ANTIVIRAL ACTIVITY OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEASE INHIBITORS IN A SINGLE-CYCLE OF INFECTION - EVIDENCE FOR A ROLE OF PROTEASE IN THE EARLY PHASE
K. Nagy et al., ANTIVIRAL ACTIVITY OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEASE INHIBITORS IN A SINGLE-CYCLE OF INFECTION - EVIDENCE FOR A ROLE OF PROTEASE IN THE EARLY PHASE, Journal of virology, 68(2), 1994, pp. 757-765
The antiviral activities of two substrate-based inhibitors of human im
munodeficiency virus type 1 (HIV-1) protease, UK-88,947 and Ro 31-8959
, were studied in acute infections. H9 and HeLaCDC-4LTR/beta-gal cells
were infected either with HTV-1(IIIB) or a replication-defective viru
s, HIV-gpt(HXB-2). Both inhibitors were capable of blocking early step
s of HIV-1 replication if added to cells prior to infection. Partial i
nhibition was also obtained by addition of inhibitor at the time of or
as late as 15 min after infection. The inhibitors were ineffective if
added 30 min postinfection. The inhibitory effects were studied by cD
NA analysis,vith PCR followed by Southern blot hybridization and by in
fectivity assays allowing quantitation of HIV-1 in a single cycle of r
eplication. When UK-88,947-treated H9 cells were coinfected with HIV-1
and human T-cell leukemia virus type I only the replication of HIV-I
was inhibited, demonstrating viral specificity. Pretreating the infect
ious virus stocks with the inhibitors also prevented replication, indi
cating that the inhibitors block the action of the viral protease and
not a cellular protease. A panel of primer sets aas used to analyze cD
NA from cell lysates by PCR amplification at 4 and 18 h postinfection.
Four hours after infection, viral specific cDNA was detected with all
of the four primer pairs used: R/U5, nef/U3, 5' gag, and long termina
l repeat (LTR)/gag. However, after 18 h, only the R/U5 and nef/U3 prim
er pairs and not the 5' gag or LTR/gag primer pair were able to allow
amplification of cDNA. The results suggest a crucial role of HN-I prot
ease in the early phase of viral replication. Although it is not clear
what early steps are affected by the protease, it is likely that the
target is the NC protein, as referred from our previous reports of the
in situ cleavage of the nucleocapsid (NC) protein by the viral protea
se inside lentiviral capsids. The results suggest that it is not the i
nhibition of initiation and progression of reverse transcription but t
he stability of full-size unintegrated cDNA which is affected in the p
resence of protease inhibitors. Alternatively, the cleavage of the NC
protein may be required for the proper formation of preintegration com
plex and/or for its transport to the nucleus.