title: Mutations in matrix and SP1 repair the packaging specificity of a Human Immunodeficiency Virus Type 1 mutant by reducing the association of Gag with spliced viral RNA.
abstract: BACKGROUND
  The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1) sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants.
  
  
  RESULTS
  HIV-1 lacking SL1 (NLΔSL1) did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K) on day 18 postinfection and C1907T in the SP1 domain (P10L) on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1.
  
  
  CONCLUSIONS
  HIV-1 evolved to overcome a deletion in SL1 and restored infectivity by acquiring compensatory mutations in the N-terminal matrix or SP1 domain of Gag. These data shed light on the functions of the N-terminal matrix and SP1 domains and suggest that both regions may have a role in Gag interactions with spliced viral RNA.
authors.affiliation: Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York, USA.
authors.forename: Natalia
authors.initials: N
authors.lastname: Ristic
authors.forename: Mario P S
authors.initials: MP
authors.lastname: Chin
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D002460:Cell Line; D015488:HIV Antigens / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D020121:5' Untranslated Regions; D015488:HIV Antigens; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1
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