. Cellular distribution of wild-type and mutant proteins correlated with ability to block HIV-1 expression. Finally, Cav-2 also blocked HIV-1 expression. These data show that coexpression of caveolins can markedly inhibit expression of HIV proviral DNA and establish that the inhibition is mediated by the hydrophobic, membrane-associated domain.

ABSTRACT A duplication of the polypurine tract (PPT) at the center of the human immunodeficiency virus type 1 (HIV-1) genome (the cPPT) has been shown to prime a separate plus-strand initiation and to result in a plus-strand displacement (DNA flap) that plays a role in nuclear import of the viral preintegration complex. Feline immunodeficiency virus (FIV) is a lentivirus that infects nondividing cells, causes progressive CD4 + T-cell depletion, and has been used as a substrate for lentiviral vectors. However, the PPT sequence is not duplicated elsewhere in the FIV genome and a central plus-strand initiation or strand displacement has not been identified. Using Southern blotting of S1 nuclease-digested FIV preintegration complexes isolated from infected cells, we detected a single-strand discontinuity at the approximate center of the reverse-transcribed genome. Primer extension analyses assigned the gap to the plus strand, and mapped the 5′ terminus of the downstream (D+) segment to a guanine residue in a purine-rich tract in pol (AAAAGAAGAGGTA G GA). RACE experiments then mapped the 3′ terminus of the upstream plus (U+)-strand segment to a T nucleotide located 88 nucleotides downstream of the D+ strand 5′ terminus, thereby identifying the extent of D+ strand displacement and the central termination sequence of this virus. Unlike HIV, the FIV cPPT is significantly divergent in sequence from its 3′ counterpart (AAAAAAGAAAAAAGGGTGG) and contains one and in some cases two pyrimidines. An invariant thymidine located −2 to the D+ strand origin is neither required nor optimal for codon usage at this position. Although the mapped cPPTs of FIV and HIV-1 act in cis , they encode homologous amino acids in integrase.