5234 Nucleic Acids Research, 2011, Vol. 39, No. 12 potentially inhibitory features, and signiﬁcant levels of the Gag structural proteins are expressed from the viral RNAs that contain the full-length 50-UTR. It has been suggested that the HIV-1 RNA contains an internal ribosomal entry site (IRES) downstream of these inhibitory structure motifs (41,42). IRES elements were initially described in picornaviruses and have since been identiﬁed in many viral and cellular mRNAs (43–47). IRES elements usually function under circumstances in which regular cap-dependent scanning is impaired, for example, following expression of a viral factor or in response to cell stress signals, such as cell-cycle arrest or heat shock (48,49). In fact, HIV-1 has been suggested to modulate host cell translation. The viral protease may cleave the translation initiation factor eIF4GI, which is essential for cap-dependent translation initiation (50–53). In addition, the viral Vpr protein causes a G2/M cell cycle arrest (54–57), which is essential for optimal viral replica- tion and which may affect translation efﬁciency in general (58,59). Thus, the presumed HIV-1 IRES was suggested to allow optimal viral gene expression under these scanning-impaired conditions (41). It has been reported though that the host shut off observed during HIV infec- tion is caused mainly by degradation of cellular mRNAs and less so by impairment of their translation (60). The mechanism of HIV-1 translation initiation remains under debate (61). Potential IRES elements have been identiﬁed in the 50-UTR and Gag sequences (41,42). In the latter case, ribosomal scanning in the 30–50 direction was proposed to allow synthesis of the Gag and Gag-Pol proteins from the upstream Gag start codon. The two studies seem to contradict each other: the Brasey paper described no IRES activity in the Gag ORF and the Buck paper did not ﬁnd such activity in the 50-UTR. Moreover, other groups have not been able to conﬁrm either data (62,63). We used a different approach to gain further insight into the translation initiation mechanism on HIV-1 mRNAs. We addressed ribosomal scanning in the 50-UTR by introducing upstream AUGs (uAUGs). Scanning ribosomal 40S subunits will recognize the uAUG if scanning initiated upstream. Translation will commence on the introduced ORF followed by ribosomal release. When scanning occurs over the entire 50-UTR, translation from the downstream authentic Gag start codon will be reduced irrespective of the location of the introduced uAUG codons (Kozak, 1984). In the case that HIV-1 50-UTR harbours an IRES, the introduced uAUGs will affect gene expression only if they are located down- stream of the IRES, similarly as was shown for picornaviruses (64–66). By placing uAUG codons at dif- ferent positions across the 50-UTR, the window of riboso- mal scanning and the position of a putative IRES element can thus be determined. MATERIALS AND METHODS DNA constructs this luciferase reporter is a fusion product of the amino-terminal 25 Gag amino acids, the Flag peptide and the ﬁreﬂy luciferase protein. Protein expression is under control of the HIV-1 LAI 50-LTR promoter and 50-UTR sequences. To optimize cloning procedures we deleted the Flag sequences and the NcoI site at the luciferase start codon with a polymerase chain reaction (PCR) on pLTR-gag-ﬂag-luc with primers TA033 and TA056 (Table 1). The PCR fragment was digested with HindIII and BspHI and ligated in the HindIII– NcoI-digested pLTR-gag-ﬂag-luc. This PCR mutagenesis created a SalI site upstream of the luciferase initiation codon and generated the plasmid pLTR-25-gag-luc that was used for subsequent mutagenesis. uAUG and uUAG mutations were introduced by PCR mutagenesis. Oligonucleotide primers are listed in Table 1. Forward primers were used in combination with TA056 and reverse primers with TA033. PCR products were used as overlapping templates in a PCR reaction with primers TA033 and TA056. The resulting PCR products were digested with HindIII and SalI, cloned into the pLTR-25-gag-luc vector and sequenced. The constructs with eight individually inserted uAUG codons were designated A1–A8 and those with a stop codon at the same position U1–U8. The constructs A1–A5 and A2–A5 were created by di- gesting the A1, A2 and A5 luciferase constructs with NcoI and SalI. The A5 fragment was cloned into the A1 and A2 vectors to construct A1–A5 and A2–A5, respectively. The U1–U5 and U2–U5 were created similarly with the U1, U2 and U5 constructs; however, digestions were per- formed with AvrII and SalI. The construct A2LF was created by PCR mutagenesis using primers TA110 and TA056, and pLTR-25-gag-luc as the template, resulting in the insertion of a G residue at position 124 of the HIV-1 leader sequence. The PCR fragment was digested with NcoI and SalI, and ligated into pLTR-A2-25-gag-luc as described above. The U2-LF control construct was created similarly using primers TA231 and TA056 in the PCR and HindIII and SalI as restriction sites for further construction. The insertional mutagenesis was conﬁrmed by sequence analysis for all constructs. Plasmid pRL-CMV expresses the Renilla luciferase reporter gene under the control of the CMV promoter (Promega). pcDNA3-Tat (67) expresses the HIV-1 LAI Tat protein under the control of the CMV promoter and is a derivate of the pcDNA3 vector (Invitrogen). The HIV-1 molecular clone pLAI has been described previous- ly (68). Cell culture C33A cells, a human cervix carcinoma cell line, were grown in Dulbecco’s MOD Eagle medium (DMEM) sup- plemented with 10% FBS, non-essential amino acids (Invitrogen), 20 mM glucose, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 C and 5% CO2. Transfection assays The plasmid pLTR-gag-ﬂag-luc was used for HIV-1 50-UTR mutagenesis (25). The protein expressed from Reporter gene expression driven by the wild-type (wt) and mutant 50-UTR was analysed in C33A cells. Cells were